Research Report - Nikolaus-Fiebiger-Zentrum für Molekulare Medizin

Nikolaus-Fiebiger-Zentrum 

für 

Molekulare Medizin 

Research Report 

2011

Publisher 

Nikolaus-Fiebiger-Center for Molecular Medicine 

Glückstrasse 6 

D-91054 ERLANGEN 

Tel.: + 49 (9131) 85 29109 

Fax: + 49 (9131) 85 29111 

Acting Director: Prof. Dr. Jürgen Behrens 

Email: jbehrens@molmed.uni-erlangen.de 

Editing and Layout 

Dr. Jürgen Behrens and Angela Döbler 

Cover Layout 

Angela Döbler

PREFACE 

Our present report covers the period from 2009 to 2012 and documents the scientific 

achievements at the Nikolaus-Fiebiger Center which precipitated in a number of high 

impact publications in both basic and applied research. The report also highlights the 

personal turnover at our institution. In 2011, we were pleased to welcome Prof. Dr. 

Dominik Müller, expert in hypertension research as the successor of Prof. von der 

Mark at the chair of Experimental Medicine I. Also two new IZKF junior researchers, 

Dr. Beate Winner and Dr. Jens Titze who have their research focus in clinical neurobiology 

and kidney physiology, respectively, started in 2011. They replace Dr. Reinhard 

Voll, now Director of the Division of Rheumatology and Clinical Immunology, Freiburg, 

and Dr. Michael Wiesener, now Professor at the Medical Clinic 4, Erlangen. Several of 

the clinical research group leaders left to take up new positions, including Dr. Falk 

Nimmerjahn, now Professor of Genetics, Erlangen, Dr. Diana Dudziak, now professor 

of Dendritic Cell Biology, Dematology, Erlangen. We are grateful to all previous and 

current members for their cooperation, their assistance in the organisation of the 

house and most importantly for contributing to the high scientific standard of our centre 

by their outstanding publications. 

The goal of our centre is to promote scientific excellence and to foster interactions between 

clinical and basic researcher. A number of publications in internationally distinguished 

and high-impact journals can be found in the individual reference lists. Moreover, 

the center is a meeting place for students taking courses in Molecular Medicine, 

Medicine and Biology and remains attractive for a large number of PhD students and 

PostDocs, frequently from foreign countries. Core facilities in the house, in particular 

the state-of-the art cell sorting facility are used by many scientists from other institutions 

of the University Erlangen. Altogether these various interactions contribute to the 

lively atmosphere of the NFZ. 

On behalf of the steering committee of the NFZ I would like to take the opportunity to 

thank the University and University Clinics for their support, the Advisory Board of the 

NFZ for help in evaluation our research and the many research units from the Medical 

Faculty for their continued interest in our house. 

Erlangen, August 2012 Prof. Jürgen Behrens 

Acting chairman of the NFZ

TABLE OF CONTENTS 

SCIENTIFIC ADVISORY BOARD 1 

GENERAL OVERVIEW 2 

RESEARCH UNITS 5 

DEPARTMENT OF EXPERIMENTAL MEDICINE I 6 

DEPARTMENT OF EXPERIMENTAL MEDICINE II 26 

DIVISION OF MOLECULAR IMMUNOLOGY 37 

HEMATOPOIESIS UNIT 49 

JUNIOR RESEARCH GROUPS 62 

CLINICAL PROJECT GROUPS 80 

TEACHING ACTIVITIES 166 

SEMINARS 167

SCIENTIFIC ADVISORY BOARD 

The scientific standard, productivity and interactions within the Research Center for 

Molecular Medicine are evaluated by an International Scientific Advisory Board. The 

members of the Board are nominated by the Medical Faculty. The board monitors and 

judges the quality and stringency of current and future research projects within the 

Center. Furthermore the Advisory Board evaluates the proposals by young clinicians 

for research terms and makes recommendations to the Extended Steering Committee. 

Prof. Dr. C. Huber 

Medical Clinic III 

Div. of Hematology 

University of Mainz 

55101 Mainz, Germany 

Prof. Dr. K. Knight 

Professor and Chair 

Department of Microbiology 

& Immunology 

Loyola University of Chicago 

Maywood, IL 60153, U.S.A. 

Prof. Dr. D. Heinegard 

Dept. Of Cell and Molecular Biology 

University of Lund 

Box 94 

22100 Lund, Sweden 

Prof. Dr. F. Melchers 

Max-Planck-Institute 

Of Infection Biology 

Charitéplatz 1 

10117 Berlin 

1

GENERAL OVERVIEW 

Overall Structure 

The Nikolaus-Fiebiger-Center of Molecular Medicine is a research institution of the 

Medical Faculty at the Friedrich-Alexander University (FAU) of Erlangen-Nuremberg. 

The center harbours two chairs of Experimental Medicine I and II (Connective Tissue 

Research and Molecular Tumor Research, respectively), a division of Molecular 

Immunology as part of the Department of Internal Medicine III, a division of Genetics 

which belongs to the Department of Genetics of the Science Faculty, as well as two 

junior research groups of the Interdisciplinary Clinical Research Center (IZKF) of the 

Medical Faculty. The intention of the research center is to strengthen biomedical 

research in the Medical School by stimulating cooperations between basic and clinical 

researchers and by giving young clinicians the opportunity to carry out competitive 

biomedical research projects under the infrastructure of a modern research center. 

The Nikolaus-Fiebiger-Center of Molecular Medicine is well equipped with modern 

research facilities required for cell and molecular biological research and offers a 

variety of biochemical, immunological and cell biological seminars, guest lectures and 

common graduate student seminars. Central equipment such as DNA sequencing, 

fluorescence activated cell sorting, confocal laser microcopy, surface plasmon 

resonance are accessible to all scientists and technical personell. All scientists of the 

center are encouraged to seek interactions and cooperations with clinicians inside and 

outside the center, and to participate in the maintenance of the central facilities and 

equipment. 

Steering Committee 

The two full professors of Experimental Medicine, the associate professor and the 

Speaker of the Interdisciplinary Center for Clinical Research (IZKF) are members of 

the Steering Committee that is responsible for the scientific and administrative 

organization of the Research Center. The Steering Comittee elects a speaker every 2 

years. Young clinicians and scientists who are members of the Medical School or the 

University Hospital and whose research is supported by extramural grants are 

encouraged to apply for a maximum of up to 3 years for research space in the center. 

Decisions on their applications are made by the Extended Steering Committee (see 

Scheme) based on recommendations of the Scientific Advisory Board. These research 

groups have access to all central large equipment, library, computers, isotope 

laboratory and other central facilities; in turn, it is expected that they will participate in 

research seminars, journal clubs and teaching. 

2

Financial Concept (Running Costs) 

Running costs of the Research Center (electricity, water, heating, gas, cleaning, 

building maintenance, salaries for central administrative and technical personell etc.) 

are shared by the University and the University Hospital. Running cost for central large 

equipment, equipment maintenance and renewal, liquid nitrogen, carbon dioxide, 

central computer software, library, isotope waste, dish washing, animal costs will be 

shared among the users. 

♦ Library 

♦ Seminar rooms 

Facilities and Equipment 

Central laboratories and facilities 

♦ S2 Biological Safety Laboratory 

♦ Isotope laboratory (S2) for experiments with 125 Iodine, 51 Cr, 45 Ca etc 

♦ Temporary animal rooms 

♦ Confocal microscope 

Central major equipment 

♦ High-speed cell sorter, analytical fluorescence-activated cell sorters 

♦ DNA Sequencer 

♦ BIAcore Surface Plasmon Resonance 

♦ Phosphoimager 

♦ Luminoimager 

♦ Preparative liquid chromatography (BioPerseptive) 

♦ Microinjection unit 

♦ Liquid nitrogen cell storage system 

4

RESEARCH UNITS 

� Department of Experimental Medicine I 

Head: Prof. K. von der Mark until 3/2011 

Prof. D. Müller from 4/2011 

� Department of Experimental Medicine II 

(Molecular Tumor Research) 

Head: Prof. J. Behrens 

� Division of Molecular Immunology 

Department of Internal Medicine III 

Head: Prof. H.-M. Jäck 

� Hematopoiesis Unit, Chair of Genetics, 

Department of Biology 

Faculty of Natural Sciences II 

Head: Prof. T. Winkler 

� Junior Research Groups of the IZKF 

Heads: PD Dr. R. Voll* 

PD Dr. M. Wiesener* 

PD Dr. B. Winner # 

PD Dr. J. Titze # 

� Clinical Research Groups 

PD Dr. J. Distler* Prof. A. Bozec # 

Prof. D. Dudziak* PD Dr. K. Gelse # 

Prof. F. Nimmerjahn* PD Dr. U. Kneser # 

PD Dr. J. Titze* Dr. G. Krönke # 

Prof. R. Voll* Dr. M. Stock # 

Prof. H. Taubert # 

Prof. E. Ullrich # 

PD Dr. M. Wiesener # 

5 

* previous 

# current

DEPARTMENT OF EXPERIMENTAL MEDICINE I 

Matrix Biology/ Molecular Pathology 

Head Klaus von der Mark, Dr. rer.nat 

(till 3/2011) Prof. of Experimental Medicine 

Address: Department of Experimental Medicine I 

Glückstr. 6 

D-91054 Erlangen 

Phone: + 49 (9131) 85 29124 

Fax: + 49 (9131) 85 26341 

E-mail: kvdmark@molmed. uni-erlangen.de 

Homepage: www.em1.molmed.uni-erlangen.de 

Supporting Staff 

Irene Bielmeier, Secretary 

Postdoctoral Fellows 

. 

Jung Park, Dr,med. dent. 

Helga von der Mark, Dr.rer. nat 

Takako Sasaki, Ph.D. 

Michael Stock, Dr. rer. nat. 

Cordula Surman-Schmitt, Dr. rer. nat. 

Nicole Eitzinger 

( � 10(2008) 

(��9/2008 

(11/2008 �) 

(��10/2010 

(��9/2011 

Doctoral Students (Biology) 

6 

PhD 8/2011

.Svitlana Golovchenko 

Doctoral Students (Molecular Medicine) 

( 10/2011) 

Diploma Students 

Catharina Müller(2008) 

Svitlana Ryabova-Golovchenko (2010) 

MatthiasGebhard (2011) 

Eva Bauer 

Friederike Pausch 

Britta Schlund 

Technicians 

General 

The legal time as head of the department of Experimental Medicine I ended for Prof. 

K. von der Mark with his official retirement date at Oct. 1, 2008. Until the installment 

of the new head of the Department, Prof. Dominik Müller, in April 2011, K. von der 

Mark continued to conduct research and teaching in the Department, but with 

strongly reduced staff and student numbers. With retirement of Dr. Helga von der 

Mark, the integrin research project was terminated, but collaborative studies with Dr. 

Jung Park, now Childrens Hospital, FAU (Prof. Holm Schneider) and Prof. Schmuki, 

Dept. of Material Sciences, FAU, on translational aspects of Titandioxide nanotubes 

are still going on. The research work of K. von der Mark focuses on two topics: 1) the 

role of hypertrophic chondrocytes in controlling subchondral trabecular bone 

formation by deleting �-catenin with a BACCol10Cre –deleter mouse, and 2) cell 

fate tracing and transdifferentiation of hypertrophic chondrocytes to osteoblasts using 

a BAC Col10Cre;ROSA-YFP reporter mice. The research projects on the new 

cartilage-specific proteins UCMA and the Wnt –inhibitor Wif 1 were continued under 

the supervision of Dr. M. Stock, now as research staff member in Internal Medicine 

III (Prof. G.Schett). 

A new research project on the role of fibulin-4 was started by Dr. Takako Sasaki in 

Nov. 2008 who received an independent research grant from the DFG in 2010. 

7

Research projects: 

I. Analyzing the role of hypertrophic chondrocytes in endochondral bone 

formation using BACCol10Cre deleter mice (K.von der Mark) 

II. Cellular response to nanoscale patterns of Titandioxide surfaces (J.Park) 

III. Control of collagen crosslinking and connective tissue stability by fibulin-4 

(Takako Sasaki) 

IV. Characterization of new cartilage matrix proteins (Michael Stock) 

Research 

I. Analyzing the role of hypertrophic chondrocytes in endochondral bone 

formation using BACCol10Cre deleter mice (P.I. :Klaus von der Mark) 

A) Deletion of �- catenin in hypertrophic growth plate chondrocytes impairs 

trabecular bone formation 

Svitlana Golovchenko, Sonja Gebhard, Britta Schlund, Matthias Gebhardt, Andreas 

Hess, Klaus von der Mark 

In numerous studies it has been shown that canonical Wnt signalling plays a central 

but complex role in the regulation of chondrogenic and osteogenic differentiation 

during skeletal development Continuous Wnt/ �-catenin signalling in nascent 

cartilaginous skeletal elements blocks chondrocyte hypertrophy and endochondral 

ossification, whereas �-catenin overexpression at later stages stimulates hypertrophy 

and Col10a1 expression, mediated by binding to the Runx2 promoter and activating 

thereby expression of Col10a1. Furthermore, activation of Wnt/ �-catenin signalling in 

mature chondrocytes stimulated the expression of markers of late hypertrophy 

including MMP13 and VEGF and the expression other MMP genes. 

The role of Wnt/ �-catenin signalling in bone development is even more complicated 

in light of report showing that Wnt signalling is also involved in the control of bone 

remodelling by regulating the expression RANKL and osteoprotegerin in osteoblasts, 

thereby controlling osteoclast differentiation. Since RANKL and OPG are also 

expressed in hypertrophic chondrocytes, we tested the hypothesis that inhibition of �catenin 

signalling in hypertrophic chondrocytes might interfere with the RANKL/OPG 

balance and thus affect trabecular bone formation in growing bones. 

For this purpose, we specifically deleted the �-catenin gene in hypertrophic 

chondrocytes by crossing a ctnnb1 fl/fl mouse with a BAC-Col10a1-Cre deleter 

mouse. Surprisingly, the Cre-positive, ctnnb1 -/- mice (BCat-ko mice) revealed 

morphologically normal growth plate cartilage, but a substantial deficiency in 

trabecular bone, with increasing loss during postnatal development. Collagen I 

immunostaining and Alcian Blue staining for proteoglycans showed that not only the 

osteoid seam of bone trabeculae was missing, but also the calcified cartilage core, 

indicating excess of osteoclast activity. This notion was confirmed by TRAP staining 

8

and in situ hybridisation analysis of MMP9 which revealed a dramatically enhanced 

concentration of osteoclasts at the cartilage-bone marrow border in BCat-ko mice. 

Quantitative analysis of mRNA isolated from hypertrophic chondrocytes or whole 

bones of BCat–ko mice and wt littermates indicated significantly enhanced RANKL 

mRNA levels in BCat ko samples. In light of recent reports indicating that not 

osteoblasts, but osteocytes and hypertrophic chondrocytes are the main sources for 

RANKL synthesis, our findings support the provocative concept that it is the �-catenin 

in hypertrophic chondrocytes which predominantly controls the extent of trabecular 

bone formation in postnatal and juvenile development by regulating RANKL 

expression. 

Fig.1a,b: In situ hybridization analysis of newborn wild type and BACCol10Cre; ctnnb1 -/- 

(= BCat-ko) embryonic littermates (2d) shows that ��catenin mRNA expression is 

substantially reduced in the BCat-ko hypertrophic zones (b) as compared to wt cartilage 

(a), but equal in proliferating chondrocytes and in the bone marrow zone. c,d: Alcian blue 

staining marks growth plate cartilage and the calcified cartilage in subchondral trabeculae of 

wt mice (c), and demonstrates substantial loss of trabeculae in BCat-ko (d). (P7 tibia). e,f: 

Staining for TRAP reveals a high concentration of osteoclasts (Blue arrows) at the erosion 

zone of hypertrophic cartilage in BCat-ko mice (f), while in the wt spongiosa osteoclasts are 

evenly distributed between cartilage erosion zone and bone trabeculae (e). (P17, 

humerus).g) Real Time PCR analysis mRNA levels of hypertrophic chondrocytes isolated 

from growth plates of wt and BCAT-ko mice (P5) confirm reduction of �-catenin mRNA and 

indicate enhanced RANKL expression in Cat-ko hypertrophic chondrocytes. 

B) Do trabecular osteoblasts originate from hypertrophic chondrocytes? 

Matthias Gebhardt, Cordula Surmann-Schmitt, Jung Park, Britta Schlund, Klaus von der 

Mark 

Although the above findings indicate that the lack of trabecular bone caused by 

inactivation of the �-catenin gene in hypertrophic chondrocytes is due to enhanced 

9

osteoclast activity, we cannot rule out the possibility that the deletion of �-catenin in 

hypertrophic chondrocytes also impairs the differentiation of osteoblasts. This would 

be in line with a hypothesis by H. Roach et al (1996) who proposed 

transdifferentiation of terminally differentiated hypertrophic chondrocytes at the 

cartilage-bone marrow interface into osteoblasts, although the general 

understanding is that terminally differentiated chondrocytes die by apoptosis. To test 

this hypothesis, our BAC-Col10Cre deleter mice were mated to ROSA26LacZ 

reporter mice, and LacZ expression was used to trace the cell fate of hypertrophic 

chondrocyte. In fact, while X-gal staining of BAC Col10cre; ROSA26LacZ embryos 

labeled exclusively hypertrophic chondrocytes as expected, in postnatal stages 

LacZ activity was also seen in bone marrow and even in osteoblasts-like cells lining 

endosteal bone trabeculae. In order to exclude unspecific expression of Cre in cells 

other than hypertrophic chondrocytes, e.g. in bone marrow cells or osteoblast 

precursors, resulting in unspecific recombination of the floxed ROSA;LacZ locus, 

Cre expression in BACCol10Cre;ROSALacZ mice was analyzed by in situ 

hybridization. Cre signals were only seen hypertrophic chondrocytes, but not in 

other cells or tissues of the mouse embryo. 

To confirm the osteogenic phenotype of the LacZ-positive cells in the bone marrow, 

BACCol10Cre;ROSAYFP mice were generated and analyzed for expression of YFP 

expression osteogenic markers such as collagen I, osteocalcin and osterix. 

Immunofluorescence analysis revealed YFP and collagen I positive cells lining 

subchondral trabeculae, confirming their osteogenic phenotype. Also the 

immunofluorescence analysis of endosteal cells obtained after flushing long bones 

of 2-3 wk old BACCol10Cre;ROSA-YFP mice with collagenase showed YFP 

positive cells staining for collagen I and osteocalcin. These results strongly support 

the notion that not all terminal differentiated hypertrophic chondrocytes die by 

apoptosis, but may – at least to some extent – transdifferentiate to trabecular 

osteoblasts. 

a b 

c 

d e 

Figure 2: X-gal staining detects LacZ reporter gene activity in BACCol10Cre; ROSA26- 

LacZ transgenic mice in hypertrophic chondrocytes in E14.5-E18.5 embryos (a), in 

postnatal stages (P6) also in bone marrow and trabecular osteoblasts (c,d, arrows). A 

similar reactivity is seen in vertebrae (D) and long bones (e) of BACCol10Cre; ROSA-YFP 

mice. 

10

The mechanism of this transdifferentiation process remains to be elucidated. We 

have preliminary evidence that after opening of the chondrocyte lacunae by 

osteoclasts hypertrophic chondrocytes de-differentiate to bone marrow stem cells 

which may then differentiate into osteoblasts precursor cells and other 

mesenchymal cells. Studies to confirm this hypothesis are under investigation. 

Fig.3 Fig. 4 

Figure 3. YFP positive endosteal and bone marrow cells express osteoblast marker 

gene mRNA. Adherent bone marrow and endosteal cells were isolated from long bones of 

P21 BAC Col10 Cre;Rosa26-YFP reporter mice by bone flushing with collagenase P and 

cultured for two weeks. (A) YFP positive cells were sorted by FACS. Gating for viable cells 

(89.5% of total cells) is illustrated in the upper panel (pink circle). Lower panel: analysis of 

viable cells for YFP fluorescence reveals a clearly defined population of positive cells 

(12.4%). (B) RT-PCR analysis of RNA isolated from YFP + and YFP - cells after sorting and 

reverse transcription. Expression of osteoblast markers was analyzed using specific primers 

for the marker genes osterix, Runx2, osteocalcin, and Col1a1). Ppia (cyclophilin a) was used 

as a housekeeping gene control. Gel electrophoresis of PCR products shows, that YFP 

positive cells express high message levels of all four osteoblast marker genes (green box). 

Cal.: calvarial cDNA. 

Figure 4: Hypertrophic chondrocytes are an additional source of osteoblasts 

Reporter gene expression is initiated in hypertrophic chondrocytes (HCs). The progeny of 

HCs continues reporter gene expression and can be detected as YFP positive trabecular 

osteoblasts (OBs). Some HCs are eliminated from the growth plate by apoptosis. 

Additionally, osteoblast precursor cells (OBP) invade the cartilage template and give rise to 

trabecular osteoblasts. Grey: cartilage matrix. Brown: bone trabeculae. Sand: bone marrow. 

PCs: proliferating chondrocytes. RCs: resting chondrocytes 

II: Synergistic control of mesenchymal stem cell differentiation by 

nanoscale surface geometry and immobilized growth factors on TiO2 

nanotubes ( P.I.: Dr. Jung Park) 

Park J, Bauer S, Pittrof A, Killian MS, Schmuki P, von der Mark K. 

11

Previously we have shown that cellular responses of bone marrow mesenchymal 

stem cells including adhesion, proliferation, migration, differentiation and apoptosis 

on titandioxide nanotube surfaces are strictly dependent on the nanoscale diameter 

of the tubular surfaces, with an optimum response on 15 nm diameter TiO2 

nanotubes (Park et al, 2007). In subsequent studies we asked the question whether 

combined environmental signals provided by nanoscale topography and by growth 

factors control cell behavior of mesenchymal stem cells (MSCs) in a synergistic or 

simply additive manner. Differentiation of MSCs to osteoblasts, chondrocytes and 

endothelial cells was studied on vertically aligned TiO2 nanotubes of size 15 and 100 

nm with and without immobilized bone morphogenetic protein-2 (BMP-2) and other 

growth factors such as EGF. Covalent immobilization of these growth factors onto the 

oxide surfaces was achieved by N,N-carbonyldiimidazole (CDI) coupling via binding 

to amine groups of the proteins either directly or via a spacer, namely 11-hydroxyundecylphosphonic 

acid (PhoA). Although BMP-2 coating stimulates both 

chondrogenic and osteogenic differentiation of MSCs, the response strongly 

depended on the surface nanoscale geometry of the BMP-2-coated nanotubes. 

Chondrogenic differentiation was strongly supported on 100 nm BMP-2-coated 

nanotubes, but not on 15 nm nanotubes, which induce spreading and dedifferentiation 

of chondrocytes. A similar response was observed with primary 

chondrocytes, which maintained their chondrogenic phenotype on BMP-2-coated 100 

nm nanotubes, but de-differentiated on 15 nm nanotubes. In contrast, osteogenic 

differentiation was greatly enhanced on 15 nm but not on 100 nm BMP-2-coated 

nanotubes as shown previously. Furthermore, covalent immobilization of BMP-2 

rescued MSCs from apoptosis occurring on uncoated 100 nm TiO2 nanotube 

surfaces. Thus, combined signals provided by BMP-2 immobilized to a defined lateral 

nanoscale spacing geometry seem to contain environmental cues that are able to 

modulate a lineage-specific decision of MSC differentiation and cell survival in a 

synergistic manner. 

III. The role of fibulin-4 in development and homeostasis of elastic 

components of the skeletal connective tissue (P.I. Dr.Takako Sasaki) 

Takako Sasaki, Eva Bauer, K.von der Mark 

The goal of this project is to elucidate the role of fibulin-4, a component of fibrillin 

microfibrils, in skeletal development and homeostasis. A recent gene targeting study 

has revealed fibulin-4 to be an essential molecule for the elastic fiber assembly 

during embryonic development. Mice deficient in fibulin-4 die perinatally due to 

cardiovascular and lung abnormalities, and similar phenotypes were reported for 

fibrillin-1 and lysyl oxidase (LOX) deficient mice. It has been proposed that fibrillin-1 

and fibulin containing microfibrils have an important role in sequestering cytokines, 

since perturbation of this function contributes to the pathogenesis of the disease. 

Thus, a hypomorphic in-frame deletion in the fibrillin-1 mutant mice, an accepted 

model of Marfan syndrome, leads to dysregulation of TGF-� activation and signaling. 

12

Increased TGF-� signaling in the cardiovascular system has also been found in mice 

hypomorphic for fibulin-4 and in the patient positive FBLN4 mutation. Recently, we 

found fibulin-4 expression also in fetal growth plate cartilage. Preliminary data on 

Fbln4 null mice showing reduced bone mass and increased bone fragility, and 

mutations found in human patients indicate that fibulin-4 may be also involved in 

skeletal development. The goal of this study is to obtain further insight into molecular 

changes causing the observed of skeletal alterations in fibulin-4 deficient mice such 

as limb contracture, detached distal phalanges and reduced bone mass. 

Results: 

Biochemical analyses on bone revealed that collagen I from fibulin-4 null mice was 

less cross-linked compared with those from wild type and heterozygous mice. 

Reduction of proteolytic activation of lysyl oxidase (LOX) which is essential for 

collagen crosslinking was detected not only in osteoblast culture but also in 

chondrocyte and fibroblast culture from fibulin-4 null mice. The mechanism how 

fibulin-4 is involved in LOX activation will be elucidated in vitro. It is still necessary to 

quantify the crosslink markers hyroxylyslpyridinoline (HP)/lysylpyridinoline (LP) of 

some more samples. 

Fig.5 SDS Page of collagens extracted with 

acid form bones of fibulin-4 k.o. mice 

shows significantly enhanced amounts of 

soluble collagen chains (a1(I) and a2(I)) , 

indicating impaired crosslink formation, 

while collagens from wild type or 

heterozygote bones remain mostly insoluble 

2) There is ample evidence in the literature for a role of fibulin-4 in extracellular 

assembly of collagen and elastin. Therefore, particular attention was paid to fibulin-4 

binding proteins such as LTBPs and lysyloxidases in order to elucidate the 

mechanisms by which the absence of fibulin-4 leads to abnormalities in mice and 

human. To this aim, four mutations in fibulin-4 found in human patients were 

introduced in recombinant fibulin-4. The C276Y mutant fibulin-4 was not secreted 

from transfected 293 cells indicating that this cysteine residue is important for the 

protein folding. Other mutants can be secreted and purified except R279C mutant. 

These data suggest that the homozygous missense mutation of C276Y and the 

compound heterozygosity resulted in null mutation of fibulin-4 therefore those 

patients died very early, similar to fibulin-4 null mice. It will be tested whether cells 

other than 293 cells can secrete mutant fibulin-4. Some of fibulin-4 mutants exhibited 

reduced binding activities with different ligands. The E57K and E126K mutants were 

more susceptible to the proteases tested, suggesting that these mutation resulted in 

loss of calcium, causing instability in the conformation of EGF-like domain . 

13

The proposed studies will provide novel insights into the role of fibulin-4 in skeletal 

system as well as in the development and homeostasis of cardiovascular tissue. 

IV. Characterization of new cartilage matrix proteins 

(P.I.: Dr .Michael Stock) 

1. mWif-1 is expressed at cartilage-mesenchyme interfaces and impedes 

Wnt3a-mediated inhibition of chondrogenesis 

Cordula Surmann-Schmitt, Nathalie Widmann, Uwe Dietz, Bernhard Saeger, Nicole 

Eitzinger, Yukio Nakamura, Marianne Rattel, Richard Latham, Christine Hartmann, 

Helga von der Mark, Georg Schett, Klaus von der Mark and Michael Stock 

Skeletal development is controlled by a complex network of growth factors. Important 

signalling cascades regulating chondrocyte differentiation and maturation include 

signals mediated by the TGFβ/BMP puderfamily of growth factors, fibroblast growth 

factors (FGF), the Ihh/PTHrp system and Wnt factors. Wnt proteins are involved in 

the regulation of all steps of cartilage development. Their activity to a large extent 

regulated at the extracellular level by factors like the Dkk family, sFRPs, Cerberus 

and Wnt inhibitory factor 1 (Wif-1). 

In this study we provide evidence that Wif-1 is highly expressed at cartilagemesenchyme 

interfaces of the early developing skeleton. In fetal and postnatal 

skeletal development, Wif-1 expression is mainly confined to a zone of only a few cell 

layers in the upper hyaline layer of epiphyseal and articular cartilage. Significant Wif- 

1 expression was also detected in trabecular bone. In order to identify cartilagerelated 

Wnt factors that Wif-1 might interact with in vivo, we performed 

coimmunoprecipitation and pull-down assays using recombinant Wif-1 and Wnt 

factors. Thereby, we could identify specific binding of Wif-1 to Wnt3a, Wnt4, Wnt5a, 

Wnt7a, Wnt9a and Wnt11. We could demonstrate that Wif-1 was able to block Wnt3a 

mediated activation of the canonical Wnt signalling pathway, probably mediated by 

14

Figure 6: Wif-1 blocks Wnt3a activity 

The chondrogenic cell line 4C6 was stimulated as indicated with 25 µl/ml or 100 µl/ml Wnt3aconditioned 

medium in the presence or absence (Ctrl.) of 10 µg/ml recombinant Wif-1. 

Wnt3a-dependent accumulation and nuclear translocation of β-catenin was detected by 

immunofluorescence 5 hours after stimulation. 

B: Mouse limb-bud cells were grown in micromass cultures and treated with Wnt3a and Wif-1 

as indicated. After RNA extraction, Col2a1 mRNA levels were determined by real-time PCR 

using cyclophilin A levels for normalisation. Values are means ± s.d. 

physical interaction of Wif-1 and Wnt3a. Consequently, Wif-1 impaired growth of 

mesenchymal precursor cells and neutralised Wnt3a-mediated inhibition of 

chondrogenesis in micromass cultures of embryonic chick limb bud cells. 

These results identify Wif-1 as a novel extracellular Wnt modulator in cartilage 

biology. 

2. The Wnt Antagonist Wif-1 interacts with CTGF and Inhibits CTGF 

Activity 

Cordula Surmann-Schmitt, Takako Sasaki, Takako Hattori, Nicole Eitzinger, Georg 

Schett, Klaus von der Mark, and Michael Stock 

A recent study showed that shifted, the Drosophila orthologue of Wif-1, is involved in 

the control of hedgehog signalling. Therefore, we hypothesized that in mammals Wif- 

1 may also be associated with signalling pathways other than the Wnt cascade. 

Since Wif-1 exerts its regulatory role in Wnt signalling by direct interaction with Wnt 

ligands, we searched for novel protein interaction partners of Wif-1, which are 

involved in signalling pathways other than Wnt signalling. Therefore, we performed a 

yeast-two-hybrid approach to identify such novel protein interactions. Thereby, we 

identified the matricellular signalling molecule connective tissue growth factor 

(CTGF/CCN2) as a potential Wif-1-interacting protein. We confirmed that Wif-1 

physically binds to connective tissue growth factor (CTGF/CCN2) in vitro, 

predominantly by interaction with the C-terminal cysteine knot domain of CTGF. In 

vivo such an interaction appears also likely since the expression patterns of these 

two secreted proteins overlap in peripheral zones of epiphyseal cartilage. In 

chondrocytes CTGF has been shown to induce the expression of cartilage matrix 

genes such as aggrecan (Acan) and collagen2a1 (Col2a1). Here we could 

demonstrate that Wif-1 is capable to interfere with CTGF-dependent induction of 

Acan and Col2a1 gene expression in primary murine chondrocytes. Conversely, 

CTGF does not interfere with Wif-1-dependent inhibition of Wnt signalling. 

These results indicate that Wif-1 may be a multifunctional modulator of signalling 

pathways in the cartilage compartment. 

3. Ucma is not Necessary for Normal Development of the Mouse Skeleton 

Nicole Eitzinger, Cordula Surmann-Schmitt, Michael Bösl, Georg Schett, Klaus 

Engelke, Andreas Hess, Klaus von der Mark, and Michael Stock 

15

We recently introduced Ucma (Upper zone of growth plate and Cartilage Matrix 

Associated protein), a highly conserved tyrosine,sulphated, secreted protein of Mw 

17 kDa, which is expressed by juvenile chondrocytes. Initial in vitro experiments 

indicated that Ucma may be involved in skeletal development by affecting 

differentiation of pre-osteoblasts. Here we present the generation and analysis of a 

Ucma-deficient mouse strain, which should help to identify the physiological role of 

Ucma. Ucma gene-deficient mice were generated by introducing a lacZ/neoRcassette 

into the first exon of the Ucma gene. This mutation results in the complete 

loss of Ucma mRNA and protein expression. As expected due to the cartilage- 

specific expression of Ucma, these mice are viable and fertile. Surprisingly, however, 

Ucma-deficient mice appear to develop normally. Neither ossification and calcification 

of the skeleton, nor cartilage development were affected by Ucma-deficiency. This is 

particularly surprising since in a recent study, the knockdown of Ucma in zebrafish 

resulted in a severely disturbed formation of craniofacial cartilage. 

Our earlier studies have shown that Ucma is most prominently expressed perinatally, 

a finding that supported our hypothesis that Ucma may play a role in skeletal 

development. In the study presented here we could show that Ucma is additionally 

expressed in the cartilage of ribs and growth plate in adult mice. This may indicate 

that Ucma might be involved in skeletal homeostasis and in the mechanical 

properties of the skeleton during challenging conditions such as ageing or disease. 

Publications 

2012 

Eitzinger N, Surmann-Schmitt C, Bösl M, Schett G, Engelke K, Hess A, von der 

Mark K, Stock M. Ucma is not necessary for normal development of the mouse 

skeleton. Bone. 2012 Mar;50(3):670-80. Epub 2011 Dec 2. PubMed PMID: 22155508. 

Park J, Bauer S, Pittrof A, Killian MS, Schmuki P, von der Mark K. Synergistic 

control of mesenchymal stem cell differentiation by nanoscale surface geometry 

and immobilized growth factors on TiO2 nanotubes. Small. 2012 Jan 9;8(1):98-107. 

doi: 10.1002/smll.201100790. Epub 2011 Nov 18. PubMed PMID: 22095845. 

Surmann-Schmitt C, Sasaki T, Hattori T, Eitzinger N, Schett G, von der Mark K, 

Stock M. The Wnt antagonist Wif-1 interacts with CTGF and inhibits CTGF activity. 

J Cell Physiol. 2012 May;227(5):2207-16. doi: 10.1002/jcp.22957. PubMed PMID: 

21928342. 

2011 

Bauer S, Park J, Pittrof A, Song YY, von der Mark K, Schmuki P. Covalent 

functionalization of TiO2 nanotube arrays with EGF and BMP-2 for modified 

behavior towards mesenchymal stem cells. Integr Biol (Camb). 2011 

Sep;3(9):927-36. Epub 2011 Aug 10. PubMed PMID: 21829821. 

Gelse K, Klinger P, Koch M, Surmann-Schmitt C, von der Mark K, Swoboda B, 

Hennig FF, Gusinde J. Thrombospondin-1 prevents excessive ossification in 

16

cartilage repair tissue induced by osteogenic protein-1. Tissue Eng Part A. 2011 

Aug;17(15-16):2101-12. Epub 2011 Jun 1. PubMed PMID: 21513464. 

Klinger P, Surmann-Schmitt C, Brem M, Swoboda B, Distler JH, Carl HD, von der 

Mark K, Hennig FF, Gelse K. Chondromodulin 1 stabilizes the chondrocyte phenotype and 

inhibits endochondral ossification of porcine cartilage repair tissue. 

Arthritis Rheum. 2011 Sep;63(9):2721-31. doi: 10.1002/art.30335. PubMed PMID: 

21391200. 

2010 

Hattori T, Müller C, Gebhard S, Bauer E, Pausch F, Schlund B, Bösl MR, Hess A, 

Surmann-Schmitt C, von der Mark H, de Crombrugghe B, von der Mark K. SOX9 is a 

major negative regulator of cartilage vascularization, bone marrow formation and 

endochondral ossification. Development. 2010 Mar;137(6):901-11. PubMed PMID: 

20179096. 

Dragu A, Schnürer S, Surmann-Schmitt C, von der Mark K, Stürzl M, Unglaub F, 

Wolf MB, Leffler M, Beier JP, Kneser U, Horch RE. Gene expression analysis of 

ischaemia and reperfusion in human microsurgical free muscle tissue transfer. J 

Cell Mol Med. 2011 Apr;15(4):983-93. doi: 10.1111/j.1582-4934.2010.01061.x. 

PubMed PMID: 20345846. 

Belluoccio D, Etich J, Rosenbaum S, Frie C, Grskovic I, Stermann J, Ehlen H, 

Vogel S, Zaucke F, von der Mark K, Bateman JF, Brachvogel B. Sorting of growth 

plate chondrocytes allows the isolation and characterization of cells of a 

defined differentiation status. J Bone Miner Res. 2010 Jun;25(6):1267-81. PubMed 

PMID: 20200945. 

Krönke G, Uderhardt S, Kim KA, Stock M, Scholtysek C, Zaiss MM, et al. R-spondin 1 

protects against inflammatory bone damage during murine arthritis by modulating the Wnt 

pathway. Arthritis Rheum. 2010 Aug; 62(8):2303-2312. 

2009 

Bauer S, Park J, Faltenbacher J, Berger S, von der Mark K, Schmuki P. Size 

selective behavior of mesenchymal stem cells on ZrO(2) and TiO(2) nanotube 

arrays. Integr Biol (Camb). 2009 Sep;1(8-9):525-32. Epub 2009 Jun 19. PubMed 

PMID: 20023767. 

Bruckner-Tuderman L, von der Mark K, Pihlajaniemi T, Unsicker K. Cell 

interactions with the extracellular matrix. Cell Tissue Res. 2010 Jan;339(1):1-5. 

PubMed PMID: 19902257. 

von der Mark K, Park J, Bauer S, Schmuki P. Nanoscale engineering of 

biomimetic surfaces: cues from the extracellular matrix. Cell Tissue Res. 2010 

Jan;339(1):131-53. Epub 2009 Nov 7. Review. PubMed PMID: 19898872. 

Surmann-Schmitt C, Widmann N, Mallein-Gerin F, von der Mark K, Stock M. 

Stable subclones of the chondrogenic murine cell line MC615 mimic distinct stages 

of chondrocyte differentiation. J Cell Biochem. 2009 Oct 15;108(3):589-99. PubMed 

PMID: 19670270. 

Park J, Bauer S, Schmuki P, von der Mark K. Narrow window in nanoscale 

dependent activation of endothelial cell growth and differentiation on TiO2 

nanotube surfaces. Nano Lett. 2009 Sep;9(9):3157-64. PubMed PMID: 19653637. 

17

Park J, Bauer S, Schlegel KA, Neukam FW, von der Mark K, Schmuki P. TiO2 

nanotube surfaces: 15 nm--an optimal length scale of surface topography for cell 

adhesion and differentiation. Small. 2009 Mar;5(6):666-71. PubMed PMID: 19235196. 

von der Mark K, Bauer S, Park J, Schmuki P. Another look at "Stem cell fate 

dictated solely by altered nanotube dimension". Proc Natl Acad Sci U S A. 2009 

Jun 16;106(24):E60; author reply E61. Epub 2009 Jun 8. PubMed PMID: 19506261; 

Surmann-Schmitt C, Widmann N, Dietz U, Saeger B, Eitzinger N, Nakamura Y, 

Rattel M, Latham R, Hartmann C, von der Mark H, Schett G, von der Mark K, Stock 

M. Wif-1 is expressed at cartilage-mesenchyme interfaces and impedes 

Wnt3a-mediated inhibition of chondrogenesis. J Cell Sci. 2009 Oct 15;122(Pt 

20):3627-37. Epub 2009 Sep. 

18

DEPARTMENT OF EXPERIMENTAL MEDICINE I 

(Hypertension-Induced Target Organ Damage) 

Head: Dominik N. Müller, Dr. rer. nat. 

Professor of Experimental Medicine 

Address: Department of Experimental Medicine I 

Glückstr. 6 


Telefone: + 49 (9131) 85 29100 

Fax: + 49 (9131) 85 26341 

E-mail: dmueller@molmed.uni-erlangen.de 

Homepage: http://www.molmed.uni-erlangen.de/ 

Head 

Dominik N. Müller, Prof. Dr. rer. nat. 


Dr. rer. nat. Katrina Binger 

Dr. rer. nat. Kristina Tanneberger 

Dr. rer. nat. Agnes Schröder 

Eva Bauer 


Irena Bielmeier, Secretary 


Technicians 

19 

Dr. med. Anke Dahlmann 

Dr. med. Wolfgang Freisinger 

Britta Schlund


Hypertension is the primary risk factor for cardiovascular disease, the major 

cause of death worldwide. Our group’s major research interests are the reninangiotensin 

system (RAS), the immune system and how both systems cause 

hypertension-induced target organ damage. In a translational approach, the Müller 

lab focuses on the vessels, hearts, kidneys with our recent work extending these 

concepts to analyze how epigenetic factors, such as high salt, influence immune cells 

and target organ damage. 

Projects 

The immune system, salt and hypertension-induced target organ damage 

Our research is aimed at better understanding the role of innate and adaptive 

immunity in hypertension-induced organ damage. T cells, macrophages, and 

dendritic cells all harbor the AT1 receptor. Ang II stimulates T cell proliferation and 

dendritic cell migration. We showed that mice lacking the transcription factor Id2, a 

pivotal factor for Langerhans dendritic cells, are resistant to Ang II-induced 

hypertension and sequelae. We also observed that regulatory T cells modulate Ang 

II-induced cardiac damage to a point of therapeutic utility. Since our data suggested 

an interaction of the renin-angiotensin system, immune system and target organ 

damage (Figure), we are investigating the role of angiotensin II in autoimmunity. 

In collaboration with Ralf Linker (Univ. Erlangen), we investigated whether blockade 

of the renin-angiotensin system improves non-cardiovascular autoimmunity. Our 

results suggest that by 

blocking the aspartyl 

protease renin, ACE 

and the AT1 receptor, 

autoimmune 

encephalitis in rodents 

can be effectively 

inhibited. 

We are now 

extending our 

immunological studies 

to the investigation of 

inflammatory 

activation in the 

interstitium by 

hyperosmolarity through sodium. This line of investigation was initiated with Jens 

Titze (Univ. Erlangen/ Vanderbilt Univ.), with whom we have an intensive 

collaboration. With dietary sodium excess, sodium accumulates in the skin and 

activates the osmotic stress gene tonicity-responsive enhancer binding protein 

(TonEBP/NFAT5) in macrophages. TonEBP activity in macrophages results in 

secretion of VEGF-C promoting the clearance of hypertonic fluid from the interstitium. 

This circuit is critically dependent on macrophages and their ability to ward off 

hypertension in case of excess sodium supply. We have initiated a program to 

determine how hypertonicity induced by deranged sodium chloride storage affects 

the differentiation of T cells and macrophages. 

20

Cardiovascular and non-cardiovascular functions of the (pro)renin receptor 

The (pro)renin receptor (PRR) is a relatively newly discovered member of the 

renin-angiotensin system (RAS). Initially, PRR was believed to directly contribute to 

cardiovascular disease by activating the RAS and several signaling cascades. 

However, recent publications in Science have shown that PRR plays an essential 

and non-RAS related role in the activation of Wnt signal transduction and cellular 

development. In light of this, our understanding of the role of the prorenin receptor 

(PRR) in physiology and pathology has changed dramatically and we are now 

focused on determining these non-RAS functions of PRR. 

In collaboration with Michael Bader (MDC), we have determined that complete 

knockout of PRR in mouse embryonic stem cells, fails to generate chimeras when 

injected into blastocysts, indicating an important role for this protein in cellular 

development. We have subsequently initiated several PRR tissue-specific conditional 

knockout models. Generation of podocyte-specific PRR knockout mice (cKO) 

resulted in the death of the animals ~2-3 weeks after birth. Within 14 days, these 

cKO animals developed nephrotic syndrome and albuminuria, due to podocyte foot 

process fusion (Figure) and cytoskeletal changes. 

Figure legend EM shows normal epithelial cells and foot processes in control animals (left). cKO mice 

(right) developed foot process fusion (c=capillary, p=podocyte). 

Our in vivo and in vitro findings indicated a functional block in autophagosomelysosome 

fusion and overload of the proteasome protein degradation machinery. 

These results suggest that the PRR is essential for podocyte function and survival by 

maintaining autophagy and protein turnover machinery. We are now investigating the 

effect of PRR deletion in other cell types, namely T cells, pancreatic b-cells and 

renin-producing cells. 

We are also focused on understanding the molecular mechanism by which 

PRR is important for cellular homeostasis and development. The PRR was initially 

proposed to have no homology to other proteins, however it is now apparent that the 

nucleotide sequence of PRR is identical to that of ATP6AP2; a gene identified as an 

21

accessory protein of the H+ vacuolar-ATPase (V-ATPase). The V-ATPase is 

responsible for establishing and maintaining intracellular pH gradients along the 

secretory and endocytic pathways in all cell types. Acidification of vacuoles along 

these pathways is essential for many cellular functions including the processing and 

secretion of proteins, receptor endocytosis and recycling, and membrane fusion 

events, which are essential for processes such as autophagy. We are undertaking 

experiments to determine the protein-protein interactions important for PRR to 

maintain this function, and its role in Wnt signal activation. 

Interestingly, the PRR also exists as a soluble receptor (sPRR), and it has 

been hypothesized that this sPRR may have an important biological role. We have 

developed a sensitive ELISA to quantitate sPRR in biological fluids, and we are 

currently investigating if sPRR levels may be an important biomarker for 

cardiovascular disease and cancer. 

CV 

Dominik N. Müller studied pharmacy at the Free University of Berlin (1991-1992). 

He completed his Ph.D. thesis in 1996 and achieved faculty rank (Habilitation) for 

Experimental Medicine in 2004. After his postdoctoral work at Franz-Volhard Clinic 

and Max-Delbrück-Center Berlin (1996-2004), he was appointed a Delbrück Fellow at 

the MDC (2004-2010). In 2011, Dominik N. Müller was recruited to a W3 (full) 

professorship for Experimental Medicine at the University of Erlangen. 

Publications (2010-2012) 

Original Articles 

Five most important publications since 2007 

Riediger, F., Quack, I., Qadri, F., Hartleben, B., Park, J.K., Potthoff, S.A., Sohn, D., 

Sihn. G., Rousselle, A., Fokuhl, V., Maschke, U., Purfürst, B., Schneider, W., Rump, 

L.C., Luft, F.C., Dechend, R., Bader, M., Huber, T.B., Nguyen, G. and Müller, D.N. 

(2011) Prorenin receptor is essential for podocyte autophagy and survival. J Am Soc 

Nephrol. 22, 2193-2202. 

Stegbauer, J., Lee, D.H., Seubert, S., Ellrichmann, G., Manzel, A., Kvakan, H., 

Müller, D.N., Gaupp, S., Rump, L.C., Gold, R.,Linker, R.A. (2009). Role of the reninangiotensin 

system in autoimmune inflammation of the central nervous system. Proc 

Natl Acad Sci U S A 106, 14942-14947. 

Machnik, A., Neuhofer, W., Jantsch, J., Dahlmann, A., Tammela, T., Machura, K., 

Park, J.-K., Beck, F.-X., Müller, D.N., Derer, W., Goss, J., Ziomber, A., Dietsch, P., 

Wagner, H., van Rooijen, N., Kurtz, A., Hilgers, K.F., Alitalo, K., Eckardt, K.U., Luft, 

22

F.C., Kerjaschki, D., Titze, J. (2009) Macrophages regulate salt-dependent volume 

and blood pressure by a VEGF-C dependent buffering mechanism. Nat Med 15, 545- 

52. 

Kvakan, H., Kleinewietfeld, M., Qadri, F., Park, J.-K., Fischer, R., Schwarz, I., Rahn, 

H.-P., Plehm, R., Wellner, M., Elitok, S., Gratze, P.., Dechend, R, Luft, F.C., and 

Müller, D.N. (2009) Regulatory T cells ameliorate angiotensin II-induced cardiac 

damage. Circulation. 119, 2904-2912. 

Henke, N., Schmidt-Ullrich, R., Dechend, R., Park, J.-K., Qadri, F., Wellner, M., Obst, 

M., Gross, V., Dietz, R., Luft, F.C., Scheidereit, C., Müller, D.N. Vascular endothelial 

cell-specific NF-kB suppression attenuates hypertension-induced renal damage. Circ 

Res. 2007; 101:268-76. 

Publications since 2010 

2012 

Markó L, Kvakan H, Park J-K, Qadri F, Spallek B, Binger KJ, Bowman EP, 

Kleinewietfeld M, Fokuhl V, Dechend R, Muller DN. Interferon gamma (IFN-γ) 

signaling inhibition ameliorates Ang II-induced cardiac damage Hypertension 2012 

(in press) 

Kopp, C., Linz, P., Wachsmuth, L., Dahlmann, A., Horbach, T., Schofl, C., Renz, W., 

Santoro, D., Niendorf, T., Muller, D.N., Neininger, M., Cavallaro, A., Eckardt, K.-U., 

Schmieder, R.E., Luft, F.C., Uder, M., Titze, J. (2012). 23Na Magnetic Resonance 

Imaging of Tissue Sodium. Hypertension 59 167-172 

Finckenberg, P., Eriksson, O., Baumann, M., Merasto, S., Lalowski, M.M., Levijoki, 

J., Haasio, K., Kyto, V., Muller, D.N., Luft, F.C., Oresic., M, Mervaala, E. (2012). 

Caloric Restriction Ameliorates Angiotensin II-Induced Mitochondrial Remodeling and 

Cardiac Hypertrophy. Hypertension 59:76-84. 

2011 

Riediger, F., Quack, I., Qadri, F., Hartleben, B., Park, J.K., Potthoff, S.A., Sohn, D., 

Sihn, G., Rousselle, A., Fokuhl, V., Maschke, U., Purfürst, B., Schneider, W., Rump, 

L.C., Luft, F.C., Dechend, R., Bader, M., Huber, T.B., Nguyen, G., Müller D.N. 

(2011). Prorenin receptor is essential for podocyte autophagy and survival. J Am 

Soc Nephrol 22, 2193-2202. 

Park, J.B., Kim, B.K., Kwon, Y.W., Muller, D.N., Lee, H.C., Youn, S.W., Choi, Y.E., 

Lee, S.W., Yang, H.M., Cho, H.J., Park, K.W., Kim, H.S. (2011). Peroxisome 

proliferator-activated receptor-gamma agonists suppress tissue factor overexpression 

in rat balloon injury model with Paclitaxel infusion. PLoS One 6, e28327. 

Batenburg, W.W., Lu, X., Leijten, F., Maschke, U., Muller, D.N., and Danser, A.H. 

(2011). Renin- and prorenin-induced effects in rat vascular smooth muscle cells 

overexpressing the human (pro)renin receptor: does (pro)renin-(pro)renin receptor 

interaction actually occur? Hypertension 58, 1111-1119. 

23

Searle, J., Mockel, M., Gwosc, S., Datwyler, S.A., Qadri, F., Albert, G.I., Holert, F., 

Isbruch, A., Klug, L., Muller, D.N., Dechend, R., Muller, R., Vollert, J.O., Slagman, A., 

Mueller, C., Herse, F. (2011). Heparin strongly induces soluble fms-like tyrosine 

kinase 1 release in vivo and in vitro--brief report. Arterioscler Thromb Vasc Biol 31, 

2972-2974. 

Falck, J.R., Wallukat, G., Puli, N., Goli, M., Arnold, C., Konkel, A., Rothe, M., Fischer, 

R., Muller, D.N., and Schunck, W.H. (2011). 17(R),18(S)-epoxyeicosatetraenoic acid, 

a potent eicosapentaenoic acid (EPA) derived regulator of cardiomyocyte 

contraction: structure-activity relationships and stable analogues. J Med Chem 54, 

4109-4118. 

Wenzel, K., Rajakumar, A., Haase, H., Geusens, N., Hubner, N., Schulz, H., Brewer, 

J., Roberts, L., Hubel, C.A., Herse, F., Hering, L., Qadri, F., Lindschau, C., Wallukat, 

G., Pijnenborg, R., Heidecke, H., Riemekasten, G., Luft, F.C., Müller, D.N., Lamarca, 

B., Dechend, R. (2011). Angiotensin II type 1 receptor antibodies and increased 

angiotensin II sensitivity in pregnant rats. Hypertension 58, 77-84. 

Herse, F., Fain, J.N., Janke, J., Engeli, S., Kuhn, C., Frey, N., Weich, H.A., 

Bergmann, A., Kappert, K., Karumanchi, S.A., Luft, F.C., Müller, D.N., Staff, A.C., 

Dechend, R. (2011). Adipose tissue-derived soluble fms-like tyrosine kinase 1 is an 

obesity-relevant endogenous paracrine adipokine. Hypertension 58, 37-42. 

Riemekasten, G., Philippe, A., Nather, M., Slowinski, T., Muller, D.N., Heidecke, H., 

Matucci-Cerinic, M., Czirjak, L., Lukitsch, I., Becker, M., Kill, A., van Laar, J.M., Catar, 

R., Luft, F.C., Burmester, G.R., Hegner, B., Dragun, D. (2011). Involvement of 

functional autoantibodies against vascular receptors in systemic sclerosis. Ann 

Rheum Dis 70, 530-536. 

Hoff, U., Lukitsch, I., Chaykovska, L., Ladwig, M., Arnold, C., Manthati, V.L., Fuller, 

T.F., Schneider, W., Gollasch, M., Müller, D.N., Flemming, B., Seeliger, E., Luft, 

F.C., Falck, J.R., Dragun, D., Schunck, W.H. (2011). Inhibition of 20-HETE synthesis 

and action protects the kidney from ischemia/reperfusion injury. Kidney Int 79, 57- 

65. 

2010 

Schmerbach, K., Pfab, T., Zhao, Y., Culman, J., Mueller, S., Villringer, A., Müller, 

D.N., Hocher, B., Unger, T., and Thoene-Reineke, C. (2010). Effects of aliskiren on 

stroke in rats expressing human renin and angiotensinogen genes. PLoS One 5, 

e15052. 

Biala, A., Martonen, E., Kaheinen, P., Levijoki, J., Finckenberg, P., Merasto, S., 

Louhelainen, M., Müller, D.N., Luft, F.C., and Mervaala, E. (2010a). Levosimendan 

improves cardiac function and survival in rats with angiotensin II-induced 

hypertensive heart failure. Hypertens Res 33, 1004-1011. 

Arnold, C., Markovic, M., Blossey, K., Wallukat, G., Fischer, R., Dechend, R., Konkel, 

A., von Schacky, C., Luft, F.C., Müller, D.N., Rothe, M., Schunck, W.H. (2010). 

24

Arachidonic acid-metabolizing cytochrome P450 enzymes are targets of {omega}-3 

fatty acids. J Biol Chem 285, 32720-32733. 

Verlohren, S., Geusens, N., Morton, J., Verhaegen, I., Hering, L., Herse, F., 

Dudenhausen, J.W., Müller, D.N., Luft, F.C., Cartwright, J.E., Davidge, S.T., 

Pijnenborg, R., Dechend, R. (2010). Inhibition of trophoblast-induced spiral artery 

remodeling reduces placental perfusion in rat pregnancy. Hypertension 56, 304-310. 

Hering, L., Herse, F., Geusens, N., Verlohren, S., Wenzel, K., Staff, A.C., Brosnihan, 

K.B., Huppertz, B., Luft, F.C., Müller, D.N., Pijnenborg, R., Cartwright, J., Dechend, 

R. (2010). Effects of circulating and local uteroplacental angiotensin II in rat 

pregnancy. Hypertension 56, 311-318. 

Wenzel, K., Wallukat, G., Qadri, F., Hubner, N., Schulz, H., Hummel, O., Herse, F., 

Heuser, A., Fischer, R., Heidecke, H., Luft F.C., Müller, D.N., Dietz, R., Dechend, R. 

(2010). Alpha1A-adrenergic receptor-directed autoimmunity induces left ventricular 

damage and diastolic dysfunction in rats. PLoS One 5, e9409. 

Machnik, A., Dahlmann, A., Kopp, C., Goss, J., Wagner, H., van Rooijen, N., Eckardt, 

K.U., Müller, D.N., Park, J.K., Luft, F.C., Kerjaschki, D., Titze, J. (2010). 

Mononuclear phagocyte system depletion blocks interstitial tonicity-responsive 

enhancer binding protein/vascular endothelial growth factor C expression and 

induces salt-sensitive hypertension in rats. Hypertension 55, 755-761. 

Mervaala, E., Biala, A., Merasto, S., Lempiainen, J., Mattila, I., Martonen, E., 

Eriksson, O., Louhelainen, M., Finckenberg, P., Kaheinen, P., Müller, D.N., Luft, 

F.C., Lapatto, R., Oresic, M. (2010). Metabolomics in angiotensin II-induced cardiac 

hypertrophy. Hypertension 55, 508-515. 

25

DEPARTMENT OF EXPERIMENTAL MEDICINE II 

(Molecular Tumour Research) 

Head: Jürgen Behrens, Dr. rer. nat. 


Address: Department of Experimental Medicine II 

Glückstr. 6 


Telefone: + 49 (9131) 85 29109 

Fax: + 49 (9131) 85 29111 

E-mail: jbehrens@molmed.uni-erlangen.de 

Homepage: www.em2.molmed.uni-erlangen.de 

Martin Sachs, Dr. rer. nat. 

Jean Schneikert, Dr. rer. nat. 

Head 

Jürgen Behrens, Prof. Dr. rer. nat. 



Angela Döbler, Secretary 


26 

Michel Hadjihannas, Dr. rer. nat. 

Ingrid Wacker, Dr. rer. nat.

Katharina Brauburger 

° PhD received 



Sandra Mattauch° 

Astrid Pfister° 


Martina Brückner 

Gabriele Daum 

* part of the time reported 

Dominic Bernkopf 

Kristina Tanneberger° 

Technicians 

27 

Shree Harsha Vijaya° 

Birgit Saffer 

Sabine Lukat*


Our group analyses molecular mechanisms that control tumour development and 

progression, focussing on oncogenic Wnt and Activin signalling. 

Functional analysis of Wnt signaling components 

Wnts are a family of secreted glycoproteins that bind to frizzled seventransmembrane 

span receptors and LRP coreceptors. Intracellularly, the Wnt 

signaling cascade blocks degradation of β-catenin in proteasomes and thereby leads 

to accumulation of β-catenin in the cytoplasm. β-Catenin then enters the nucleus and 

activates the expression of Wnt-specific target genes by interacting with LEF-1/TCF 

transcription factors. Conductin (also named Axin2), and its relative axin act as 

scaffolding components that simultaneously bind to β-catenin, the serine/threonine 

kinases GSK3β and CK1ε, and APC. In this complex GSK3β and CK1ε 

phosphorylate β-catenin at serine and threonine residues, which leads to 

ubiquitination and subsequent proteasomal degradation of β-catenin. It is suggested 

that axin proteins block Wnt signaling by assembling the essential components of the 

β-catenin degradation pathway. In colorectal tumours mutations of the tumour 

suppressor APC (adenomatous polyposis coli) lead to stabilization of β-catenin and 

constitutive signaling to the nucleus independent of Wnts. APC is a co-factor 

indispensable for axin-mediated degradation of β-catenin. APC binds to β-catenin 

and axin/conductin via multiple interaction domains thereby supporting β-catenin 

degradation. Mutations of β-catenin have been observed that also lead to 

stabilization of β-catenin. Thus, aberrant activation of the Wnt signaling pathway is 

considered a major oncogenic mechanism for many tumour types. 

In the past years we have identified and functionally characterized several key 

components of the pathway using our highly efficient yeast two hybrid screening 

system. Among these are Lef-1 and conductin/axin2 as interaction partners of βcatenin, 

and more recently the Amer family of APC interactors, as well as EB1 as an 

interaction partner of Amer2. 

Role of conductin/axin2 in the cell cycle 

Michel Hadjihannas, Martina Brückner 

We found that conductin levels are regulated during the cell cycle with lowest levels 

present during the G1/S phase and highest during G2/M. Following exit from mitosis 

conductin expression levels decline in parallel with those of mitotic regulators, such 

as cyclin B1. In line, Wnt/β-catenin target genes are low at G2/M and high at G1/S, 

28

and β-catenin phosphorylation oscillates during the cell cycle in a conductindependent 

manner. Conductin is degraded by the anaphase-promoting 

complex/cyclosome cofactor CDC20. Knockdown of CDC20 blocks Wnt signalling 

through conductin. CDC20-resistant conductin inhibits Wnt signalling and attenuates 

colony formation of colorectal cancer cells. We propose that CDC20-mediated 

degradation of conductin regulates Wnt/β-catenin signalling for maximal activity 

during G1/S (Hadjihannas et al., 2012). 

We also found that conductin localizes at the centrosomes by binding to the centrioleassociated 

component C-Nap1. Knockout or knockdown of conductin leads to 

premature centrosome separation which is abolished by knockdown of β-catenin. 

Conductin promotes phosphorylation of the amino-terminal serine (Ser 33/37) and 

threonine (Thr 41) residues of centrosome-associated β-catenin. β-Catenin mutated 

at these residues causes centrosomal separation, whereas a phospho-mimicking 

mutant of β-catenin does not. Treatment with Wnts and inhibition of glycogen 

synthase kinase 3 block β-catenin phosphorylation and induce centrosomal splitting. 

These data indicate that Wnt signalling and conductin regulate centrosomal cohesion 

by altering the phosphorylation status of β-catenin at the centrosomes (Hadjihannas 

et al., 2010). 

Functional dissection of APC mutants truncated in colorectal cancer 

Jean Schneickert, Eva Kohler, Shree Harsha Vijaya Chandra 

The tumour suppressor Adenomatous Polyposis Coli (APC) is truncated in most 

colon cancers, but is not completely lost. It is not clear why colon cancer cells retain 

the truncated APC fragment. The mutations affecting both APC alleles are 

interdependent, the position of the first APC mutation determining where the second 

hit will occur. This results in a complex pattern of mutation distribution in the APC 

sequence that translates into the stabilization of β−catenin that in turn feeds the 

affected cells with a permanent mitogenic signal. We found a new APC domain, the 

β−catenin inhibitory domain (CID) of APC located between the second and third 20 

amino acid repeats and therefore present in many truncated APC products found in 

human tumours. In truncated APC, the CID is absolutely necessary to down-regulate 

the transcriptional activity and the level of β−catenin, even when an axin/conductin 

binding site is present (Kohler et al., 2009). 

The four 15 amino acid repeats (15R) and the seven 20 amino acid repeats (20R) of 

APC are beta-catenin-binding sites, but their role in beta-catenin degradation has 

remained unclear. We showed that binding of β-catenin to the 15R of APC is 

necessary and sufficient to target β-catenin for degradation. The first 15R displays 

the highest affinity for beta-catenin in the 15R-20R module. The analysis of the 

distribution of truncating mutations along the APC sequence in colorectal tumours 

from FAP patients revealed that the first 15R is one target of the positive selection of 

mutations that lead to tumour development (Kohler et al., 2010). 

29

We found that the transcriptional repressor C-terminal binding protein (CtBP) 

promotes the oligomerization of truncated APC through binding to the 15 amino acid 

repeats of truncated APC. CtBP can bind to either first, third or fourth 15 amino acid 

repeats, but not to the second. CtBP-mediated oligomerization requires both 

dimerization domains of truncated APC as well as CtBP dimerization. This suggests 

that the sensitivity of truncated APC to oligomerization by CtBP constitutes an 

essential facet of tumour development (Schneikert et al., 2011). 

RNA interference was used to down-regulate truncated APC in several colorectal 

cancer cell lines expressing truncated APCs of different lengths, thereby performing 

an analysis covering most of the mutation cluster region. The consequences on 

proliferation in vitro, tumour formation in vivo and the level and transcriptional activity 

of β-catenin were investigated. Down-regulation of truncated APC results in an 

inhibition of tumour cell population expansion in vitro in 6 cell lines out of 6 and 

inhibition of tumour outgrowth in vivo as analysed in one of these cell lines, HT29. 

Down-regulation of truncated APC is accompanied by an up-regulation of the 

transcriptional activity of β-catenin and in most cases β-catenin levels, indicating that 

truncated APC can still modulate wnt signalling through controlling the level of βcatenin. 

Thus, truncated APC is an essential component of colorectal cancer cells, 

required for cell proliferation, possibly by adjusting β-catenin signalling to the "just 

right" level (Vijaya Chandra et al., 2012). 

The AMER family of Wnt pathway regulators 

Kristina Tanneberger, Astrid Alzner, Katharina Brauburger, Martin Sachs 

Amer 1, Amer2 and Amer3 constitute a new family of Wnt pathway regulators that 

are characterized by the presence of conserved APC binding domains which interact 

with the armadillo repeats of APC. We have initially identified Amer1 in a yeast two 

hybrid screen for interaction partners of APC and have cloned the other family 

members as open reading frames from database information. 

Amer1 

We previously identified Amer1 (APC membrane recruitment 1) as a novel 

membrane-associated protein that interacts with APC and recruits it away from 

microtubule ends to the plasma membrane. The N-terminus of Amer1 contains two 

distinct phosphatidylinositol(4,5)-bisphosphate [PtdIns(4,5)P(2)]-binding domains 

composed of clusters of lysines, which mediate its localization to the plasma 

membrane. 

Amer1 is identical to the tumour suppressor WTX, which is mutated in a proportion of 

Wilms tumours. Amer1/WTX acts as an inhibitor of Wnt signaling by inducing βcatenin 

degradation. Amer1 directly interacts with the armadillo repeats of β-catenin 

30

via a domain consisting of repeated arginine-glutamic acid-alanine (REA) motifs, and 

assembles the β-catenin destruction complex at the plasma membrane by recruiting 

β-catenin, APC, and Axin/Conductin. Deletion or specific mutations of the membrane 

binding domain of Amer1 abolish its membrane localization and abrogate negative 

control of Wnt signaling. Knockdown of Amer1 leads to the activation of Wnt target 

genes, preferentially in dense compared with sparse cell cultures, suggesting that 

Amer1 function is regulated by cell contacts. Amer1 stabilizes Axin and counteracts 

Wnt-induced degradation of Axin, which requires membrane localization of Amer1. 

The data suggest that Amer1 exerts its negative regulatory role in Wnt signaling by 

acting as a scaffold protein for the β-catenin destruction complex and promoting 

stabilization of Axin at the plasma membrane (Tanneberger et al., 2011a). 

Phosphorylation of the Wnt receptor low-density lipoprotein receptor-related protein 6 

(LRP6) by glycogen synthase kinase 3β (GSK3β) and casein kinase 1γ (CK1γ) is a 

key step in Wnt/β-catenin signalling, which requires Wnt-induced formation of 

phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). We found that Amer1 is 

essential for the activation of Wnt signalling at the LRP6 receptor level. Knockdown 

of Amer1 reduced Wnt-induced LRP6 phosphorylation, Axin translocation to the 

plasma membrane and formation of LRP6 signalosomes. Overexpression of Amer1 

promotes LRP6 phosphorylation, which requires interaction of Amer1 with 

PtdIns(4,5)P(2). Amer1 translocates to the plasma membrane in a PtdIns(4,5)P(2)dependent 

manner after Wnt treatment and is required for LRP6 phosphorylation 

stimulated by application of PtdIns(4,5)P(2). Amer1 binds CK1γ, recruits Axin and 

GSK3β to the plasma membrane and promotes complex formation between Axin and 

LRP6. We propose a mechanism for Wnt receptor activation by which generation of 

PtdIns(4,5)P(2) leads to recruitment of Amer1 to the plasma membrane, which acts 

as a scaffold protein to stimulate phosphorylation of LRP6. 

Altogether our data show that Amer1 has a dual functional role in Wnt signalling 

acting as an activator at the level of the Frizzled/LRP receptor complex and as an 

inhibitor by assembling the β-catenin destruction complex (Fig. 1). Of note, Amer1 

makes use of similar interaction partners, namely axin and GSK3 for both activities 

(Tanneberger et al., 2011b). 

31

Fig. 1 Dual functional role of Amer1 in Wnt signalling. Amer1 recruits the b-catenin 

destruction complex to the plasma membrane by binding to PIP2 which promotes b-catenin 

degradation and blocks Wnt signalling. When Wnts bind to Frizzled/LRP6 receptors more 

PIP2 is generated probably in vicinity to the receptors, Amer1 becomes recruited and 

stimulates phosphorylation of LRP6 receptors through interaction with GSK3 and CK1γ. 

Thus, Amer1 can act both as a repressor and an activator of the pathway. 

Amer2 

Amer2 (APC membrane recruitment 2; FAM123A) is a direct interaction partner of 

APC, related to Amer1/WTX. We showed that Amer2 recruits APC to the plasma 

membrane by binding to phosphatidylinositol 4,5-bisphosphate lipids via lysine-rich 

motifs and that APC links β-catenin and the destruction complex components axin 

and conductin to Amer2. Knockdown of Amer2 increased Wnt target gene expression 

and reporter activity in cell lines, and overexpression reduced reporter activity, which 

required membrane association of Amer2. In Xenopus embryos, Amer2 is expressed 

mainly in the dorsal neuroectoderm and neural tissues. Down-regulation of Amer2 by 

specific morpholino oligonucleotides altered neuroectodermal patterning, which could 

be rescued by expression of a dominant-negative mutant of Lef1 that interferes with 

β-catenin-dependent transcription. Our data characterize Amer2 for the first time as a 

negative regulator of Wnt signaling both in cell lines and in vivo and define Amer 

proteins as a novel family of Wnt pathway regulators. 

In yeast two hybrid screen using a C-terminal fragment of Amer2 as a bait we found 

that Amer2 is a direct interaction partner of EB1, which is a key factor in the 

32

organisation of the microtubule cytoskeleton. Of note, EB1 interacts with APC to 

stabilise microtubules. Amer2 binds to EB1 via specific S/TxIP motifs and recruits it to 

the plasma membrane. Coexpression of Amer2 and EB1 generates stabilised 

microtubules whereas knockdown of Amer2 leads to destabilisation of microtubules. 

Knockdown of Amer2, APC, or EB1 reduces cell migration, and morpholino-mediated 

downregulation of Xenopus Amer2 blocks convergent extension cell movements 

suggesting that the Amer2/EB1/APC complex regulates cell migration by altering 

microtubule stability (Pfister et al., JBC in revision). The Amer2 knock-out in mice has 

recently been generated in our lab but shows no obvious phenotypes, possibly due to 

compensation by Amer2. We are currently generating double-knockout animals of 

Amer1 and Amer2 for further characterization. 

Fig. 2 Scheme of Amer2 interactions with APC and EB1 in microtubule stabilization. 

Amer3 

Amer3 does not interact with the plasma membrane and is mainly located in the 

cytoplasm and occasionally in the cell nucleus. Moreover, in contrast to Amer1 and 

Amer2 this protein acts as an activator rather than inhibitor of Wnt signalling. 

Ongoing work aims at identifying the precise function of Amer3 and its interplay with 

the other Amers in the Wnt pathway. 

33

HIF target gene expression in renal cell carcinomas 

A key role for Activin B in cellular transformation after loss of the von Hippel- 

Lindau (VHL) tumour suppressor 

Ingrid Wacker, Martin Sachs, 

Clear cell renal cell carcinoma (clear cell RCC) is the most frequent tumour disease 

in the kidney. Most of these tumours contain mutations in the VHL tumour suppressor 

gene, which leads to stabilization of HIF1α and Hif2α, and causes increased 

transcription of HIF target genes. We found that VHL suppresses key features of cell 

transformation solely through downregulation of the HIF-dependent expression of 

Activin B, a member of the TGFβ superfamily. Activin B expression is repressed by 

restoration of VHL in VHL-deficient RCC cells and upregulated by hypoxia. RCC 

tumour samples show increased expression of Activin B as compared to normal 

kidney. Importantly, knockdown of Activin B reduces tumour growth of RCC cell lines 

in nude mice. Our data indicate that Activin B is a key mediator of VHL/HIF induced 

transformation in RCC (Wacker et al., 2009). We are currently searching for ways to 

block Activin B signalling using biological reagents in order to prevent RCC 

tumourigenesis. 

Liprinα4 is a novel HIF1α target gene 

Sandra Mattauch 

Liprin-α1 to liprin-α4 constitute a family of cytoplasmic proteins, which have been 

found in various multiprotein complexes. For liprin-α1 roles in synapse formation and 

cell spreading were described but other liprin family members are not well 

characterized. We found that liprin-α4 is upregulated in human clear cell renal cell 

carcinomas as compared to normal kidney tissue and that it is regulated the VHL/HIF 

system. The liprin-α4 gene promoter is directly activated by binding of the hypoxiainducible 

factor 1α (HIF-1α) to HRE consensus binding sites as shown by reporter 

assays and chromatin immunoprecipitations. RNAi mediated knockdown of liprin-α4 

leads to reduced E-cadherin and β-catenin levels at cell junctions and to dissociation 

of epithelial cell contacts. Our data describe for the first time liprin-α4 as a hypoxiainduced 

gene potentially involved in cell-cell adhesion (Mattauch et al., 2010). 

34

2012 



Vijaya Chandra, S.H., Wacker, I., Appelt, U.K., Behrens, J., and Schneikert, J. 

(2012). A common role for various human truncated adenomatous polyposis 

coli isoforms in the control of Beta-catenin activity and cell proliferation. PLoS 

One 7, e34479. 

Pfister, A.S., Tanneberger, K., Schambony, A., and Behrens, J. (2012). Amer2 

protein is a novel negative regulator of Wnt/beta-catenin signaling involved in 

neuroectodermal patterning. J Biol Chem 287, 1734-1741. 

Hadjihannas, M.V., Bernkopf, D.B., Bruckner, M., and Behrens, J. (2012). Cell cycle 

control of Wnt/beta-catenin signalling by conductin/axin2 through CDC20. 

EMBO Rep 13, 347-354. 

2011 

Tanneberger, K., Pfister, A.S., Kriz, V., Bryja, V., Schambony, A., and Behrens, J. 

(2011a). Structural and Functional Characterization of the Wnt Inhibitor APC 

Membrane Recruitment 1 (Amer1). J Biol Chem 286, 19204-19214. 

Tanneberger, K., Pfister, A.S., Brauburger, K., Schneikert, J., Hadjihannas, M.V., 

Kriz, V., Schulte, G., Bryja, V., and Behrens, J. (2011b). Amer1/WTX couples 

Wnt-induced formation of PtdIns(4,5)P2 to LRP6 phosphorylation. EMBO J 30, 

1433-1443. 

Schneikert, J., Brauburger, K., and Behrens, J. (2011). APC mutations in colorectal 

tumours from FAP patients are selected for CtBP-mediated oligomerization of 

truncated APC. Hum Mol Genet 20, 3554-3564. 

2010 

Schietke, R., Warnecke, C., Wacker, I., Schodel, J., Mole, D.R., Campean, V., 

Amann, K., Goppelt-Struebe, M., Behrens, J., Eckardt, K.U., et al. (2010). The 

lysyl oxidases LOX and LOXL2 are necessary and sufficient to repress Ecadherin 

in hypoxia: insights into cellular transformation processes mediated 

by HIF-1. J Biol Chem 285, 6658-6669. 

Mattauch, S., Sachs, M., and Behrens, J. (2010). Liprin-alpha4 is a new hypoxiainducible 

target gene required for maintenance of cell-cell contacts. Exp Cell 

Res 316, 2883-2892. 

Kohler, E.M., Brauburger, K., Behrens, J., and Schneikert, J. (2010). Contribution of 

the 15 amino acid repeats of truncated APC to beta-catenin degradation and 

selection of APC mutations in colorectal tumours from FAP patients. 

Oncogene 29, 1663-1671. 

Hadjihannas, M.V., Bruckner, M., and Behrens, J. (2010). Conductin/axin2 and Wnt 

signalling regulates centrosome cohesion. EMBO Rep 11, 317-324. 

35

2009 

Wacker, I., Sachs, M., Knaup, K., Wiesener, M., Weiske, J., Huber, O., Akcetin, Z., 

and Behrens, J. (2009). Key role for activin B in cellular transformation after 

loss of the von Hippel-Lindau tumour suppressor. Mol Cell Biol 29, 1707-1718. 

Rotte, A., Bhandaru, M., Foller, M., Biswas, R., Mack, A.F., Friedrich, B., Rexhepaj, 

R., Nasir, O., Ackermann, T.F., Boini, K.M., et al. (2009). APC Sensitive 

Gastric Acid Secretion. Cell Physiol Biochem 23, 133-142. 

Nasir, O., Wang, K., Foller, M., Gu, S., Bhandaru, M., Ackermann, T.F., Boini, K.M., 

Mack, A., Klingel, K., Amato, R., et al. (2009). Relative resistance of SGK1 

knockout mice against chemical carcinogenesis. IUBMB Life 61, 768-776. 

Kohler, E.M., Chandra, S.H., Behrens, J., and Schneikert, J. (2009). Beta-catenin 

degradation mediated by the CID domain of APC provides a model for the 

selection of APC mutations in colorectal, desmoid and duodenal tumours. 

Hum Mol Genet 18, 213-226. 

TRAINING OF GRADUATE AND MEDICAL STUDENTS 

Doctoral theses (Biology) 

Astrid Pfister 

Klonierung und funktionelle Charakterisierung von AMER2 

Katharina Brauburger 

Molekulare Funktionsanalyse von APC 

Sandra Mattauch 

Identifizierung und Charakterisierung von HIF-Zielgenen im Nierenzellkarzinom 

Shree Harsha Vijaya Chandra 

Blockade des Wachstums von Dickdarmkrebs durch RNA Interferenz 

Doctoral theses (Molecular Medicine) 

Dominic Bernkopf 

Molekularer und funktioneller Vergleich von Axin und Conductin/Axin2 

Kristina Tanneberger 

Molekulare Charakterisierung des Tumoursuppressors AMER1/WTX 

36

DIVISION OF MOLECULAR IMMUNOLOGY 

(Department of Internal Medicine III) 

Head: Hans Martin Jäck, Dr. rer. nat. 

Professor of Immunology 

Address: Division of Molecular Immunology 

Glückstr. 6 


Telefone: + 49 (9131) 85 35912 

Fax: + 49 (9131) 85 39343 

E-mail: hjaeck@molmed.uni-erlangen.de 

Homepage: www.molim.uni-erlangen.de 

Dirk Mielenz, Dr. rer. nat. 

Jürgen Wittmann, Dr. rer. nat. 

Wolfgang Schuh, Dr. rer. nat. 

Head 

Hans-Martin Jäck, Prof. Dr. rer. nat. 

Professor of Immunology 


Elisabeth Lang, Secretary 


37 

Barbara Fürnrohr, Dr. rer. nat. 

Rebecca Winkelmann, Dr. rer. nat. 

Martina Porstner, Dr. rer. nat.

Andreas Brandl* 

Sebastian Dütting* 

Sandra Schreiber* 

* PhD received 


Pavitra Purohit 

Julia Schmid 

Sebastian Brachs 

Patrick Daum 

Carmen Kroczek* 

Stefanie Thiele* 

Anke Lang* 

Doctoral Students (Molecular Medicine & Medicine) 

Martina Porstner* 

Rebecca Winkelmann* 


Edith Roth 

Christiane Lang 

Heike Danzer 

Sandra Hagen 

Technicians 

Manuela Hauke 

Lena Sandrock 

Katrin Weiß 

Professor Emeritus 

Prof. Dr. med. Dr. 

mult. h.c. Joachim R. 

Kalden 

38 

Alexandra Klej 

Uwe Appelt


Our research concentrates on molecular mechanisms that guide the development of 

antibody-producing B cells and control the adaptive humoral immunity in healthy 

organisms. In addition, we are interested in strategies to target pathologic plasma 

cells in autoimmune disease and in patients with multiple myeloma. 

Nonsense-mediated mRNA decay in lymphocytes 

Jürgen Wittmann, Carmen Baumgarten, Manuela Hauke, Heike Danzer and Hans- 

Martin Jäck 

Identification of novel factors involved in nonsense-mediated mRNA decay in 

higher eukaryotes 

Nonsense-mediated mRNA decay (NMD) is an essential eukaryotic pathway that 

recognizes and degrades erroneous mRNAs that contain a premature termination 

codon (nonsense mRNAs). By doing so, a negative influence on physiologic 

functions within a cell by faulty mRNAs is prevented, and error-free gene expression 

is guaranteed. Three evolutionary conserved proteins, UPF1, UPF2 and UPF3, play 

important roles in NMD. In particular, UPF2 fulfils a central role as adaptor protein, 

because it interacts with UPF1 and UPF3. However, not much is known about the 

biochemical role of UPF2 and its precise function in the different effector arms of 

NMD. We are therefore planning to identify interaction partners of key NMD factors 

by immunoprecipitation followed by mass spectrometric analysis to shed some light 

on mechanistic details of UPF function during NMD. 

Collaboration with Henning Urlaub (Göttingen) 

Function of the nonsense-mediated mRNA decay pathway during murine 

lymphocyte development 

As a result of the programmed rearrangements that generate the diverse 

immunoglobulin repertoire, nonsense codons are frequently generated in B- and Tcell 

receptor genes. Therefore, during B- and T-cell-development, a large number of 

lymphoid precursors with non-functional mRNAs are produced, which have to be 

detected and degraded early by the NMD pathway to avoid the production of 

deleterious products. To start addressing the role of NMD during lymphocyte 

development, we have generated two different conditional knock-out mice for one of 

the key factors of NMD. Although NMD is predicted to be important during 

embryogenesis, our conditional knock-out strategy allows us to study the role of NMD 

not only in lymphocyte development, but also in disease prevention in pre-selected 

tissues in the adult. Therefore, we will address presently unanswered fundamental 

questions about the role of NMD in B cell development and the basic NMD 

mechanism. 

Collaboration with Bo Porse (Copenhagen) 

39

microRNAs in lymphocytes in health and disease 

Jürgen Wittmann, Markus Zwick, Ina Wirries, Sandra Schreiber, Patrick Daum, 

Andreas Brandl, Martina Porstner, Manuela Hauke, Heike Danzer and Hans-Martin 

Jäck 

Role of microRNAs in B cell development in health and disease 

MicroRNAs (miRNAs) are a class of small endogenous non-coding RNAs that 

regulate gene expression at the post-transcriptional level by targeting partially 

sequence-complementary mRNAs. To investigate the role of miRNAs in B cell 

development, we used deep sequencing analysis of sorted primary B cells of several 

developmental stages to profile their miRNome. These studies allowed us to 

determine a set of marker miRNAs characteristic of the respective developmental 

stage. In addition, stage-specific novel miRNAs and other small RNA classes were 

discovered, opening up new research avenues for elucidating the fine-tuning of gene 

expression in murine B cell development. 

To expand the known regulatory function of miRNAs in early hematopoiesis and to 

test the hypothesis that miRNAs play a role in the pre-B cell receptor (pre-BCR)mediated 

control of early B cell development, we used a tetracycline-inducible µ 

heavy chain mouse model to identify differently expressed miRNAs in pro- and pre-B 

cells by miRNA microarray- and deep sequencing analysis. Several miRNAs were 

found to be up- or downregulated after µ heavy chain induction. In silico analysis 

identified several potential mRNA targets of these miRNAs that are involved in early 

B cell development, BCR signal transduction, opening of the Ig light chain locus as 

well as proliferation and cancer development. Functional assays with overexpressed 

miRNAs and reporter gene assays revealed that they indeed regulate key processes 

in early B cell development. 

Role of microRNAs in rheumatic diseases 

Rheumatoid arthritis is a chronic, systemic, inflammatory autoimmune disease that 

has its primary target in synovial tissues. It principally attacks the joints, which 

produce an inflammatory reaction that often progresses to destruction of the articular 

cartilage and bones and may eventually result in ankylosis of the joints. The major 

goal of this project is to gain insights into the molecular circuits that control the 

development of rheumatoid- and autoimmune diseases. Our short-term aim is to 

identify prognostic, diagnostic and potentially therapeutic miRNA markers and to 

better understand the role miRNAs play in the development of rheumatoid diseases. 

Plasma cell homing and B cell homeostasis controlled by Krüppellike 

factor 2 (KLF2) 

Rebecca Winkelmann, Lena Sandrock, Wolfgang Schuh and Hans-Martin Jäck 

Plasma cells are powerful micro factories producing thousands of antibody molecules 

per second and thereby protecting the organism from harmful pathogens and 

infections. Once a B cell encounters its specific antigen, a fascinating differentiation 

program is initiated that leads to morphological cellular restructuring and converting 

40

the B cell into an antibody-producing cell, the so-called plasma cell. Plasma cells can 

be subdivided into short- and long-lived plasma cells. Short-lived plasma cells 

predominantly produce IgM and have a life-span of approximately one week; in 

contrast long-lived plasma cells continuously produce high affinity IgG or IgA 

molecules and survive for years. Long-lived plasma cells, which are generated in 

secondary lymphoid tissues, have to reach specialized survival niches formed by 

stromal and other cells in the bone marrow. Still, it is puzzling how migration, homing 

and survival of these cells are controlled. 

Krüppel-like factor 2 (KLF2/LKLF) belongs to the family of Krüppel-like transcription 

factors, which consists of at least 17 members in mammals. Krüppel-like 

transcription factors bind to GC rich DNA domains via three C-terminal zinc fingers 

and are involved in controlling proliferation and terminal differentiation of various cell 

types. KLF2 was originally discovered in lung tissue and was shown to be important 

for cardiovascular development. Apart from its role in lung and cardiovascular 

development, KLF2 plays an important role in activation and migration of T 

lymphocytes. 

Using mice with a B cell specific deletion of KLF2, we demonstrated that KLF2 is 

crucial for proper homing of antigen-specific plasma cells to the bone marrow and for 

B cell homeostasis. In KLF2 deficient animals all splenic B cell subsets including B1 

cells were present, but their numbers were increased with a clear bias for marginal 

zone (MZ) B cells. In contrast, less peyers patches harboring fewer B cells were 

found and B1 cells in the peritoneal cavity were almost absent. B cell specific deletion 

of KLF2 results in a clear reduction of plasma cells in the bone marrow, whereas 

plasma cell numbers in the spleen and the blood were fairly normal, indicating that 

KLF2 plays an important role in homing of plasma cells to the bone marrow (Fig. 1). 

Since we found diminished levels of ß7 Integrin and L-Selectin on KLF2-deficient B 

cells, we propose that KLF2 regulates plasma cell homing to the bone marrow via 

regulation of these two cell adhesion molecules. Future experiments using a plasma 

cell-specific GFP reporter mouse will clarify the role of KLF2 in humoral immunity and 

provide new ways to interfere with the generation of pathologic plasma cells in 

patients with multiple myeloma and autoimmune disease. 

Fig. 1 Elispot assay of TNP-specific IgG secreting cells in spleen and bone marrow 14 days after 

boost immunization with TNP-KLH. A triplicate of one representative experiment is shown to the left, 

and results of all analyzed littermates are summarized with one dot representing the mean value of 

triplicates of one mouse is shown to the right. 

41

Function of EFhd1 and EFhd2 in B cells and brain in vivo 

Pavitra Purohit, Sebastian Brachs, Sebastian Dütting, Carmen Kroczek, Christiane 

Lang and Dirk Mielenz 

Antigen-induced B cell activation depends on a subset of antigen-B cell receptor 

(BCR) complexes that partition into plasma membrane microdomains, thereby, 

controlling the BCR-elicited calcium (Ca 2+ ) efflux from the endoplasmatic reticulum. 

This, in turn, regulates the total cytosolic Ca 2+ concentration [Ca 2+ ]i after BCR 

stimulation. Amplitude and duration of [Ca 2+ ]i in response to BCR activation governs 

activity of pro- or anti-apoptotic transcription factors, NF-AT and NF-�B. To identify 

mechanisms mediating membrane microdomain association of the BCR, a couple of 

years ago we defined the membrane microdomain proteome of B cells by separating 

purified membrane microdomains on 2D gels, followed my mass spectrometry. 

Thereby, we identified the new Ca 2+ binding protein Swiprosin-1/EFhd2 (EFhd2). 

EFhd2 regulates proximal BCR signaling and [Ca 2+ ]i via a mechanism that involves 

low-affine (110 �M) and cooperative Ca 2+ binding to EFhd2 in a 2:1 molar ratio, as 

well as membrane microdomain association of EFhd2 and proximal BCR signaling 

proteins. In addition to enhancing proximal BCR signals, EFhd2 interferes with antiapoptotic 

NF-�B activation, thereby, acting pro-apoptotically in B cells. Thus, loss of 

EFhd2 was expected to affect B cell selection and activation. To elucidate the 

function of EFhd2 in vivo, we generated EFhd2 deficient mice (EFhd2 K.O.). 

Surprisingly, B cell development and basal immunoglobulin (Ig) titers of all 

subclasses were normal in EFhd2 K.O. mice. However, EFhd2 K.O. mice responded 

slightly better to T-independent and T-dependent immunizations. More clearly, EFhd2 

K.O. mice developed higher affine antibodies towards a T-dependent antigen after 

recall immunization. We tested whether that could be due to increased competition 

amongst B cells, which would favour selection of high-affine B cell clones by T cells. 

In fact, EFhd2 K.O. mice developed larger germinal centers after immunization with 

sheep red blood cells. Along these lines, EFhd2 K.O. mice develop anti double 

stranded DNA antibodies after 1 year of age, pointing to either a defect in negative B 

cell selection or enhanced polyclonal activation. We obtained evidence for the latter 

point through polyclonal stimulation of purified B cells in vitro with lipopolysaccharide, 

demonstrating that EFhd2 K.O. B cells are 1) pre-activated and 2) switch faster from 

this pre-activated state into the plasma cell differentiation programme. In summary, 

EFhd2 is a negative regulator of B cell activation through an as yet to be defined 

mechanism. 

In contrast to EFhd2 that is expressed in all B cell stages, the related EFhd1 

becomes down-regulated during early B cell development by the pre-BCR. Activity of 

the transcription factor Foxo1, that controls stress responses, senescence and 

expression of recombinase activating genes (rag), is controlled by the pre-BCR as 

well as Interleukin-7 (IL-7). Interestingly we found a physical interaction of 

endogenous EFhd1 with endogenous Foxo1 in the cytoplasm of the pro B cell line 

38B9, as well as in 293 cells transfected with EFhd1. EFhd2 does not interact with 

Foxo1. To circumvent down-regulation of EFhd1 by the pre-BCR we generated 

transgenic mice which ectopically express EFhd1 (EFhd1tg) both in B as well as in T 

cells. In contrast to data obtained with an in vitro differentiation system, ectopic 

expression of EFhd1 did not affect steady state early B cell development in vivo. 

However, transgenic B and T cells expressed more CD62L, a Foxo1 target gene, and 

revealed increased Foxo1 expression on the protein level. We hypothesize that 

EFhd1 regulates Foxo1 function positively. This interaction should contribute to 

42

Foxo1 activity in early B cells in the context of IL-7 and pre-BCR signaling. Loss of 

EFhd1 should induce loss of function of Foxo1, thereby, reducing rag expression and 

slowing down early B cell development. Both gain and loss of function of EFhd1 may 

only be revealed under competitive conditions. Thus, we will create mixed bone 

marrow chimeras of wt and EFhd1tg bone marrow as well as wt and EFhd1 K.O. 

bone marrow and analyze the putative contribution of EFhd1 to early B cell 

development 

Since there was evidence that EFhd2 might be involved in neurodegeneration 

mediated by the microtubule-stabilizing protein tau we tested whether EFhd2 

modulates tau function. Surprisingly, we observed that co-expression of EFhd2 with 

mutant tau proteins (tau P301S and tau �K280), that cause frontotemporal dementia 

associated with parkinsonism (FTDP), but not with wildtype tau, caused degradation 

of EFhd2, but not of the co-expressed GFP. We are currently elucidating the 

proteolytic pathway underlying this degradation process. In line with a putative 

interaction of tau and EFhd2, we detected EFhd2 with newly generated monoclonal 

antibodies in ordered presynaptic complexes in murine as well as human brain 

sections. Interestingly, the presynaptic, ordered expression of EFhd2 was completely 

abolished in barins of Alzheimer’s patients. Likewise, EFhd2 co-localized with tau and 

with tau tangles in brains of Alzheimer’s patients. Finally we observed that the 

presynaptic marker Bassoon was largely absent from synaptic structures in brains of 

EFhd2 K.O. mice whereas tau expression was stronger and more confined to 

presynaptic complexes than in wildtype mice. Taken together we hypothesize that 

EFhd2 modulates tau function to control the composition of pre-synaptic complexes. 

This might be important during onset and progression of tau-mediated 

neurodegenerative diseases and for learning or rewarding processes. 

Genetic and functional analysis of Lupus-associated risk alleles 

Katrin Weiß and Barbara Fürnrohr 

Systemic Lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease 

involving multiple organs including skin, joints, kidneys, brains, the cardiovascular 

system and serosal membranes. Immunologically, SLE is characterised by the 

presence of antinuclear autoantibodies, especially against double stranded (ds) DNA 

and nucleosomes, activation of the complement system during flares and type I 

interferon secretion. The etiology of SLE remains elusive, however, an interplay of 

genetic and environmental factors is considered to ultimately cause immune 

dysregulation, resulting in clinical symptoms. Twin studies have reported a 

concordance of 24-57% and 2-5% in monozygotic and dizygotic twins, respectively, 

therefore SLE has an estimated heritability of 66%. Recent genome-wide association 

studies together with candidate gene studies have uncovered four major candidate 

gene loci, namely the MHC class II locus IKZF1, STAT4 and ITGAM which are most 

probably of functional relevance in SLE. 

Genetic variation at the ITGAM gene has been identified as a key genetic 

susceptibility effect in human systemic lupus erythematosus (SLE). It is one of the 

strongest known genetic risk factors in SLE, and is common (minor allele frequency 

approximately 10%) in all except East Asian populations. Interest has specifically 

focussed on the rs1143679 variant, which encodes an arginine to histidine amino 

acid change at position 77 in the �-propeller of the extracellular domain in CD11b 

which is encoded by ITGAM. As part of the complement receptor CR3, CD11b is 

43

mainly expressed on phagocytes, functioning as an adhesion molecule and a 

phagocytic receptor. 

At the Department of Medical and Molecular Genetics at King’s College in London in 

the group of Professor Timothy Vyse we have studied together with Dr. Benjamin 

Rhodes ex vivo monocytes/monocyte-derived macrophages from healthy volunteers 

homozygous for the 77H variant compared with homozygous wild-type (WT) controls. 

In cells isolated from donors homozygous for 77H we observed a reduction in the 

phagocytosis of iC3b opsonised sheep erythrocytes (sRBCiC3b) in comparison to WT 

cells. 

1160 

Ch04 Ch02/C h05 Ch05 

Fig. 1 Phagocytic Assay with red-fluorescent labelled iC3b-opsonised sheep red blood cells 

internalised by green-fluorescent labelled human macrophages. 

In addition, the adhesion of 77H monocytes was reduced to a range of CR3 ligands, 

such as iC3b, fibrinogen, fibronectin and DC-SIGN. To confirm the functionality of 

R77H specific ITGAM variants were transfected into COS7 cells and defective 

phagocytosis was observed in COS7 cells expression the 77H variant. We could also 

demonstrate differences in the release of Toll-like receptor-induced pro-inflammatory 

cytokines from monocytes of donors with different ITGAM genotypes following preengagement 

of CR3 using sRBCiC3b. The R77H variant therefore impairs a broad 

range of CR3 effector functions in human monocytes. 

CR3 ligation is likely to be important in immune complex and apoptotic cell clearance, 

both of which may contribute to the pathogenesis of SLE. Our future experiments will 

include phagocytic assays with monocytes from donors with different ITGAM 

genotype using fluorescent labelled apoptotic cells. Protein purification and structural 

analysis of the individual CD11b variants will hopefully unravel the functional 

differences observed in adhesion and phagocytosis. 

Collaboration with Prof. Timothy Vyse and Dr. Ben Rhodes (King’s College London) 

44

2009 


Original Articles (members are underlined) 

Trageser, D., Iacobucci, I., Nahar, R., Duy, C., Von Levetzow, G., Klemm, L., Park, 

E., Schuh, W., Gruber, T., Herzog, S., Kim, Y.M., Hofmann, W.K., Li, A., 

Storlazzi, C.T., Jäck, H.M., Groffen, J., Martinelli, G., Heisterkamp, N., Jumaa, 

H., and Müschen, M. (2009). Pre-B cell receptor-mediated cell cycle arrest in 

Philadelphia chromosome-positive acute lymphoblastic leukemia requires 

IKAROS function. J Exp Med, 206 (8): 1739-1753. 

Urbonaviciute, V., Meister, S., Fürnrohr, B.G., Frey, B., Gückel, W., Schett, G., 

Herrmann, M., and Voll, R.E. (2009). Oxidation of the alarmin high-mobility 

group box 1 protein (HMGB1) during apoptosis. Autoimmunity. 42 (4):305-7. 

2010 

Danke, C., Grünz, X., Wittmann, J., Schmidt, A., Agha-Mohammadi, S., Kutsch, O., 

Jäck, H. M., Hillen, W., and Berens, C. (2010). Adjusting transgene expression 

levels in lymphocytes with a set of inducible promoters. J Gene Med, 12:501- 

515. 

Duy, C., Yu, J.J., Nahar, R., Swaminathan, S. , Kweon, S.M., Polo, J.M., Valls, E., 

Klemm, L., Shojaee, S., Cerchietti, L., Schuh, W., Jäck, H.M., Hurtz, C., 

Ramezani-Rad, P., Herzog, S., Jumaa, H., Koeffler, H.P., De Alborán, I.M., 

Melnick, A.M., Ye, B.H., and Müschen, M. (2010). BCL6 is critical for the 

development of a diverse primary B cell repertoire. J Exp Med, 207 (6), pp. 

1209-1221. 

Fürnrohr, B.G., Wach, S., Haslbeck, M., Weber, C.K., Stach, C.M., Hueber, A., Graef, 

D., Spriewald, B., Manger, K., Herrmann, M., Kaufmann, K.M., Summer, G.F., 

James, J.A., Goodmon, E., Kelly, J.A., Schett, G., Winkler, T., Harley, J.B., 

and Voll, R.E. (2010). Hsp70 polymorphisms are genetically associated with 

systemic lupus erythematosous. Ann Rheum Dis, 69 (11):1983-9. 

Hakkim, A., Fürnrohr, B.G., Amann, K., Laube, B., Abu Abed, U., Brinkmann, V., 

Herrmann, M., Voll, R.E., and Zychlinsky, A. (2010). Impairment of neutrophil 

extracellular trap degradation is associated with lupus nephritis. PNAS, 107 

(21):9813-8. 

Kroczek, C., Lang, C., Brachs, S., Grohmann, M., Dutting, S., Schweizer, A., 

Nitschke, L., Feller, H. M., Jäck, H.M. and Mielenz, D. (2010). Swiprosin- 

1/EFhd2 controls B cell receptor signaling through the assembly of the B cell 

receptor, Syk, and phospholipase C gamma2 in membrane rafts. J Immunol, 

184:3665-3676. 

45

Lang, V. R., Mielenz, D., Neubert, K., Bohm, C., Schett, G., Jäck, H.M., Voll, R.E., 

and Meister, S. (2010). The early marginal zone B cell-initiated T-independent 

type 2 response resists the proteasome inhibitor bortezomib. J Immunol, 

185:5637-5647. 

Metzner, M., Schuh, W., Roth, E., Jäck, H.M., and Wabl, M. (2010). Two forms of 

activation-induced cytidine deaminase differing in their ability to bind agarose. 

PloS one, 5 (1). 

Weber, C. K., Haslbeck, M., Englbrecht, M., Sehnert, B., Mielenz, D., Graef, D., 

Distler, J. H., Mueller, R. B., Burkhardt, H., Schett, G., Voll, R. E., and 

Fürnrohr, B. G. (2010). Antibodies to the endoplasmic reticulum-resident 

chaperones calnexin, BiP and Grp94 in patients with rheumatoid arthritis and 

systemic lupus erythematosus. Rheumatology (Oxford), (12):2255-63. 

2011 

Avouac, J., Fürnrohr, B.G., Tomcik, M., Palumbo, K., Zerr, P., Horn, A., Dees, C., 

Akhmetshina, A., Beyer, C., Distler, O., Schett, G., Allanore, Y., Distler, J.H. 

(2011). Inactivation of the transcription factor STAT4 prevents inflammationdriven 

fibrosis in systemic sclerosis animal models. Arthritis Rheum, 63 

(3):800-9. 

Brandl, A., Wittmann, J., and Jäck, H. M. (2011). Increased retroviral titers of miRNAencoding 

retroviruses by inhibition of the RNaseIII enzyme Drosha. Eur J 

Immunol, 41:549-551. 

Dedman, A. M., Majeed, Y., Tumova, S., Zeng, F., Kumar, B., Munsch, C., Bateson, 

A. N., Wittmann, J., Jäck, H. M., Porter, K. E., and Beech, D. J.. (2011). 

TRPC1 transcript variants, inefficient nonsense-mediated decay and low upframeshift-1 

in vascular smooth muscle cells. BMC Mol Biol, 12:30. 

Marschall, J.S., Wilhelm, T., Schuh, W., and Huber, M. (2011). MEK/Erk-based 

negative feedback mechanism involved in control of Steel Factor-triggered 

production of Krüppel-like factor 2 in mast cells. Cell Signal, (4):879-88. 

Rosenbaum, S., Kreft, S., Etich, J., Frie, C., Stermann, J., Grskovic, I., Frey, B., 

Mielenz, D., Pöschl, E., Gaipl, U., Paulsson, M., and Brachvogel, B. (2011). 

Identification of novel binding partners (annexins) for the cell death signal 

phosphatidylserine and definition of their recognition motif. J Biol Chem, 

286:5708-5716. 

Weber, C.K., Mielenz, D., Voll, R.E., and Fürnrohr, B.G. (2011). Comment on: 

Antibodies to the endoplasmic reticulum-resident chaperones calnexin, BiP 

and Grp94 in patients with rheumatoid arthritis and systemic lupus 

erythematosus: Reply. Rheumatology (Oxford), 50:629-631. 

46

Winkelmann, R., Sandrock, L., Porstner, M., Roth, E., Mathews, M., Hobeika, E., 

Reth, M., Kahn, M.L., Schuh, W., and Jäck, H.M. (2011). B cell homeostasis 

and plasma cell homing controlled by Krüppel-like factor 2. PNAS, 108 (2), pp. 

710-715. 

2012 

Buettner, M., Lang, A., Tudor, C.S., Meyer, B., Cruchley, A., Barros, M.H., Farrell, P., 

Jäck, H.M., Schuh, W., and Niedobitek, G. (2012). Lytic EBV infection in 

epithelial cells but not in B lymphocytes is dependent on Blimp1. J Gen Virol, 

[Epub ahead of print]. 

Hagen, S., Brachs, S., Kroczek, C., Fürnrohr, B.G., Lang, C., and Mielenz, D. (2012). 

The B cell receptor-induced calcium flux involves a calcium mediated positive 

feedback loop. Cell Calcium, [ePub 06Feb]. 

Preußner, M., Schreiner, S., Hung, L.H., Porstner, M., Jäck, H.M., Benes, V., Rätsch, 

G., and Bindereif, A. (2012). HnRNP L and L-like cooperate in multiple-exon 

regulation of CD45 alternative splicing. Nucleic Acids Res. 2012 Mar 8. [Epub 

ahead of print] 

Books and Reviews 

Dütting, S., Brachs, S., and Mielenz, D. (2011). Fraternal twins: Swiprosin-1/EFhd2 

and Swiprosin-2/EFhd1, two homologous EF-hand containing calcium binding 

adaptor proteins with distinct functions. Cell Commun Signal, 9:2. 

Wittmann, J., and Jäck, H.M. (2011). microRNAs in rheumatoid arthritis: midget 

RNAs with a giant impact. Ann Rheum Dis, 70:Suppl 1:i92-i96. 

Wittmann, J., Jäck H.M., and Mashreghi, M.F. (2011). microRNAs in B- and T cells 

as regulators of inflammation. Z Rheumatol, 70:507-510. 

Rhodes B., Fürnrohr B.G., and Vyse, T.J. (2011). C-reactive protein in rheumatology: 

biology and genetics. Nat Rev Rheumatol. 7 (5):282-9. 

Sestak, A.L., Fürnrohr, B.G., Harley J.B., Merrill, J.T., and Namjou, B. (2011). The 

genetics of systemic lupus erythematosus and implications for targeted 

therapy. Ann Rheum Dis, 70 Suppl 1:i37-43. 

Wittmann, J., and Jäck, H.M. (2010). Serum microRNAs as powerful biomarkers in 

disease. Biochim Biophys Acta – Reviews on Cancer, 1806:200-207. 

Wittmann, J., and Jäck, H.M. (2010). New surprises from the deep - The family of 

small regulatory RNAs increases. ScientificWorldJournal, 10:1239-1243. 

47



Andreas Brandl 

MicroRNA-vermittelte Kontrolle der Antigen-induzierten B-Zell-Differenzierung und 

deren Rolle in der B-Zell-Entwicklung 

Sebastian Dütting 

Charakterisierung von Swiprosin-2/EFHD1 und dessen Funktion während der frühen 

B-Zellentwicklung 

Carmen Kroczek 

Funktionelle Analyse von Swiprosin-1/EFhd2 im proximalen B-Zellrezeptor Signalweg 

Anke Lang 

Mechanismen der lytischen Reaktivierung des Epstein-Barr Virus 

Sandra Schreiber 

Die Rolle von microRNAs in der frühen B-Zellentwicklung 

Stefanie Thiele 

Die Rolle der microRNAs und Dicer in Entzündungszellen von 

Atemwegserkrankungen 


Rebecca Winkelmann 

The function of Krüppel-like factor 2 in B cell development 

Martina Porstner 

Untersuchungen zur Rolle von microRNAs in der Plasmazelldifferenzierung und dem 

Multiplen Myelom 

48

Hematopoiesis Unit 

Chair of Genetics – Department Biology 

Head: Thomas Winkler, Dr. rer. nat. 

Professor for Genetics 

Address: Hematopoiesis Unit 

Glückstr. 6 

D-91054 ERLANGEN 

Telefone: + 49 (9131) 85 29136 

Fax: + 49 (9131) 85 29106 

E-mail: thomas.winkler@molmed.rz.uni-erlangen.de 

Homepage: http://www.genetik.nat.uni-erlangen.de 

Ute Wellmann, Dr. rer. nat. * 


Sven Brenner ° 

Agnes Giniewski 

Kristin Schröder 


Tanja Fisch* 

Markus Mroz* 


Head 

Thomas Winkler, Prof. Dr. rer. nat. 

Professor for Genetics 



Sonja Pötzsch ° 

Martina Seefried 

Technicians 

49 

Sabrina Sell 

Florian Weisel ° 

Andrea Schneider 

Hannes Tittlbach*


Our research focus lies on the selection processes at different stages of the 

development of B-lymphocytes. We investigate the genetic mechanisms for the early 

development of B-lymphocytes, the mechanisms for the loss of self-tolerance in the 

autoimmune disease systemic lupus erythematosus and antibody-mediated 

protection against cytomegalovirus. In our research we predominantly work with 

mouse models that we also create in our laboratory by the techniques of gene 

targeting (knock-out mice, knock-in mice, transgenic mice). 

B cell development and regulation of VDJ-recombination 

During the differentiation of B-lymphocytes the genes for the antibody molecules are 

assembled in a unique process called VDJ-recombination. VDJ-recombination is 

responsible for the enormous diversity of antibody molecules. This diversity is 

necessary to defend all possible invaders of our body. Whereas the molecular 

mechanism of the VDJ-recombination is understood in very much detail today, the 

regulation of this process is less well understood. All 7 different antigen-receptor 

gene loci (3 for immunoglobulins and 4 for T-cell-receptors) use the same VDJrecombination 

machinery, but still the expression of the different genes is tighly 

regulated. Antibody receptors are expressed only on B-lymphocytes and T-cell 

receptors only on T-lymphocytes. In addition, during development of B-lymphocytes 

the genes for the heavy chain of the immunoglobulin is recombined before the genes 

for the light chain and additional regulation exists that only one antigen-receptor is 

expressed on each B-lymphocyte. 

Our research focus is to investigate the accessibility of the immunoglobulin heavy 

chain genes for the VDJ-recombinase. The epigenetic mechanisms for the regulation 

of the chromatin state at the immunoglobulin loci is analyzed by modern methods 

(ChIP-on-ChiP, ChIP-Seq). In addition we try to identify the essential regulatory 

sequences at the immunoglobulin locus by gene-targeting. 

Regulation of VDJ-recombination in precursor B-lymphocytes 

Agnes Giniewski and Thomas Winkler 

VDJ recombination is strictly confined to the early precursors of T- and B-cells, where 

only T-cells recombine the T-cell-receptor loci and only B-cells recombine the B-cellreceptor 

loci. The differential regulation of VDJ recombination remains unknown as 

the Rag1 and Rag2 enzymes that are critical for this reaction are expressed in early 

B-cells as well as in early T-cells. It has been proposed, that the differentially 

regulated accessibility of the T- and B-cell receptor loci is the basis of this regulation. 

As chromatin accessibility is regulated by histone modifications, we generated a high 

50

esolution map of histone modifications at the complete IgH-locus (app. 2.8 Mb) from 

early B-cell progenitors. Chromatin immunoprecipitation (ChIP) experiments using 

primary pro-B cells from Rag -/- mice and specific antibodies for acetylated histone H3 

and several different histone H3 methylations were performed. For the quantification 

of the results we use custom-made high-density arrays for a high-resolution analysis 

of histone modifications of the complete IgH locus (ChIP-on Chip analysis). The IgH 

Eµ intron enhancer region from the switch region to the first D segment (DQ52) is 

found to be enriched for the two activating histone modifications H3Ac and 

H3K4Me2. We also have indications that within the VH gene cluster histone 

acetylation and histone H3 K4 methylation is limited to the VH gene segments. 

In the ChIP-on Chip analysis we discovered three new regions in the intergenic part 

of the distal VH cluster (we call them IVARs), which are associated with high levels of 

active chromatin marks (H3Ac and H3K4Me2/3) only in pro B cells but not in pro T 

cells. 

To elucidate the role of the IVARs in VH -> DJH recombination we were generating a 

knock out mouse, which is lacking the IVAR #3 element. By analyzing the VDJrecombination 

repertoire using high-throughput next generation sequencing we found 

that the usage of distal VH gene segments is altered in mutant mice, supporting our 

hypothesis that the IVAR elements are involved in the VDJ-recombination of distal 

VH-elements. 

A hypomorph mutation of the B cell receptor 

Sven Brenner and Thomas Winkler 

As a result from a gene targeting experiment in which we wanted to introduce a GFPreporter 

gene into the immunoglobulin locus, we incidentally created a hypomorphic 

IgH-gene allele. Due to a poly-adenylation site introduced into the enhancer intron 

the levels of Igµ heavy chain mRNA and protein are reduced approximately 5-fold. In 

heterozygous mice B cells expressing the targeted IgH allele are undetectable, 

although VDJ recombination at the targeted allele is normal. 

We could show for these novel gene-targeted mice that hypomorphic expression of 

µHC leads to augmented selection processes at all stages of B-cell development, 

noticeably at the expansion of pre-B cells, the positive selection of immature B 

lymphocytes in the bone marrow and the selection into the follicular, marginal zone 

and B1 B lymphocyte compartments in peripheral lymphoid organs. Immature as well 

as mature follicular and marginal zone B lymphocytes in the peripheral lymphoid 

organs express lower levels of the receptor for B-cell activating factor (BAFF). In 

addition, hypomorphic expression of the BCR favors receptor editing. Together, our 

results highlight the critical importance of pre-BCR and BCR receptor levels for the 

normal development of B-lymphocyte-subpopulations in the context of intact VDJ 

recombination and a diverse antibody repertoire. 

51

During the last 3 years we had successful collaborations with Dr. Lars Nitschke (Dep. 

Biology, Genetics, Erlangen). 

The role of B lymphocytes in the chronic autoimmune disease 

systemic Lupus erythematosus 

SLE is a multisystem autoimmune disease characterized by autoantibodies against 

nuclear antigens, particularly antibodies against native (double-stranded) DNA (antidsDNA). 

Glomerulonephritis is one of the most serious manifestations of the disease. 

The incidence of glomerulonephritis in SLE patients is about 40-60%. The nephritis 

that accompanies SLE is mediated by immune complexes that deposit in the kidney 

and initiate an inflammatory response leading to organ damage and failure. The 

presence of anti-dsDNA antibodies correlates with nephritis in both human patients 

and mice with a spontaneous lupus like disease. Our research focus is on both the 

mechanisms of the generation of anti-dsDNA antibodies and on the role of antidsDNA 

antibodies in the pathogenesis of lupus nephritis. 

Molecular mechanisms of glomerular deposition of autoantibodies and 

pathogenesis of lupus nephritis 

Ute Wellmann, and Thomas Winkler, Technician: Markus Mroz 

A monoclonal anti-dsDNA antibody from a SLE patient binds in vivo to the 

mesangium upon injection in mice. Recently we have identified a crossreactivity of 

this antibody with nucleolin, a multifunctional protein that had been shown to be 

displayed on the cell surface of endothelial cells. We can show that nucleolin-specific 

rabbit antisera also bind specifically in the glomerulus of mice upon injection 

supporting the notion that nucleolin is accessible for autoantibodies in the 

glomerulus. Furthermore, we found a strong correlation of anti-nucleolin 

autoantibodies with more severe glomerulonephritis in several mouse models for 

SLE. In addition sera from SLE patients with severe glomerulonephritis contain antinucleolin 

autoantibodies in elevated titres. 

To further elucidate the potential pathogenetic role of anti-nucleolin (crossreactive) 

autoantibodies we eluted immune complexes from kidneys from proteinuric SLE mice 

(NZB/W mice). The eluted antibodies were highly enriched for anti-nucleolin reactivity 

further supporting the pathogenic role of these autoantibodies. 

(Collaborations with Dr. Ole-Petter Rekvig, University Tromsö, Norway) 

The evolution of anti-dsDNA autoantibodies in germinal centers 

Kristin Kruse, Ute Wellmann and Thomas Winkler, Technician: Andrea Schneider 

and Markus Mroz 

52

It has been proposed that the anti-double-stranded DNA (dsDNA) response in 

patients with systemic lupus erythematosus (SLE) is antigen driven and that DNA or 

nucleosomes select anti-DNA reactive, somatically mutated B cells. Recently we 

showed that high-affinity binding to dsDNA and nucleosomes is acquired by somatic 

replacement mutations in a stepwise manner, presumably in germinal center 

reactions. Importantly, autoreactivity seems to by acquired de novo as antibodies that 

have been backmutated to the germline sequence lack any autoreactivity towards 

DNA (Wellmann et al. 2005, PNAS 102: 9258-63). Nucleosomes accumulating due to 

defects in clearing of apoptotic debris, might positively select high affinity anti-DNA B 

cells. 

To directly test this hypothesis we were generating knock-in mice expressing a 

backmutated, non-autoreactive, lupus autoantibody. Only 3 somatic mutations are 

necessary and sufficient to create high affinity anti-dsDNA reactivity in this antibody. 

We targeted the BCR heavy- and light chain loci to obtain 33.C9gl heavy chain and 

33.C9gl light chain double knockin mice. As expected, B cells expressing the 

revertant B-cell receptor are developing normally and show no evidence for 

tolerization. Upon immunization with a surrogate phage antigen the transgenic B cells 

form germinal centers and undergo somatic hypermutation. The three critical somatic 

hypermutations, which are necessary and sufficient for the acquisition of high affinity 

anti-dsDNA binding, were not observed in a large collection of sequences analyzed. 

In accordance with this, anti-DNA autoantibodies do not develop, even after repetitive 

immunizations and when FDCs were MFG-E8 deficient. These results strongly 

suggest a self-tolerance checkpoint by deletion of autoreactive clones during or after 

the germinal center reaction. 

(Collaboration with Dr. Martin Herrmann, Med. Dept. III, Erlangen) 

We had further successful collaborations with Dr. Reinhard Voll at the NFZ on the 

role of plasma cells in SLE (Starke et al., 2011). 

Antibody-based immune protection against Cytomegalovirus 

Infections with Cytomegalovirus are usually asymptomatic and 50 – 90 % of the 

human population is persistently infected with this herpes virus. Severe disease 

becomes apparent when the immune system of the infected individual is suppressed, 

for example after transplantation. In addition, human cytomegalovirus is an important 

congenital infection. 

We aim at the better understanding of humoral immune-responses against the virus 

to develop new therapeutic concepts and potential therapeutic antibodies for 

treatment. We carry out all research in this field in close collaboration with the group 

of Prof. Michael Mach at the Virology Institute. 

For our research we use the human cytomegalovirus (HCMV) as well as its close 

relative in the mouse, the murine cytomegalovirus (MCMV). 

53

Adoptive transfer of memory B cells as a new cell based therapy for infection 

with Cytomegalovirus after transplantation 

Florian Weisel, Martina Seefried, Anna Bootz*, Monika Dietz*, Astrid Karbach*, Julia 

Winkler # , Andreas Mackensen # , Michael Mach*, Thomas Winkler, Technicians: 

Andrea Schneider, Hannes Tittlbach 

* Institute for Clinical and Molecular Virology 

# Department of Internal Medicine 5, Hematology and Oncology 

Severe disease associated with cytomegalovirus (CMV) infection is still a major 

problem in transplant patients. Support of the patient’s immune defense against the 

virus is therefore a major goal in transplantation medicine. We use the murine model 

of CMV (MCMV) to investigate the potential of a cell-based strategy to support the 

humoral antiviral immune response. We have previously shown that a transfer of 

memory B cells was effective in protecting from an ongoing viral infection indicating a 

therapeutic potential of virus specific memory B cells. In the recent period for this 

report we analyzed the effector mechanisms for the protective effect of anti-CMV 

antibodies. We have clear data that Fc-receptor (FcR) mediated mechanisms, 

presumably antibody dependent cellular cytotoxicity (ADCC) play a major role in the 

protective effect of antibodies in this viral disease. Determination of viral load in 

organs (Fig. 1A, B) and analysis of survival (Fig. 1C) after serotherapy confirmed that 

FcγR-deficient mice are not protected by administration of serum from MCMVimmune 

donors. Further experiments with individual FcγRs revealed a complex and 

partly redundant function of the different FcγRs. Whereas genetic deletion or antibody 

blockade of either FcγRIII or FcγRIV alone have any measurable effect on antibody 

protection, a combined defect of FcγRIII and FcγRIV had strong effects on protective 

function of antibodies. With regard to effector cells we have the surprising finding that 

NK cells, which are believed to be efficient effectors for ADCC, have surprisingly no 

major role in antibody-mediated protection in CMV-infection as NK cell-depleted 

animals were fully protected by serum therapy. Preliminary results indicate that 

certain monocyte subpopulations that can be specifically depleted by clodronate 

liposomes are important for antibody protection. We are currently studying the 

function of these monocyte subpopulation and the role of FcγRI in antibody-mediated 

protection in CMV-infection. 

54

Fig. 1 Fc_Rs are essential for antibody-mediated protection from MCMV infection. 

Mice were infected with 10 5 plaque forming units MCMV157luc and treated with 100µl serum from 

naïve or MCMV-immune donors. (A) Bioluminescence imaging of mice 10 days after serum treatment. 

The pseudocolor scale indicates viral load. (B) Relative viral load in organs in mice 14days after 

serotherapy. Luciferase activity was measured in organ homogenates and relative light units (RLU)/15 

µg protein are shown. (C) Survival curve of 5 mice/group after serotherapy. 

Our data provide evidence that a cell based strategy using memory B-lymphocytes to 

support the humoral immune response can be effective to combat infectious 

pathogens in severely immunodeficient hosts, like patients after stem cell 

transplantation. To bring this strategy into the clinics, the preclinical technology 

development including registration and implementation according to AMG – 

guidelines were mandatory prerequisites for the initiation of a phase I/II clinical trial, 

particularly as no experience was available for the GMP-qualified production of Blymphocytes. 

We therefore developed protocols for the production of a GMP-qualified 

B-lymphocyte product from leukapheresis material during the last 2 years by using 

the CliniMacs-technology in cooperation with Miltenyi-Biotec. A two-step separation 

strategy was developed and validated under GMP-conditions in a validated GMPlaboratory 

(Department of Transfusion Medicine). The manufacturing license for the 

B-cell product will soon be obtained. 

As a second prerequisite for the initiation of a first-in-human phase I/IIa clinical trial 

we developed a clinical protocol for a phase I/II study in accordance with GCP 

guidelines. An adoptive transfer of B-lymphocytes after allogeneic stem cell 

55

transplantation represents a novel, world-wide first-in-man cellular therapy. 

Therefore, this therapy has to be evaluated in a Phase I/IIa clinical trial primarily for 

safety and feasibility. The B-lymphocyte product will be the investigational medicinal 

product (IMP) in this trial. After extensive discussion with stem cell transplantation 

specialists we considered that a phase I/II trial with patients at risk for HCMVreactivation 

shortly after stem cell transplantation would potentially provide 

inconclusive data, especially regarding safety, which has to be the primary endpoint 

of the study. We therefore intend a transfer of the B-lymphocyte product 

approximately 4-5 months after transplantation when B-lymphocyte numbers are still 

significantly reduced in patients receiving matched-unrelated grafts and revaccination 

of the patient is routinely performed in the clinic. For the patient, a 

significant benefit for the reconstitution of the humoral immune function would be 

expected by the transfer of the B-lymphocyte product. At the same time, routinely 

performed re-vaccinations would offer the unique possibility to assess the function of 

the transferred B-lymphocytes from the donor. For the immune monitoring of patients 

in the clinical trial we already developed methods for the analysis of the frequencies 

of antigen-specific memory B-lymphocytes and plasma-blasts for various viral 

antigens and vaccine antigens. 

Currently we complete the documents for the trial protocol, the investigator brochure 

and the IMP dossier for the application to Paul-Ehrlich-Institure with the help of the 

Center of Clinical Studies (CCS) of the University hospital. These preclinical and 

clinical aspects of our research are performed in close collaboration with Dr. Julia 

Winkler and Dr. Andreas Mackensen, Department of Internal Medicine 5 and Miltenyi 

Biotec and are supported by the BayImmuNet. 

Furthermore, we refined our murine model further including viral reactivation from 

latency as well as induction of graft-versus-host disease (GVHD) after bone marrow 

transplantation to adopt it as close as possible to the clinical situation. Using this 

model, we were able to demonstrate a vital importance of antibodies in prevention of 

virus reactivation following bone marrow transplantation. When B-cell deficient mice 

unable to produce antibodies in the serum were infected with MCMV, a rapid 

clearance of the virus was observed in the B-cell deficient mice, as expected, 

because these mice contain a normal T-cell compartment being able to control 

infection to latency. When these recipients were treated by irradiation and 

transplanted with autologous or allogeneic bone marrow a massive reactivation of 

MCMV was observed in B-cell deficient bone marrow recipient mice whereas mice 

with serum antibodies only showed a low level of reactivation of the virus Fig. 2). By 

day 30 after transplantation the virus in surviving B-cell deficient animals was under 

control, due to the newly formed immune system derived from the bone marrow 

transplant. 

When a graft-versus-host reaction was induced in this transplant setting we observed 

a massive and uncontrolled reactivation of MCMV, even in the presence of antibodies 

(data not shown). These preliminary data demonstrate a major clinical problem in 

56

stem cell transplantations for the first time in the mouse model and will enable us to 

refine our therapy concepts under these clinically relevant conditions. 

Fig. 2 Antibodies are important for prevention of MCMV reactivation after bone marrow 

transplantation 

C57Bl/6 mice and B-cell deficient JH -/- were infected with 10 5 plaque forming units of MCMV157luc 

and transplanted with bone marrow after lethal irradiation. Bioluminescence imaging of mice after 

primary infection, latency (d24) and after bone marrow transplantation. 

These experiments were in part (GVHD-model) performed in close collaboration with 

the group of Dr. Evelyn Ulrich, Department of Internal Medicine 5. 

Neutralizing human antibodies against HCMV 

Sonja Pötzsch and Thomas Winkler, Technicians: Tanja Fisch, Hannes Tittlbach and 

Uwe Appelt 

Human cytomegalovirus (HCMV) infections can lead to clinically manifested, often 

life-threatening disease among immunocompromised hosts, in particular solid organ 

or bone marrow transplant recipients. Owing to lack of immune response in these 

patients, virus replication and dissemination occurs uncontrollably. Therefore, 

57

passive immunization for prevention or amelioration of CMV disease in high-risk 

individuals is a major goal not only in transplant medicine. The administration of CMV 

neutralizing monoclonal antibodies could be a feasible approach and has shown to 

be very effective in the mouse model. 

In addition, infection during pregnancy has been recognized as a major problem for 

the newborn. Congenital (present at birth) CMV infection causes more long-term 

problems and childhood deaths than Down syndrome, fetal alcohol syndrome, and 

neural tube defects. Antibodies delivered to pregnant mothers at risk would have the 

potential to prevent severe sequelae, such as hearing loss and other CNS defects in 

about 30,000 children which are born with congenital CMV infection each year 

(numbers from CDC for the USA, http://www.cdc.gov/cmv/trends-stats.html). 

Target antigens of CMV neutralizing antibodies usually are glycoproteins in the viral 

membrane, the most immunogenic being glycoprotein B (gB). Glycoprotein B (gB) is 

a well conserved protein among herpesviruses. In the case of HCMV, gB is a major 

target for the virus neutralizing humoral immune response. Two antigenic domains 

(AD) on gB which are targets of neutralizing antibodies have been identified and 

characterized in some detail. AD-1 is located between aa 552-635 and AD-2 between 

aa 68-77 of gB strain AD169. A third antigenic domain (AD-3) which is located 

intravirally and which is not target of neutralizing antibodies has also been identified. 

We have used data from the recently 

established 3D structure of HSV-1 gB for 

molecular modelling of HCMV gB and 

based on the model we have identified 

two domains on HCMV gB which 

resemble structural compact domains on 

the protein surface that could be targets of 

antibodies. Domain 1 comprises aa 132- 

343 and domain 2, a discontinuous 

sequence, comprises aa 116-132 + 344- 

440 of gB strain AD169 (Fig. 3) 

Fig. 3 Antigenic domains (ADs) on HCMV 

glycoprotein B 

(3D model generated by H. Sticht) 

In a comprehensive screening of over 800 

human monoclonal antibodies binding to 

gB derived from normal blood donors we identified several strongly neutralizing 

antibodies reacting against newly defined epitopes on domain 1 and domain 2 (AD-5 

and AD-4, respectively. These new antibodies certainly represent lead candidates for 

further preclinical and clinical development for therapeutic applications in prenatal 

HCMV infections and HCMV reactivation in transplant patients. 

(Collaboration with Dr. Michael Mach, Institute for Virology, Erlangen, Dr. Heinrich 

Sticht, Bioinformatics, Erlangen and 4-Antibody AG, Basel, Switzerland). 

58

2011/12 



Schietke, R.E., T. Hackenbeck, M. Tran, R. Gunther, B. Klanke, C.L. Warnecke, K.X. 

Knaup, D. Shukla, C. Rosenberger, R. Koesters, S. Bachmann, P. Betz, G. Schley, J. 

Schodel, C. Willam, T. Winkler, K. Amann, K.U. Eckardt, P. Maxwell, and M.S. 

Wiesener. 2012. Renal Tubular HIF-2alpha Expression Requires VHL Inactivation 

and Causes Fibrosis and Cysts. PLoS ONE 7:e31034. 

Sitte, S., J. Glasner, J. Jellusova, F. Weisel, M. Panattoni, R. Pardi, and A. Gessner. 

2012. JAB1 is essential for B cell development and germinal center formation and 

inversely regulates Fas ligand and Bcl6 expression. J Immunol 188:2677-2686 

Pötzsch, S., N. Spindler, A.K. Wiegers, T. Fisch, P. Rucker, H. Sticht, N. Grieb, T. 

Baroti, F. Weisel, T. Stamminger, L. Martin-Parras, M. Mach, and T.H. Winkler. 2011. 

B Cell Repertoire Analysis Identifies New Antigenic Domains on Glycoprotein B of 

Human Cytomegalovirus which Are Target of Neutralizing Antibodies. PLoS Pathog 

7:e1002172. 

Brenner, S., D. Drewel, T. Steinbart, F. Weisel, E. Hartel, S. Pötzsch, H. Welzel, A. 

Brandl, P. Yu, G.C. Mudde, A. Schweizer, L. Nitschke, and T.H. Winkler. 2011. A 

hypomorphic IgH-chain allele affects development of B-cell subsets and favours 

receptor editing. Embo J 30:2705-2718. 

Ackermann, J.A., D. Radtke, A. Maurberger, T.H. Winkler, and L. Nitschke. 2011. 

Grb2 regulates B-cell maturation, B-cell memory responses and inhibits B-cell Ca(2+) 

signalling. Embo J 30:1621-1633. 

Starke, C., S. Frey, U. Wellmann, V. Urbonaviciute, M. Herrmann, K. Amann, G. 

Schett, T. Winkler, and R.E. Voll. 2011. High frequency of autoantibody-secreting 

cells and long-lived plasma cells within inflamed kidneys of NZB/W F1 lupus mice. 

Eur J Immunol 41:2107-2112 

2010 

Furnrohr, B.G., S. Wach, J.A. Kelly, M. Haslbeck, C.K. Weber, C.M. Stach, A.J. 

Hueber, D. Graef, B.M. Spriewald, K. Manger, M. Herrmann, K.M. Kaufman, S.G. 

Frank, E. Goodmon, J.A. James, G. Schett, T.H. Winkler, J.B. Harley, and R.E. Voll. 

2010. Polymorphisms in the Hsp70 gene locus are genetically associated with 

systemic lupus erythematosus. Ann Rheum Dis 69:1983-1989. 

Jellusova, J., U. Wellmann, K. Amann, T.H. Winkler, and L. Nitschke. 2010. CD22 x 

Siglec-G double-deficient mice have massively increased B1 cell numbers and 

develop systemic autoimmunity. J Immunol 184:3618-3627. 

59

Weisel, F.J., U.K. Appelt, A.M. Schneider, J.U. Horlitz, N. van Rooijen, H. Korner, M. 

Mach, and T.H. Winkler. 2010. Unique Requirements for Reactivation of Virus- 

Specific Memory B Lymphocytes. J Immunol 185:4011-4021. 

2009 

Burkhardt, C., S. Himmelein, W. Britt, T. Winkler, and M. Mach. 2009. Glycoprotein N 

subtypes of human cytomegalovirus induce a strain-specific antibody response 

during natural infection. J Gen Virol 90:1951-1961. 


Almqvist, N., T.H. Winkler, and I.L. Martensson. 2011. Autoantibodies: Focus on anti- 

DNA antibodies. Self Nonself 2:11-18. 

Kruse, K., C. Janko, V. Urbonaviciute, C.T. Mierke, T.H. Winkler, R.E. Voll, G. Schett, 

L.E. Munoz, and M. Herrmann. 2010. Inefficient clearance of dying cells in patients 

with SLE: anti-dsDNA autoantibodies, MFG-E8, HMGB-1 and other players. 

Apoptosis 15:1098-1113. 

60



Florian Weisel 

Reaktivierung Virus-spezifischer Gedächnis B Zellen 

Sven Brenner 

Selection processes guided by B-cell receptor signal strength 

Sonja Pötzsch 

Neutralizing human monoclonal antibodies against Cytomegalovirus 

Kristin Kruse 

Die Evolution von anti-DNA Autoantikörpern in Keimzentren 

Agnes Giniewski 

Charakterisierung eines neuen regulatorischen Elementes im IgH-Genlokus 

Martina Seefried 

Immunological mechanisms of reactivation of murine CMV after bone marrow 

transplantion 

Sabrina Sell 

The protective role of γ/δ T-lymphocytes in murine CMV infection 

61

IZKF Junior Group III 

BMBF Research Group Neurosciences 

Head: Beate Winner, PD Dr. med. 

Address: IZKF Junior Group III 

Glückstr. 6 


Telefone: + 49 (9131) 85 39301 

Fax: + 49 (9131) 85 39311 

E-mail: beate.winner@med.uni-erlangen.de 

Homepage: 

http://www.izkf.uk-erlangen.de/e37/e38/e87/e1593/index_ger.html 

Head 

Beate Winner, PD Dr. med. 

Research associates Doctoral students (Biology) 

Francesc Perez-Branguli, PhD* 

Iryna Prots, Dr. rer. nat. 

Haixin Zhang, MD/ PhD* 

Master Students (M)/ 

Student helpers (S) 

Stefanie Brey (M) * 

Naime Denguir (S) 

Theresa Halder (S) 


62 

Steven Havlicek 

Himanshu Mishra 

Technicians 

Daniela Gräf* 

Domenika Saul* 

Holger Wend


The overall goal of research in our laboratory is to model neurodegenerative 

diseases in stem cell based models. Specifically we investigate neurodegeneration 

and regeneration in neurites. The two diseases studied are Parkinson’s disease (PD) 

and hereditary spastic paraplegia (HSP), a specific genetic form of motor neuron 

disease. 

Hereditary spastic paraplegia (HSP) and other motor neuron diseases 

We are specifically interested in understanding the involvement of selected hereditary 

spastic paraplegia (HSP) proteins in axonal degeneration. HSP is a genetic condition 

leading to corticospinal tract degeneration. HSPs selectively involve a lengthdependent 

axonopathy from long projecting corticospinal motorneurons (CSMN), 

while sparing the neuronal cell bodies. We study CSMN to understand the molecular 

mechanisms crucial for axonal maintenance and degeneration. The models we use 

are human induced pluripotent stem cell (hiPSC)-derived neurons generated from 

HSP patient’s fibroblasts (SPG4 and SPG11). The overall aim is to understand the 

normal function of selected HSP proteins, to determine how abnormality of these 

functions lead to the disease and to use this knowledge to rationally design new 

therapeutic strategies for the disorder. 

63

Figure 1: Induced pluripotent stem cells (iPSC) to model neurodegenerative 

diseases. Human iPSC from patients (e.g. HSP) and controls are generated after 

reprogramming of somatic tissue into iPSCs. Neural progenitor cells (NPC) are 

generated from these iPSCs and are further differentiated into neurons and/or glial 

cells. The neuronal phenotype is analyzed. Once a distinct disease-related 

phenotype is identified, drug-screening platforms can be developed to test 

compounds. Therapeutic compounds could emerge from the screenings, potentially 

benefiting neurologic patients. 

Understanding aggregation in Parkinson’s disease (PD) 

Protein aggregation of mis-folded proteins is associated with several 

synucleinopathies. Lewy bodies, an aggregated fibrillar form of multiple proteins 

(among them α-synuclein), are the major pathological hallmark in PD. During the 

recent year we have shown in vivo that small oligomeric species of the protein, rather 

than its aggregated fibrillar counterpart, are the toxic culprit in a rat model of 

synucleinopathies. We are investigating the impact of oligomerisation of α-synuclein 

in human in vitro models. Specifically, we are interested in studying the mechanism 

and functional consequences of oligomerization for neurite degeneration, axonal 

transport, and cellular membranes. 

Important results 

In vivo demonstration that oligomers are toxic 

We tested, whether Glu -> Lys mutations in the core area of α-synuclein interfere 

with the formation of α-synuclein fibrils and form oligomers instead. Several single 

point mutations in the core region (30-110) of α-synuclein have been investigated for 

their tendency to form fibrils and oligomers when compared with wild-type (WT) α- 

synuclein. In collaboration with Roland Riek, ETH Zürich, these results were obtained 

by incubating the different (artificial) variants of α-synuclein in a cell-free system. α- 

synuclein variants were analyzed by electron microscopy (EM), which indicated that 

these artificial mutants in the core region (30-110) were able to form oligomers but 

did not form fibrils. Interestingly, some preparations of these artificial mutants did not 

show amyloid fibrils even after long-term incubation, but formed very pronounced, 

ring/pore-like structures resembling large oligomers. We found two α-synuclein single 

point mutations, E35K and E57K, that showed a strongly decreased tendency of fibril 

formation when compared with WT α-synuclein, as evidenced by time-resolved 

amyloid formation. Neither variant E35K nor E57K showed amyloid fibrils under 

64

certain conditions, but they formed very pronounced ring/pore-like structures. In 

contrast, human WT and familial α-synuclein variants formed amyloid fibrils as 

expected. We injected lentiviral vectors of the artificial mutants into the rat substantia 

nigra and examined survival of dopaminergic neurons after 3 weeks. Our data 

indicate, that an enormous loss of dopaminergic cells in the substantia nigra of rats 

upon 3 weeks of lentiviral infection is observed for the artificial α-synuclein variants 

that form large quantities of oligomers, whereas the variant that forms fibrils very 

quickly is less toxic. 

The correlation between the extent of oligomer formation in vitro and toxicity in vivo is 

apparent. Furthermore, there is a lack of correlation between the tendency of amyloid 

fibril formation in vitro and toxicity in vivo and between the amount of monomer in 

vitro and toxicity in vivo. Combining the in vitro and in vivo experiments, we suggest 

that the in vivo-derived SDS-stable oligomers may be the building blocks for a larger 

alpha-synuclein oligomer that causes toxicity. In conclusion, this structure-toxicity 

relationship study of human α-synuclein strongly supports the notion that α-synuclein 

oligomers are the toxic species in PD and that the amyloid fibrils are not directly toxic, 

despite being present in the Lewy bodies, which are the pathological hallmark of PD 

(published in PNAS 2011). 

We are currently investigating the role of α-synuclein oligomers for axonal transport, 

membrane stability and the effect of α-synuclein on dendrites in stem cell based 

rodent and human models. 

Figure 2: In vitro aggregation of wild-type α-synuclein (B) compared to artificial 

oligomer-forming mutants (e.g. A) in a cell-free system. EM shows an abundance of 

fibrils in the wild-type α-synuclein (B), whereas only pore-like oligomers are found in 

the artificial oligomerizing mutant (A). Lentivirus injection into the rat substantia nigra 

of adult rats (C: paradigm): decrease in TH-positive cells following injection with the 

65

oligomerizing mutant (α-syn mut, F) compared to wild-type α-synuclein (α-syn, E) 

and GFP control (D, PNAS 2011). 

Figure 3: Neuronal differentiation of dopaminergic neurons from human embryonic 

stem cell (hESC)-derived neural precursor cells (NPCs). (A) Neurons were 

differentiated from NPCs over 6 weeks using neurotrophic factors and characterized 

by immunofluorescence for neuronal markers (B) and by electrophysiology (C, 

collaboration with Dr. Tobias Huth, Institute of Physiology and Pathophysiology, 

Head: Prof. Dr. Christian Alzheimer). 

Publications (2010-current) 

Bracko O, Singer T, Aigner S, Knobloch M, Winner B, Ray J, Clemenson GD Jr, Suh 

H, Couillard-Despres S, Aigner L, Gage FH, Jessberger S (2012). Gene expression 

profiling of neural stem cells and their neuronal progeny reveals IGF2 as a regulator 

of adult hippocampal neurogenesis. J Neurosci. 32(10):3376-87. 

Kohl Z, Winner B, Ubhi K, Rockenstein E, Mante M, Münch M, Barlow C, Carter T, 

Masliah E, Winkler J (2012) Fluoxetine rescues impaired hippocampal neurogenesis 

in a transgenic A53T synuclein mouse model. Eur J Neurosci. 35(1):10-9. 

Winner B, Kohl Z, Gage FH. (2011) Neurodegenerative disease and adult 

neurogenesis. Eur J Neurosci. 33(6):1139-51. 

Winner B, Jappelli R, Maji SK, Desplats PA, Boyer L, Aigner S, Hetzer C, Loher T, 

Vilar M, Campioni S, Tzitzilonis C, Soragni A, Jessberger S, Mira H, Consiglio A, 

Pham E, Masliah E, Gage FH, Riek R. (2011) In vivo demonstration that alphasynuclein 

oligomers are toxic. Proc Natl Acad Sci U S A. 108(10):4194-9. 

McCrone P, Payan CA, Knapp M, Ludolph A, Agid Y, Leigh PN, Bensimon G; 

NNIPPS Study Group (2011) The economic costs of progressive supranuclear palsy 

and multiple system atrophy in France, Germany and the United Kingdom. PLoS 

One. 6(9):e24369. 

Payan CA, Viallet F, Landwehrmeyer BG, Bonnet AM, Borg M, Durif F, Lacomblez L, 

Bloch F, Verny M, Fermanian J, Agid Y, Ludolph AC, Leigh PN, Bensimon G; 

NNIPPS Study Group (2011), Disease severity and progression in progressive 

supranuclear palsy and multiple system atrophy: validation of the NNIPPS— 

Parkinson Plus Scale. PLoS One. 6(8):e22293. 

Rolland Y, Vérin M, Payan CA, Duchesne S, Kraft E, Hauser TK, Jarosz J, Deasy N, 

Defevbre L, Delmaire C, Dormont D, Ludolph AC, Bensimon G, Leigh PN; NNIPPS 

66

Study Group (2011). A new MRI rating scale for progressive supranuclear palsy and 

multiple system atrophy: validity and reliability. J Neurol Neurosurg Psychiatry. 

82(9):1025-32. 

Winner B, Melrose HL, Zhao C, Hinkle KM, Yue M, Kent C, Braithwaite AT, 

Ogholikhan S, Aigner R, Winkler J, Farrer MJ, Gage FH (2011). Adult neurogenesis 

and neurite outgrowth are impaired in LRRK2 G2019S mice. Neurobiol Dis., 

41(3):706-16. 

Marchetto MC, Winner B, Gage FH (2010). Pluripotent stem cells in 

neurodegenerative and neurodevelopmental diseases. Hum Mol Genet. R71–R76. 

Glass CK, Saijo K, Winner B, Marchetto MC, Gage FH (2010). Mechanism 

underlying inflammation in neurodegeneration. Cell. 19;140(6):918-34. 

Kohl Z, Regensburger M, Aigner R, Kandasamy M, Winner B, Aigner L, Winkler J. 

(2010) Impaired adult olfactory bulb neurogenesis in the R6/2 mouse model of 

Huntington’s disease. BMC Neurosci. 11:114. 

Kandasamy M, Couillard-Despres S, Raber KA, Stephan M, Lehner B, Winner B, 

Kohl Z, Rivera FJ, Nguyen HP, Riess O, Bogdahn U, Winkler J, von Hörsten S, 

Aigner L (2010). Stem cell quiescence in the hippocampal neurogenic niche is 

associated with elevated transforming growth factor-beta signaling in an animal 

model of Huntington disease. J Neuropathol Exp Neurol. 69(7):717-28. 

Doctoral (Biology) 

TRAINING OF GRADUATE AND MASTER STUDENTS 

Steven Havlicek 

Modeling familial motor neuron disease (SPG4) by the use of induced pluripotent 

stem cells 

Himanshu Mishra 

Modeling familial motor neuron disease (SPG11) by the use of induced pluripotent 

stem cells 

Master (Molecular Medicine) 

Stefanie Brey 

Impact of oligomerisation of α-synuclein on membranes in human neural in vitro 

models 

67

INTERDISCILINARY CENTER OF CLINICAL 

RESEARCH 

Junior Research Group 2 (IZKF – N2) 

Head: Jens Titze, Prof. Dr. med. 

Address: IZKF – N2 

Glückstr. 6 


Telefone: + 49 (9131) 85 39300 

Fax: + 49 (9131) 85 36386 

E-mail: jens.titze@uk-erlangen.de 

jens.m.titze@vanderbilt.edu 

Homepage: http://www.izkf.ukerlangen.de/e1615/inhalt1880/IZKF_JB11_05.2012.pdf 

Agnes Schröder, Dr. rer. nat. 

Peter Linz, Dr. rer. nat. 

Natalia Rakova, Dr. 


Head 

Jens Titze, Prof. Dr. med. 


Doris Bittner, Secretary 


68 

Anke Dahlmann, Dr. med. 

Christoph Kopp, Dr. med. 

Sven Brenner, Dr. rer. nat.

Kathrin Jüttner 


Florian Eicher 


Ulrike Goller 

Jenny Goß 



Diana Friedrich 

Doctoral Students (Medicine) 

Kathrin Dörfelt 

Technicians 

Sabrina Cabric 

69


Primary essential hypertension is the major cardiovascular disease risk factor. 

Dietary salt intake is a putative driving force of blood pressure elevation; however, 

the mechanisms of this effect remain unclear. The central nervous system, heart and 

blood vessels, and kidney are primary participants and the kidney is the putative 

grand regulator of salt disposition and blood pressure. The overall goal of this work is 

to address whether or not local regulation of skin electrolyte metabolism is important 

for blood pressure control. Clinical evidence from pilot studies in humans and 

preliminary experimental data accumulated in our laboratory, support this hypothesis. 

Our data point to macrophage-derived vascular endothelial growth factor C (VEGF-C) 

as a crucial factor controlling skin electrolyte homeostasis. VEGF-C promotes 

interstitial electrolyte clearance through the cutaneous lymph capillary network. 

Macrophages induce hyperplasia of subcutaneous lymph capillaries after sensing 

local Na + or Cl - overload in the interstitium. The sensing function is accomplished by 

binding of the transcription factor tonicity-enhancer binding protein (TonEBP) to the 

promoter region of the VEGF-C gene. The cells exert their regulatory function by 

increasing VEGF-C expression and secretion. Blockade of this VEGF-C response 

from macrophages leads to skin electrolyte accumulation and arterial hypertension. 

Significance and Background 

The importance of a constant milieu intérieur, maintained through extracellular 

electrolyte and water content, has long been appreciated for blood pressure control. 

The underlying concept is that extracellular fluids readily equilibrate, resulting in a 

homogenous and uniform composition of the extracellular fluid space. The 

predominant role of the kidney for internal environment composition and blood 

pressure control relies on the idea that renal regulation of blood composition is 

sufficient for concomitant maintenance of interstitial fluid composition. Any inability of 

the body to excrete Na + is thus regarded as a major pathophysiological component 

for development of hypertension – and traditionally considered due to perturbations 

of renal function (Fig. 1A). 

We have previously shown that Na + is stored independent of water in the skin. This 

finding resulted in a hypothesis-driven research program in which we searched for 

extrarenal mechanisms for regulation of local interstitial electrolyte and water 

homeostasis. Approximately 70-80% of the extracellular fluids are interstitial and 

thereby not directly controlled by renal salt and water excretion. Therefore, 

identification of novel local extrarenal regulatory mechanisms for interstitial fluid 

70

homeostasis at the skin tissue level would provide a novel research avenue towards 

maintenance of electrolyte and water homeostasis and introduce new potential 

therapeutic targets for salt, water, and blood pressure disorders. 

We first identified a potential mechanism for storage and release of Na + from the 

skin. We have shown that a high-salt diet results in specific changes in the negative 

charge density of the extracellular matrix in the skin interstitium. Dietary salt led to 

increased glycosaminoglycan (GAG) polymerization and sulfatation in the skin 

interstitium. The resulting increase in the negative interstitial charge density was 

paralleled with Na + storage in the skin. In reverse, a low-salt diet led to reduced GAG 

polymerization and sulfatation, and the resulting decrease in the negative GAG 

charge density was paralleled by release of Na + from the skin. We interpreted these 

findings in a way that the skin interstitium actively modulated its extracellular GAG 

charge density in order to store or release Na + from an interstitial reservoir. Because 

Na + would electrostatically interact with GAGs, this storage process could result in 

water-free, osmotically inactive Na + storage in the skin. We concluded that the skin 

interstitium holds the capacity to locally control its electrolyte composition via 

extrarenal regulatory mechanisms. 

We then found that Na + storage was additionally paralleled by macrophage infiltration 

into the Na + -loaded interstitium (Fig. 1B), where the cells exert an important 

extrarenal regulatory cascade for interstitial electrolyte homeostasis and systemic 

blood pressure control. In the Na + -loaded skin, macrophages express the 

osmoprotective transcription factor TonEBP, suggesting that Na + storage results in 

A B 

Fig. 1 (A) Traditional research approach for body electrolyte balance and blood pressure 

homeostasis, based on the oversimplified concept of passive body fluid equilibrium in closed 

systems where known forces are balanced and electrolyte concentrations are not remarkably 

different between blood volume and interstitial volume (equilibrium). Suggesting that blood 

purification is sufficient for interstitial clearance of electrolytes and water, this research relies on 

investigation of renal electrolyte and water clearance mechanisms. (B) Novel research approach for 

body electrolyte balance and blood pressure homeostasis, based on the finding that interstitial 

electrolyte concentrations are higher than in blood (“skin Na + storage”). Interstitial electrolyte 

balance is not achieved by renal blood purification alone, but relies on additional extrarenal 

regulatory mechanisms within the skin interstitium. Macrophages act as local osmosensors that 

regulate local interstitial electrolyte composition via a TonEBP/VEGF-C dependent mechanism, 

enhancing electrolyte clearance via VEGF-C/VEGFR3 mediated modulation of the lymph capillary 

network in the skin. We are aware that peripheral vascular resistance in hypertension is increased 

throughout the entire body, including obviously the kidneys. Our skin storage mechanism implies an 

additional compartment that clearly impacts on general vascular resistance, and does not contradict 

the infinite-gain theory that links internal environment composition with blood pressure control. 

71

hypertonic interstitial fluid accumulation. TonEBP binds to the promoter of the VEGF- 

C gene in macrophages, resulting in gene expression and secretion of VEGF-C into 

the interstitial Na + reservoir. VEGF-C binds to vascular endothelial growth factor 

receptor 2 (VEGFR2) and increases eNOS expression in blood vessels. VEGF-C 

also binds to VEGFR3 on lymph endothelial cells, resulting in hyperplasia of 

interstitial lymph capillaries. When we blocked the physiologic macrophage-driven 

VEGF-C response experimentally, eNOS expression in blood vessels did not 

increase, the lymph capillary network showed no hyperplasia, and blood pressure 

was increased. These findings suggest that interstitial skin macrophages regulate 

blood pressure via a TonEBP/VEGF-C-dependent mechanism, either by increasing 

NO production in small blood vessels, or by enhancing local interstitial electrolyte 

clearance in the skin through the lymph capillary network (Fig. 1B). 

Our findings have added a new and unexpected research field for hypertension 

research. They suggest that skin electrolyte and water homeostasis is important for 

blood pressure regulation. We furthermore suggest that macrophages can modulate 

skin electrolyte and water metabolism via a TonEBP/VEGF-C regulatory axis, and we 

have identified the lymph capillary system in the skin as a new target for modulation 

of interstitial electrolyte composition and blood pressure. 

Macrophages regulate interstitial electrolyte composition via 

TonEBP/VEGF-C driven modulation of lymph capillaries 

We have shown that rats, in which we blocked de-novo infiltration of MPS cells into 

the skin interstitium with clodronate liposome treatment, were unable to increase 

VEGF-C in skin in response to a high-salt diet, and developed salt-sensitive 

hypertension. Clodronate liposomes are phagocytized by blood circulatory 

macrophages / MPS cells and induce apoptosis. We concluded that increased 

TonEBP / VEGF-C expression in skin in response to high salt was induced by 

circulatory monocytes/macrophages which entered the Na + overloaded 

subcutaneous interstitium. This finding was corroborated by cell culture experiments, 

where we showed that macrophages express VEGF-C in response to osmotic stress 

by TonEBP binding to the VEGF-C promoter. In additional in-vitro experiments in 

cells with TonEBP overexpression or silencing, we furthermore demonstrated that 

TonEBP promoter binding was essential for VEGF-C secretion in macrophages in 

response to osmotic stress. However, we have so far not yet demonstrated in vivo 

that TonEBP is essential for VEGF-C driven modulation of interstitial electrolyte 

composition and blood pressure. 

To test the hypothesis that TonEBP deficiency results in reduced skin VEGF-C 

levels, reduced lymph capillary density, electrolyte accumulation, and blood pressure 

increase in vivo, we have designed experiments with TonEBP (gene name: Nfat5) 

deficient mice. TonEBP-null mouse embryos die prenatally or late in gestation and 

have pronounced renal medullary atrophy and impaired cardiac development. In 

72

contrast, studies utilizing haploinsufficient TonEBP mice report normal development. 

TonEBP +/- lymphocytes proliferate normal at 330-350 mosmol/kg osmotic stress, and 

only when osmolality in cell culture media is >350 mosmol/kg is their proliferation 

impaired. No data on renal function in TonEBP +/- mice are available to the best of 

our knowledge. Our preliminary data on skin electrolyte composition suggest that 

reduced TonEBP expression in skin in TonEBP +/- mice fed a high-salt diet (Fig. 2A) 

is paralleled by reduced VEGF-C gene expression and reduced lymph capillary 

density in skin (Fig. 2B), which should lead to impaired skin electrolyte clearance. 

Indeed, TonEBP mice with high-salt diet were characterized by an almost two-fold 

increase in skin Na + and Cl - content 

(Fig. 2C), while the ratio of skin 

(Na + +K + )-to-water ratio was 

unchanged in the mice (Fig. 2D). 

We interpret these preliminary 

findings that the skin interstitium in 

mice is characterized by hypertonic 

interstitial fluid accumulation 

([Na + +K + ] = 180 mmol/L; Fig. 2D), 

and that immune cells are exposed 

to osmotic stress when leaving blood 

vessels and entering the interstitial 

Fig. 2. VEGF-C expression and skin electrolytes in 

TonEBP +/- mice. (A) TonEBP mRNA expression in 

skin in wild type (wt; n=6) and TonEBP -/+ (n=5) 

mice. (B) VEGF-C mRNA expression and average 

lymph capillary diameter in wt (n=3) and TonEBP -/+ 

(n=3) mice fed a high-salt diet. (C & D) Skin Na + 

and Cl - content, and skin (Na + +K + )-to-water ratio in 

the same mice. * P

its soluble receptor sVEGFR3). This systemic depletion of VEGF-C resulted in 

increased skin electrolyte accumulation and blood pressure. We concluded that a 

loss of lymph capillary clearance function for skin electrolytes might lead to saltsensitive 

hypertension, and that VEGF-C initiates electrolyte clearance from skin 

tissue. We tested in an animal model with selective depletion of VEGF-C driven 

lymphangiogenesis in the skin whether primary disorders in skin electrolyte and water 

metabolism result in experimental arterial hypertension. 

We reasoned that mice with over-expression of soluble VEGFR3 (sVEGFR-3) 

receptor (gene name: flt-4) under the control of the keratinocyte promoter K14 (K14- 

FLT4 mice) may serve as a model to study the effect of targeted overexpression of 

sVEGFR3 in the skin on skin electrolyte metabolism and systemic blood pressure. 

High-dose overexpression of sVEGFR3 in keratinocytes in mice homozygous for the 

transgene leads to 

embryonic death. In 

contrast, K14 FLT4 

mice with 

heterozygous 

overexpression of 

the sVEGFR3 breed 

well and show 

selective hypoplasia 

of subdermal lymph 

capillaries with only 

discrete skin edema. 

In our preliminary 

experiments, we 

found that a high-salt 

diet induced lymph 

capillary hyperplasia 

in wt mice, while 

subdermal lymph 

Fig. 3. Selective blockade of the MPS/VEGF-C regulatory axis in the 

skin by over-expression of sVEGFR3 by skin keratinocytes (VEGF-C 

trap) in K14-FLT4 mice results in exaggerated salt-sensitive 

hypertension. (A-D) Subcutaneous lymph capillaries, visualized with a 

LYVE-1 specific antibody (green) in wt (n=14) and K14-FLT4 mice 

(n=12) fed a low-salt (LSD) or a high-salt (HSD) diet for two weeks. (E) 

CD68 and VEGF-C expression in skin in the same mice. β-actin 

expression was used as a loading control. (F) eNOS and p eNOS 

expression in mouse skin. (G) Lymph capillary density in ear and mean 

arterial blood pressure in the mice. * P < 0.05 versus wt/LSD; † P < 

0.05 versus K14 Flt4/LSD; ‡ P < 0.05 versus wt/HSD. 

capillaries remained hypoplastic in heterozygous K14-FLT4 mice (Fig. 3 A-D), 

because increased VEGF-C protein expression by macrophages (Fig. 3E) was 

blocked by sVEGFR3 from skin keratinocytes which acted as a skin-specific VEGF-C 

trap. In support of this skin specificity, renal macrophage count, VEGF-C mRNA and 

protein expression, and lymph capillary density was unchanged in the same animals. 

Targeted depletion of subdermal lymphcapillaries resulted in Na + , Cl - , and water 

accumulation in skin in K14-FLT4 mice, even with low salt diet. Salt-sensitive 

hypertension was augmented in K14-FLT4 mice, and blood pressure tended to be 

higher in K14-FLT4 than in wt mice with low-salt diet (Fig. 3G). 

We conclude that targeted depletion of skin VEGF-C with hypoplasia of intradermal 

lymph capillaries result in skin electrolyte and water retention and blood pressure. 

This hypertension comes about an external regulatory mechanism within the skin, 

presumably because of reduced electrolyte and water clearance by hypoplastic skin 

74

vessels. This animal model underscores the importance of skin lymph capillaries for 

skin electrolyte homeostasis and systemic blood pressure control. Perhaps more 

importantly, this animal model provides a valuable model that is complementary to 

our animal model with pharmacological blockade of VEGF-C/VEGFR3 driven lymph 

capillary hyperplasia. 

Macrophages control skin electrolyte composition via VEGF-C 

driven electrolyte clearance by lymph capillaries 

As predicted by our model (Fig. 1B), we have shown previously that blockade of the 

macrophage driven VEGF-C response in rats prevents hyperplasia of the 

lymphcapillary network with electrolyte accumulation in the skin, inhibits an increase 

in eNOS expression in blood vessels, and leads to salt-sensitive hypertension. 

However, it has been unclear whether macrophages exert this blood-pressure 

buffering effect in response to high-salt diet by VEGF-C/VEGFR2-driven modulation 

of vascular eNOS expression (vasodilation), or by VEGF-C/VEGFR3-driven 

hyperplasia of interstitial skin lymph capillaries (enhanced electrolyte clearance). 

Other investigators have shown previously that selective VEGFR-2 blockade reduces 

vascular eNOS expression and increases blood pressure independently of salt 

intake. 

We have data suggesting that VEGF-C/VEGFR3-driven modulation of the lymph 

capillary bed is 

decisive for the blood 

pressure buffering 

mechanism of 

macrophages (Fig. 4). 

We fed mice 

(background: FVB) a 

low or a high-salt diet 

for 2 consecutive 

weeks and also treated 

Fig. 4. Selective blockade of the VEGF-C/VEGFR-3 regulatory axis in 

the skin by systemic treatment with anti-VEGFR-3 antibody (mF4- 

31c1) blocks lymph capillary hyperplasia and results in exaggerated 

salt-sensitive arterial hypertension. (A-D) Subcutaneous lymph 

capillaries, visualized with a LYVE-1 specific antibody (green) in 

untreated (n=20) and mF4-31c1-treated mice (n=24) fed a low-salt 

(LSD) or a high-salt (HSD) diet for two weeks. (E) CD68 (macrophage 

surface marker) and VEGF-C expression in skin in the same mice. βactin 

expression was used as a loading control. (F) eNOS and p 

eNOS expression in mouse skin. (G) Lymph capillary density in ear 

and mean arterial blood pressure in the mice. * P < 0.05 versus 

wt/LSD; † P < 0.05 versus K14 Flt4/LSD; ‡ P < 0.05 versus wt/HSD. 

the mice with an anti- 

VEGFR3 antibody 

(mF4-31c1, 45 μg/g 

body weight i.p. every 

48 hours). The 

resulting blockade of 

VEGF-C/VEGFR3driven 

lymph capillary 

hyperplasia allowed us 

to specifically 

investigate the role of lymph capillaries for macrophage/VEGF-C-driven blood 

75

Fig. 5. Selective blockade of the VEGF- 

C/VEGFR-3 regulatory axis in the skin by 

systemic treatment with anti-VEGFR-3 antibody 

(mF4-31c1) blocks lymph capillary hyperplasia 

and results in tissue Cl - retention, which parallels 

salt-sensitive hypertension. (A): Relationship 

between lymphcapillary density and skin Na + 

content relative to tissue dry weight (rSKNa + ). 

(B): Relationship between lymphcapillary density 

and skin water content relative to tissue dry 

weight (rSKW). (C): Relationship between 

lymphcapillary density and skin Cl - content 

relative to tissue dry weight (rSKCl - ). (D): 

Relationship between lymph capillary density in 

skin and mean arterial blood pressure (MAP, 

intra-arterial measurement and the end of the 

experiment). LSD: low-salt diet, HSD: high-salt 

diet. 

pressure control. We found that mF4- 

31c1 did not change lymph capillary 

density in low-salt diet (LSD) mice; 

however, skin lymph capillary 

hyperplasia with high-salt diet (HSD) 

was completely prevented with anti- 

VEGFR-3 treatment (Fig. 4A-D). 

VEGFR-3 blockade was characterized 

by increased macrophage recruitment 

and increased VEGF-C production in 

the skin (Fig. 4E), presumably by a 

compensatory effect of the 

marcophages to overcome VEGFR3 

blockade. As expected, high VEGF-C 

levels in both untreated and mF4- 

31c1-treated mice with HSD diet 

resulted in increased p-eNOS 

expression (Fig. 4F). We conclude 

that mF4-31c1 treatment selectively 

eliminates the macrophage/VEGF-Cdriven 

hyperplastic response of 

cutaneous lymph capillaries, while 

VEGF-C/VEGFR2-driven increase in 

eNOS expression in blood vessels 

remains intact. This selective 

blockade of the hyperplastic response 

of lymph capillaries to HSD diet increased mean arterial blood pressure by 19 mmHg 

within two weeks (Fig. 4G), despite normal p-eNOS expression (Fig. 4F). The 

findings support the idea that macrophages exert their blood-pressure buffering effect 

by VEGF-C driven lymphatic regulation of blood pressure. 

Lymph capillaries serve as a transit system for rapid movement of interstitial immune 

cells into secondary lymphatic tissue, such as lymph nodes. They also serve as a 

drainage system of interstitial fluid into the blood stream. Having shown that saltsensitive 

hypertension “goes” with subcutaneous lymph capillaries in the skin, we 

next tested whether or not blockade VEGF-C/VEGFR3-driven hyperplasia of the 

lymph capillary system resulted in increased Na + , K + , Cl - , and water content in the 

skin. This finding would support the hypothesis that macrophages induce interstitial 

electrolyte clearance via an unknown clearance process within the subcutaneous 

lymph capillary system (Fig. 1B). Therefore, we chemically analyzed electrolyte 

composition in the skin in the same mice. We found that VEGFR-3 blockade in HSD 

mice neither increased Na + (Fig. 5A), nor water content in skin tissue (Fig. 5B). 

Instead, VEGFR-3 blockade resulted in a selective increase in skin Cl - content (Fig. 

5C). This finding suggests that macrophages selectively influence Cl - metabolism in 

the skin via VEGF-C/VEGFR3-driven modulation of lymph capillary function. In 

76

Tab. 1. Skin electrolyte and water composition in comparison to 

serum electrolyte composition in the same animals. Note that 

selective blockade of the VEGF-C/VEGFR-3 regulatory axis in 

the skin by systemic treatment with anti-VEGFR-3 antibody 

(mF4-31c1) tissue led to Cl - retention and increased Cl - 

concentration at the skin tissue level, but not in serum. Skin Na + 

content was more stable compared with Cl - . Depending on 

intervention, skin Na + content was 2-4 fold higher than Cl - 

content, while serum Na + content was exactly 1.2-fold higher 

than serum Cl - . 

parallel with increased 

tissue Cl - content, blood 

pressure was elevated 

(Fig. 5D), suggesting that 

Cl - metabolism is of major 

importance for saltsensitive 

hypertension. 

This finding is in line with 

data from humans, where 

salt-sensitive 

hypertension is mainly 

linked with NaCl 

ingestion, while similar 

NaHCO3 loading does not 

increase blood pressure. 

Our results bring saltsensitive 

hypertension 

into a novel biological 

context, as we suggest 

that hyperplastic lymph capillaries specifically control Cl - clearance from 

subcutaneous skin tissue, while hyperplasia of an otherwise intact lymph capillary 

system is not coupled with Na + or water clearance. We interpret our data in a way 

that skin lymph capillaries apparently do not only collect and pump interstitial fluid, 

but also actively modulate its electrolyte concentration. The unknown underlying 

concentration mechanism is controlled by VEGF-C from skin macrophages: we found 

that modulation of lymph capillary density by anti-VEGFR3 treatment resulted in 

increased Cl - concentration in skin tissue, but not in blood (Tab. 1). 

2012 



Kopp C, Linz P, Wachsmuth L, Dahlmann A, Horbach T, Schofl C, Renz W, Santoro D, 

Niendorf T, Muller DN, Neininger M, Cavallaro A, Eckardt KU, Schmieder RE, Luft FC, Uder 

M, Titze J. (23)Na magnetic resonance imaging of tissue sodium. Hypertension. 

2012;59:167-172 

77

2011 

Slagman MC, Kwakernaak AJ, Yazdani S, Laverman GD, van den Born J, Titze J, Navis G. 

Vascular endothelial growth factor c levels are modulated by dietary salt intake in proteinuric 

chronic kidney disease patients and in healthy subjects. Nephrol Dial Transplant. 

2011;27:978-982. 

2010 

Machnik A, Dahlmann A, Kopp C, Goss J, Wagner H, van Rooijen N, Eckardt KU, Muller DN, 

Park JK, Luft FC, Kerjaschki D, Titze J. Mononuclear phagocyte system depletion blocks 

interstitial tonicity-responsive enhance binding protein/vascular endothelial growth factor c 

expression and induces salt-sensitive hypertension in rats. Hypertension. 2010;55:755-761. 

2009 

Machnik A, Neuhofer W, Jantsch J, Dahlmann A, Tammela T, Machura K, Park JK, Beck FX, 

Muller DN, Derer W, Goss J, Ziomber A, Dietsch P, Wagner H, van Rooijen N, Kurtz A, 

Hilgers KF, Alitalo K, Eckardt KU, Luft FC, Kerjaschki D, Titze J. Macrophages regulate saltdependent 

volume and blood pressure by a vascular endothelial growth factor-c-dependent 

buffering mechanism. Nat Med. 2009;15:545-552 

Heer M, Frings-Meuthen P, Titze J, Boschmann M, Frisch S, Baecker N, Beck L. Increasing 

sodium intake from a previous low or high intake affects water. Br J Nutr. 2009;101:1286- 

1294. 

2008 

Gratze P, Dechend R, Stocker C, Park JK, Feldt S, Shagdarsuren E, Wellner M, Gueler F, 

Rong S, Gross V, Obst M, Plehm R, Alenina N, Zenclussen A, Titze J, Small K, Yokota Y, 

Zenke M, Luft FC, Muller DN. Novel role for inhibitor of differentiation 2 in the genesis of 

angiotensin ii-induced hypertension. Circulation. 2008;117:2645-2656 

Ziomber A, Machnik A, Dahlmann A, Dietsch P, Beck FX, Wagner H, Hilgers KF, Luft FC, 

Eckardt KU, Titze J. Sodium-, potassium-, chloride-, and bicarbonate-related effects on blood 

pressure and electrolyte homeostasis in deoxycorticosterone acetate-treated rats. Am J 

Physiol Renal Physiol. 2008;295:F1752-1763 

78

2012 

Reviews/Comments 

Harrison DG, Marvar PJ, Titze JM. Vascular inflammatory cells in hypertension. Front 

Physiol. 2012;3:128. 

2010 

Titze J, Machnik A. Sodium sensing in the interstitium and relationship to hypertension. Curr 

Opin Nephrol Hypertens. 2010 Jul;19(4):385-92. 

Titze, J., Muller, D. N., Luft, F. C. Response to Blood Pressure Control: A Facelift for 

Macrophages? Hypertension. 2010;56(2): E24-E24 

2009 

Titze J. “Water-free sodium accumulation”. Semin Dial. 2009 May-Jun;22(3):253-5. 

Titze J, Ritz E. Salt and its effect on blood pressure and target organ damage: New pieces in 

an old puzzle. J Nephrol. 2009;22:177-189 

Titze J. Salz- und Wasserhaushalt: Neue Salzspeicher. Der Nephrologe. 2009;4:488–496 



Agnes Schröder (2009) 

Macrophages as regulators of volume and blood pressure homeostasis. 

Doctoral theses (Medical Students) 

Markus Schafflhuber (2009) 

Gycosaminoglycan polymerization and Na + storage in the skin 

Christoph Ilies (2010) 

Osmotically inactive Na + storage in the skin 

Katharina Bauer (2010) 

Internal Na + balance in desoxycorticosterone acetate-treated rats 

79

Matrix Biology Group 

(Dpt. Of Internal Medicine III) 

Head: PD Dr. med. Jörg Distler 

Address: Department of Internal Medicine III 

Krankenhausstr. 12 


Telefone: + 49 (9131) 85 39226 

Fax: + 49 (9131) 85 35467 

E-mail: joerg.distler@uk-erlangen.de 

Christian Beyer, Dr. med. * 


Head 

PD Dr. med. Jörg Distler 


-- 


80 

Alfiya Akhmetshina, Dr. rer. nat. *

Nicole Reich° 

Pawel Zerr 

Katrin Palumbo 


Maria Halter 

Anna Maria Herrmann* 

Julia Gebhardt* 


Jerome Avouac, Dr. 

med., Paris 


Clara Dees 

Alfiya Akhmetshina° 

Ekatherina Skirtladze 

Technicians 

Guest Scientists 

Michal Tomcik, Dr. 

med., Prag 

81


Our group is mainly interested in mechanisms of fibroblast activation and in the 

development of novel anti-fibrotic approaches. Tissue fibrosis can affect virtually 

every organ system and represents a major medicinal problem, because effective 

therapies are currently not available. During our time in the Nikolaus-Fiebiger Center 

from 2009 to 2010, we used systemic sclerosis (SSc), a prototypic systemic fibrotic 

disease, to investigate the role of the following pathways for fibroblast activation and 

fibrosis. We very particularly interested (i) in AP-1 signaling as a downstream 

mediator of the pro-fibrotic effects of TGFβ, (ii) in the role of morphogenic pathways 

such as hedgehog and notch-signaling in fibrosis and (iii) in cannabinoid receptors as 

potential targets for therapeutic approaches in fibrotic diseases. 

AP-1 signaling 

Transforming growth factor-β (TGF-β) is a key-player in fibrotic diseases. However, 

the molecular mechanisms by which TGF-β activates fibroblasts are incompletely 

understood. We have previously shown that the expression of the AP-1 transcription 

factor Fra-2 is upregulated in SSc and that Fra-2 stimulates the release of 

extracellular matrix. We hypothesized that other members of the AP-1 family may 

also contribute to the aberrant release of collagen in SSc. Indeed, we observed that 

the mRNA as well as the protein levels of three other members of the AP-1 family, c- 

Jun, JunD and cFos were strongly increased in fibrotic skin of SSc patients compared 

to healthy individuals. The overexpression persisted in cultured fibroblasts from SSc 

patients. c-Jun, JunD and cFos were induced by TGF-β in a time- and dosedependent 

manner. Moreover, the expression of those AP-1 family members colocalized 

with nuclear pSmad 3 in cultured fibroblasts and in fibrotic skin. Inhibition of 

canonical Smad signaling by siRNA mediated knockdown of either Smad3 or Smad4 

prevented the stimulatory effects of TGF-β and normalized the expression of c-Jun, 

JunD and cFos in SSc fibroblasts. To further evaluate the role of AP-1 signaling as a 

downstream mediator of the pro-fibrotic effects of TGF-β in vitro and in vivo, we 

employed knockout and knockdown strategies as well as selective chemical 

82

inhibitors. The small molecule inhibitor T-5224 abrogated the stimulatory effects of 

TGF-β on collagen synthesis and differentiation of resting fibroblasts into 

metabolically active myofibroblasts. Similar results were obtained upon siRNA 

mediated knockdown of cJun, JunD and cFos and in JunD deficient murine 

fibroblasts. Moreover, JunD -/- mice were protected from bleomycin induced fibrosis 

with reduced dermal thickening, decreased myofibroblast counts and lower collagen 

content of lesional skin. AP-1 signaling may also be a potential target for therapeutic 

intervention. T-5224, which is currently evaluated in clinical trials for rheumatoid 

arthritis, exerted potent anti-fibrotic effects in murine models of SSc in well tolerated, 

pharmacologically relevant doses. T-5224 prevented fibrosis in the mouse models of 

bleomycin-induced dermal fibrosis and fibrosis induced by overexpression of a 

constitutively active TGF-β receptor 1. Treatment with T-5224 dose-dependently 

reduced dermal thickening, myofibroblast counts and the collagen content in those 

models without toxic effect. Given the central role of AP-1 signaling as a downstream 

mediator of TGF- β signaling and the availability of well tolerated, selective inhibitors 

such as T-5224, AP-1 signaling might be a promising molecular target for the 

treatment of SSc and other fibrotic diseases. 

Hedgehog and Notch signaling 

Hedgehog and Notch signaling are so-called morphogenic pathways that do not only 

play major roles during embryonic development, but that are also essential to 

maintain tissue homeostasis in the adult. Aberrant activation of hedgehog- or notch 

signaling has both been implicated in the pathogenesis of various diseases in adults, 

including a variety of different malignancies. Given their important role in cellular 

differentiation, we hypothesized that hedgehog- and notch signaling may contribute 

to the activation of fibroblasts and may play a role in tissue fibrosis. 

For hedgehog signaling, we demonstrated an overexpression of the ligand sonic 

hedgehog (Shh) with accumulation of the downstream transcription factors Gli-1 and 

Gli-2 in SSc. The expression of hedgehog target genes such as ptch-1, ptch-2 and 

cyclin D were also upregulated, confirming the activation of hedgehog signaling in 

SSc. Stimulation of cultured fibroblasts with recombinant Shh induced an activated 

phenotype with differentiation of resting fibroblasts into myofibroblasts and increased 

release of collagen. Overexpression of Shh in the skin of mice induced fibrosis with 

83

accumulation of collagen, dermal thickening and myofibroblast differentiation, 

demonstrating that activation of hedgehog signaling alone is sufficient to induce 

fibrosis in vivo. Consistent with these findings, mice lacking one allele of the inhibitory 

receptor ptch-1 were more sensitive to experimental fibrosis. Conversely, inhibition of 

the activating receptor smoothened (Smo) either pharmacologically by the selective 

inhibitor LDE223 or by siRNA prevented bleomycin induced dermal fibrosis. Inhibition 

of the hedgehog pathway also exerted potent anti-fibrotic effects in the tight skin 1 

mouse model. 

We also demonstrated a role of aberrant notch signaling in the pathogenesis of SSc. 

Notch signaling was activated in SSc in vivo as well as in cultured SSc fibroblasts in 

vitro as shown by overexpression of the notch ligand Jagged-1 (Jag-1) and by 

elevated transcription of notch target genes such as hes-1. Stimulation of healthy 

dermal fibroblasts with the Jag-1-Fc induced an SSc-like phenotype with expression 

of contractile proteins and increased release of collagen. Inhibition of Notch signaling 

by the γ-secretase inhibitor DAPT or by siRNA mediated knockdown of Notch-1 

efficiently reduced the release of collagen from SSc fibroblasts. Overexpression of a 

Notch antisense construct or treatment with DAPT also prevented bleomycin-induced 

fibrosis and fibrosis in tight-skin 1 mice. Moreover, targeting Notch signaling resulted 

in almost complete regression of pre-established experimental fibrosis. Thus, we 

demonstrated that Notch signaling plays an important role in fibroblast activation and 

tissue fibrosis. 

Collectively, our results describe a central role of hedgehog and notch signaling for 

fibroblast activation in fibrosis. These findings might have direct translational 

implications because inhibitors of Smo as well as γ-secretase inhibitors are available 

and were well tolerated in clinical trials of cancer patients. 

Cannabinoid receptors 

Cannabinoids are derivates of the marijuana component Δ 9 -tetrahydrocannabinol that 

exert their effects via CB1 and CB2 receptors. Cannabinoids can be subdivided into 

three different groups according to their origin. The family of cannabinoids includes 

plant derived cannabinoids, synthetic cannabinoids and endogenous cannabinoids 

(endocannabinoids) that are synthesized within the human body. Besides their 

effects in the CNS, endocannabinoids regulate physiological and pathophysiological 

processes in non-neuronal tissues. Recent data demonstrated that cannabinoids 

84

egulate the activation of hepatic stellate cells, which contribute to the pathogenesis 

of liver fibrosis. We therefore aimed to evaluate the role of cannabinoids and their 

receptors in fibroblast activation and evaluate targeting of cannabinoid receptors as a 

therapeutic approach in fibrotic diseases. 

We demonstrated that mice deficient for CB1 (CB1 -/- ) were protected from 

bleomycin-induced dermal fibrosis with reduced dermal thickening, hydroxyproline 

content and myofibroblast counts. Inactivation of CB1 decreased the number of 

infiltrating T cells and macrophages in lesional skin. In contrast, activation of CB1 

with the selective small molecule inhibitor ACEA increased leukocyte infiltration and 

enhanced the fibrotic response to bleomycin. The phenotype of CB1 -/- mice was 

mimicked by transplantation of CB1 -/- bone marrow into CB1 +/+ mice, demonstrating 

that leukocytes mediate the pro-fibrotic effects of CB1. Consistently, knockdown of 

CB1 did not prevent fibrosis in the inflammation-independent Tsk-1 model. 

In contrast to CB1 -/- mice, CB2 -/- mice were more sensitive to bleomycin induced 

dermal fibrosis. Leukocytes counts were significantly higher in lesional skin of CB2 -/-+ 

mice than CB2 +/+ mice. Treatment with the CB2 antagonist AM-630 also increased 

dermal fibrosis, whereas the CB2 agonist JWH-133 reduced leukocyte infiltration and 

dermal fibrosis. As for CB1, the altered sensitivity of CB2 -/- mice to fibrosis was 

mediated by bone marrow derived cells rather than by resident fibroblasts, because 

the phenotype of CB2 -/- mice was mimicked by transplantation of CB2 -/- bone 

marrow into CB2 +/+ mice, but not by CB2 -/- mice transplanted with bone marrow from 

CB2 +/+ mice. Consistent with this hypothesis, neither knockdown or inhibition, nor 

activation of CB1 or CB2 in cultured fibroblasts altered fibroblast activation or the 

release of collagen. 

We demonstrated that CB1 and CB2 have opposing effects in fibrosis. CB1 

stimulates leukocyte infiltration and enhances tissue fibrosis, whereas CB2 limits 

leukocyte influx and ameliorates experimental dermal fibrosis. Since selective CB1 

antagonists as well as selective CB2 agonists are available, the endocannabiod 

system and both of its receptors might be interesting molecular targets for the 

treatment of early inflammatory stages of fibrotic diseases, whereas they are unlikely 

to be effective in later, less inflammatory stages with predominant endogenous 

activation of fibroblasts. 

85



1. Balistreri E, Garcia-Gonzalez E, Selvi E, Akhmetshina A, Palumbo K, 

Lorenzini S, Maggio R, Lucatelli M, Galeazzi M, Distler J. The cannabinoid 

WIN55, 212-2 abrogates dermal fibrosis in scleroderma bleomycin model. Ann 

Rheum Dis. 2010 Dec 21. 

2. Dieudé P, Guedj M, Truchetet ME, Wipff J, Revillod L, Riemekasten G, 

Matucci-Cerinic M, Melchers I, Hachulla E, Airo P, Diot E, Hunzelmann N, 

Mouthon L, Cabane J, Cracowski JL, Riccieri V, Distler J, Amoura Z, Valentini 

G, Camaraschi P, Tarner I, Frances C, Carpentier P, Brembilla NC, Meyer O, 

Kahan A, Chizzolini C, Boileau C, Allanore Y. Association of the CD226 

307Ser variant with systemic sclerosis: Evidence for a contribution of costimulation 

pathways in SSc pathogenesis. Arthritis Rheum. 2010 Dec 15. 

3. Dieudé P, Guedj M, Wipff J, Ruiz B, Riemekasten G, Airo P, Melchers I, 

Hachulla E, Cerinic MM, Diot E, Hunzelmann N, Caramaschi P, Sibilia J, Tiev 

K, Mouthon L, Riccieri V, Cracowski JL, Carpentier PH, Distler J, Amoura Z, 

Tarner I, Avouac J, Meyer O, Kahan A, Boileau C, Allanore Y. NLRP1 

influences the systemic sclerosis phenotype: a new clue for the contribution of 

innate immunity in systemic sclerosis-related fibrosing alveolitis pathogenesis. 

Ann Rheum Dis. 2010 Dec 13. 

4. Polzer K, Neubert K, Meister S, Frey B, Baum W, Distler JH, Gückel E, Schett 

G, Voll RE, Zwerina J. Proteasome inhibition aggravates TNF-mediated bone 

resorption. Arthritis Rheum. 2010 Dec 6. 

5. Avouac J, Fürnrohr BG, Tomcik M, Palumbo K, Zerr P, Horn A, Dees C, 

Akhmetshina A, Beyer C, Distler O, Schett G, Allanore Y, Distler JH. 

Inactivation of the transcription factor STAT4 prevents inflammation-driven 

fibrosis in systemic sclerosis animal models. Arthritis Rheum. 2010 Nov 30. 

6. Heiland GR, Zwerina K, Baum W, Kireva T, Distler JH, Grisanti M, Asuncion F, 

Li X, Ominsky M, Richards W, Schett G, Zwerina J. Neutralisation of Dkk-1 

protects from systemic bone loss during inflammation and reduces sclerostin 

expression. Ann Rheum Dis. 2010 Sep 21. 

7. Weber CK, Haslbeck M, Englbrecht M, Sehnert B, Mielenz D, Graef D, Distler 

JH, Mueller RB, Burkhardt H, Schett G, Voll RE, Fürnrohr BG. Antibodies to 

the endoplasmic reticulum-resident chaperones calnexin, BiP and Grp94 in 

patients with rheumatoid arthritis and systemic lupus erythematosus. 

Rheumatology (Oxford). 2010 Aug 17. 

8. Dallos T, Heiland GR, Strehl J, Karonitsch T, Gross W, Moosig F, Holl-Ulrich 

C, Distler JH, Manger B, Schett G, Zwerina J. Chemokine CCL17/TARC in 

Churg-Strauss syndrome. Arthritis Rheum. 2010 Jul 28. 

86

9. Marquart S, Zerr P, Akhmetshina A, Palumbo K, Reich N, Tomcik M, Horn A, 

Dees C, Engel M, Zwerina J, Distler O, Schett G, Distler JH. Inactivation of the 

cannabinoid receptor CB1 prevents leukocyte infiltration and experimental 

fibrosis. Arthritis Rheum. 2010 Jul 8. 

10. Wipff J, Dieudé P, Guedj M, Ruiz B, Riemekasten G, Cracowski J, Matucci- 

Cerinic M, Melchers I, Humbert M, Hachulla E, Airo P, Diot E, Hunzelmann N, 

Caramaschi P, Sibilia J, Valentini G, Tiev K, Girerd B, Mouthon L, Riccieri V, 

Carpentier P, Distler JH, Amoura Z, Tarner I, Degano B, Avouac J, Meyer O, 

Kahan A, Boileau C, Allanore Y. Association of KCNA5 gene polymorphism 

with systemic sclerosis-associated pulmonary arterial hypertension in the 

European Caucasian population. Arthritis Rheum. 2010 Jun 15. 

11. Avouac J, Walker U, Tyndall A, Kahan A, Matucci-Cerinic M, Allanore Y; 

EUSTAR, Miniati I, Muller A, Iannone F, Distler O, Becvar R, Sierakowsky S, 

Kowal-Bielecka O, Coelho P, Cabane J, Cutolo M, Shoenfeld Y, Valentini G, 

Rovensky J, Riemekasten G, Vlachoyiannopoulos P, Caporali R, Jiri S, Inanc 

M, Zimmermann Gorska I, Carreira P, Novak S, Czirjak L, Oliveira Ramos F, 

Jendro M, Chizzolini C, Kucharz EJ, Richter J, Cozzi F, Rozman B, Mallia CM, 

Gabrielli A, Farge D, Kiener HP, Schöffel D, Airo P, Wollheim F, Martinovic D, 

Trotta F, Jablonska S, Reich K, Bombardieri S, Siakka P, Pellerito R, Bambara 

LM, Morovic-Vergles J, Denton C, Hinrichs R, Van den Hoogen F, Damjanov 

N, Kötter I, Ortiz V, Heitmann S, Krasowska D, Seidel M, Hasler P, Van Laar 

JM, Kaltwasser JP, Foeldvari I, Juan Mas A, Bajocchi G, Wislowska M, 

Pereira Da Silva JA, Jacobsen S, Worm M, Graniger W, Kuhn A, Stankovic A, 

Cossutta R, Majdan M, Damjanovska Rajcevska L, Tikly M, Nasonov EL, 

Steinbrink K, Herrick A, Müller-Ladner U, Dinc A, Scorza R, Sondergaard K, 

Indiveri F, Nielsen H, Szekanecz Z, Silver RM, Antivalle M, Espinosa IB, 

García de la Pena Lefebvre P, Midtvedt O, Launay D, Valesini F, Tuvik P, 

Ionescu RM, Del Papa N, Pinto S, Wigley F, Mihai C, Sinziana Capranu M, 

Sunderkötter C, Jun JB, Alhasani S, Distler JH, Ton E, Soukup T, Seibold J, 

Zeni S, Nash P, Mouthon L, De Keyser F, Duruöz MT, Cantatore FP, Strauss 

G, von Mülhen CA, Pozzi MR, Eyerich K, Szechinski J, Keiserman M, 

Houssiau FA, Román-Ivorra JA, Krummel-Lorenz B, Aringer M, Westhovens 

R, Bellisai F, Mayer M, Stoeckl F, Uprus M, Volpe A, Buslau M, Yavuz S, 

Granel B, Valderílio Feijó A, Del Galdo F, Popa S, Zenone T, Ricardo 

Machado X, Pileckyte M, Stebbings S, Mathieu A, Tulli A, Tourinho T, Souza 

R, Acayaba de Toledo R, Stamp L, Solanki K, Veale D, Francisco Marques 

Neto J, Bagnato GF, Loyo E, Toloza S, Li M, Ahmed Abdel Atty Mohamed W, 

Cobankara V, Olas J, Salsano F, Oksel F, Tanaseanu CM, Foti R, Ancuta C, 

Vonk M, Caramashi P, Beretta L, Balbir A, Chiàla A, Pasalic Simic K, Ghio M, 

Stamenkovic B, Rednic S, Host N, Pellerito R, Hachulla E, Furst DE. 

Characteristics of joint involvement and relationships with systemic 

inflammation in systemic sclerosis: results from the EULAR Scleroderma Trial 

and Research Group (EUSTAR) database. J Rheumatol. 2010 Jul;37(7):1488- 

501. Epub 2010 Jun 15. 

12. Dieudé P, Guedj M, Wipff J, Ruiz B, Riemekasten G, Matucci-Cerinic M, 

Melchers I, Hachulla E, Airo P, Diot E, Hunzelmann N, Cabane J, Mouthon L, 

Cracowski JL, Riccieri V, Distler J, Meyer O, Kahan A, Boileau C, Allanore Y. 

Association of the TNFAIP3 rs5029939 variant with systemic sclerosis in the 

European Caucasian population. Ann Rheum Dis. 2010 May 28. 

87

13. Krönke G, Uderhardt S, Kim KA, Stock M, Scholtysek C, Zaiss MM, Surmann- 

Schmitt C, Luther J, Katzenbeisser J, David JP, Abdollahi-Roodsaz S, Tran K, 

Bright JM, Binnerts ME, Akhmetshina A, Böhm C, Distler JH, Joosten LA, 

Schett G, Abo A. R-spondin1 protects against inflammatory bone damage 

during murine arthritis by modulating the wnt pathway. Arthritis Rheum. 2010 

Apr 6. 

14. Reich N, Beyer C, Gelse K, Akhmetshina A, Dees C, Zwerina J, Schett G, 

Distler O, Distler JH. Microparticles stimulate angiogenesis by inducing ELR(+) 

CXC-chemokines in synovial fibroblasts. J Cell Mol Med. 2010 Mar 8. 

15. Maurer B, Stanczyk J, Jüngel A, Akhmetshina A, Trenkmann M, Brock M, 

Kowal-Bielecka O, Gay RE, Michel BA, Distler JH, Gay S, Distler O. miR-29 is 

a key regulator of collagen expression in systemic sclerosis. Arthritis Rheum. 

2010 Mar 3. 

16. Akhmetshina A, Beer J, Polzer K, Englbrecht M, Palumbo K, Dees C, Reich N, 

Zwerina J, Szucs G, Gusinde J, Nevskaya T, Distler O, Kerjaschki D, Schett 

G, Distler JH. Decreased lymphatic vessel counts in systemic sclerosis - 

Association with fingertip ulcers. Arthritis Rheum. 2010 Feb 12. 

17. Reich N, Maurer B, Akhmetshina A, Venalis P, Dees C, Zwerina J, Nevskaja 

T, Schett G, Distler JH. The transcription factor fos-related antigen-2 (Fra-2) 

regulates the production of extracellular matrix in systemic sclerosis. Arthritis 

Rheum 2010 Jan;62(1):280-90. 

18. Functional autoantibodies against serpin E2 in rheumatoid arthritis. 

Maciejewska-Rodrigues H, Al-Shamisi M, Hemmatazad H, Ospelt C, Bouton 

MC, Jäger D, Cope AP, Charles P, Plant D, Distler JH, Gay RE, Michel BA, 

Knuth A, Neidhart M, Gay S, Jüngel A.. Arthritis Rheum. 2010 Jan;62(1):93- 

104. 

19. Maurer B, Busch N, Jüngel A, Pileckyte M, Gay RE, Michel BA, Schett G, Gay 

S, Distler JH, Distler O. Fra-2 induces progressive peripheral vasculopathy in 

mice closely resembling human systemic sclerosis. Circulation. 2009 Nov 23. 

20. Kurowska-Stolarska M, Distler JH, Jüngel A, Rudnicka W, Neumann E, Pap T, 

Wenger RH, Michel BA, Müller-Ladner U, Gay RE, Maslinski W, Gay S and 

Distler O. Id-2 induced by hypoxia promotes synovial fibroblast-dependent 

osteoclastogenesis. Arthritis Rheum. 2009 Nov 30;60(12):3663-3675. 

21. Ruiz Heiland G, Aigner E, Dallos T, Sahinbegovic E, Krenn V, Thaler C, Weiss 

G, Distler JH, Datz C, Schett G, Zwerina J. Synovial immunopathology in 

hemochromatosis arthropathy. Ann Rheum Dis. 2009 Nov 23. 

22. Dieudé P, Wipff J, Guedj M, Ruiz B, Melchers I, Hachulla E, Riemekasten G, 

Diot E, Hunzelmann N, Sibilia J, Tiev K, Mouthon L, Cracowski JL, Carpentier 

PH, Distler J, Amoura Z, Tarner I, Avouac J, Meyer O, Kahan A, Boileau C, 

Allanore Y. BANK1 is a genetic risk factor for diffuse cutaneous systemic 

sclerosis and has additive effects with IRF5 and STAT4. Arthritis Rheum. 2009 

Nov;60(11):3447-54. 

88

23. Mieliauskaite D, Venalis P, Dumalakiene I, Venalis A, Distler J. Relationship 

between serum levels of TGF-beta1 and clinical parameters in patients with 

rheumatoid arthritis and Sjögren's syndrome secondary to rheumatoid arthritis. 

Autoimmunity. 2009 May;42(4):356-8. 

24. Guiducci S, Distler JH, Milia AF, Miniati I, Rogai V, Manetti M, Falcini F, Ibba- 

Manneschi L, Gay S, Distler O, Matucci-Cerinic M. Stiff skin syndrome: 

evidence for an inflammation-independent fibrosis? Rheumatology (Oxford). 

2009 May 25. 

25. Hemmatazad H, Rodrigues HM, Maurer B, Brentano F, Pileckyte M, Distler 

JH, Gay RE, Michel BA, Gay S, Huber LC, Distler O, Jüngel A. Histone 

deacetylase 7, a potential target for the antifibrotic treatment of systemic 

sclerosis. Arthritis Rheum. 2009 May;60(5):1519-29. 

26. Akhmetshina A, Dees C, Busch N, Beer J, Zimmer A, Distler O, Schett G, 

Distler JH. The cannabinoid receptor CB2 exerts anti-fibrotic effects in 

experimental dermal fibrosis. Arthritis Rheum, 2009 Apr;60(4):1129-36. 

27. Polzer K, Joosten L, Gasser J, Distler JH, Ruiz G, Baum W, Redlich K, 

Bobacz K, Smolen JS, van den Berg W, Schett G, Zwerina J. IL-1 is essential 

for systemic inflammatory bone loss. Ann Rheum Dis. 2009 Feb 5. 

28. Akhmetshina A, Venalis P, Dees C, Busch N, Zwerina J, Jüngel A, Schett G, 

Distler O, Distler JH. Treatment with imatinib does not only prevent fibrosis in 

different preclinical models of SSc, but also induces regression of established 

fibrosis. Arthritis Rheum 2009 Jan;60(1):219-24. 

29. Hanitsch LG, Burmester GR, Witt C, Hunzelmann N, Genth E, Krieg T, 

Lehmacher W, Melchers I, Meurer M, Müller-Ladner U, Schulze-Lohoff E, 

Becker M, Sunderkoetter C; Worm M, Klaus P, Rubbert A, Steinbrink K, 

Grundt B, Hein R, Scharffetter-Kochanek K, Hinrichs R, Walker K, Szeimies 

RM, Karrer S, Müller A, Seitz C, Schmidt E, Lehmann P, Foeldvári I, 

Reichenberger F, Gross L, Kuhn A, Haust M, Reich K, Böhm M, Saar P, 

Fierlbeck G, Kötter I, Lorenz HM, Blank N, Gräfenstein K, Juche A, Aberer E, 

Bali G, Fiehn C, Stadler R, Bartels V, Buslau M, Distler J, Sticherling M, 

Riemekasten G. Skin sclerosis is only of limited value to identify SSc patients 

with severe manifestations--an analysis of a distinct patient subgroup of the 

German Systemic Sclerosis Network (DNSS) Register. Rheumatology 

(Oxford). 2009 Jan;48(1):70-3. 

89

Reviews 

1. Iwamoto N, Distler JH, Distler O. Tyrosine Kinase Inhibitors in the Treatment 

of Systemic Sclerosis: From Animal Models to Clinical Trials. Curr Rheumatol 

Rep. 2010 Nov 2. 

2. Beyer C, Schett G, Distler O, Distler JH. Animal Models of Systemic Sclerosis: 

Prospects and Limitations. Arthritis Rheum. 2010 Jul 8. 

3. Beyer C, Distler JH, Distler O. Are tyrosine kinase inhibitors promising for the 

treatment of systemic sclerosis and other fibrotic diseases? Swiss Med Wkly. 

2010 Apr 26. 

4. Distler JH and Distler O. Tyrosine kinase inhibitors for the treatment of fibrotic 

diseases such as systemic sclerosis: towards molecular targeted therapies. 

Ann Rheum Dis. 2010 Jan;69 Suppl 1:i48-51. 

5. Beyer C and Distler JH. The scientific basis for novel treatments of systemic 

sclerosis. f1000 Medicine Reports 2009, 1:95. 

6. Distler JH, Beyer C, Schett G, Lüscher TF, Gay S, Distler O. Endothelial 

Progenitor Cells - Novel players in the pathogenesis of rheumatic diseases. 

Arthritis Rheum. 2009 Nov;60(11):3168-79. 

7. Abraham DJ, Krieg T, Distler J, Distler O. Overview of pathogenesis of 

systemic sclerosis. Rheumatology (Oxford). 2009 Jun;48 Suppl 3:iii3-7. 

8. Beyer C, Schett G, Gay S, Distler O, Distler JH. Hypoxia in the pathogenesis 

of systemic sclerosis. Arthritis Res Ther. 2009;11(2):220. Epub 2009 Apr 21. 



Alfiya Akhmetshina, Nicole Busch, Michal Tomcik (PhD part time in Erlangen), 

Jerome Avouac (PhD part time in Erlangen) 


Sieglinde Marquart, Constantin Huhn, Tobias Strapatsas, Christina Bergmann, Michael 

Balzer 

90

Laboratory of Dendritic Cell Biology 

(Department of Dermatology) 

Head: Diana Dudziak, Dr. rer. nat. 

Professor of Dendritic Cell Biology 

Address: Laboratory of Dendritic Cell Biology 

Department of Dermatology 

Hartmannstr. 14 


Telephone: + 49 (9131) 85 39346 

Fax: + 49 (9131) 85 39347 

E-mail: diana.dudziak@uk-erlangen.de 

Homepage: http://www.hautklinik.ukerlangen.de/e1585/e1893/e2171/index_ger.html 

Head 

Diana Dudziak, Prof. Dr. rer. nat. 

Professor of Dendritic Cell Biology 


Kirsten Neubert, Dr. rer. nat. 

Nathalie Eissing, Dr. med. vet. 


Gordon Heidkamp 

Christian Lehmann 

Anna Baranska 

Lukas Heger 

91



Kirsten Ehrenspeck 

Nathalie Eissing* 

Yang Jiao 

Technicians 

Simone Beck 

Christina Weiss 

92


Our research is focussed on the function of Dendritic cell subpopulations in the 

process of induction of immunity and maintenance of peripheral tolerance. Dendritic 

cells are very important antigen presenting cells. We are especially interested in the 

analysis of antigen uptake, antigen processing and antigen presentation by Dendritic 

cells, if and how Dendritic cell subpopulations differ in antigen processing and 

presentation, and how Dendritic cell subpopulations migrate in the lymphoid tissues 

when they have encountered their antigens under immunizing and tolerizing 

conditions. Moreover, to understand antigen processing and presentation 

mechanisms we focus on the responding T cells. Depending on the Dendritic cell 

subpopulation we found that different Dendritic cell subpopulations can induce 

different T cell responses in vivo (Dudziak et al., 2007). These findings will have 

important implications in the development of new therapeutic vaccines. 

Dendritic cells and antigen targeting 

Dendritic cells maintain peripheral tolerance and induce immunity 

The immune system controls invading pathogenic organisms by innate and adaptive 

immune mechanisms. To enable recognition of virtually any antigen, developing B 

and T cells randomly rearrange their receptors to produce unique clones with a wide 

variety of specificities. It is well accepted that this also leads to the generation of 

receptors that can recognize ‘self’ antigens. By central tolerance mechanisms at 

several checkpoints self reactive B cells are deleted in the bone marrow whereas self 

reactive T cells undergo negative selection mechanisms in the thymus where they 

are either deleted or undergo anergic mechanisms. As some of the T cells can 

escape negative selection in the thymus, checkpoints in the periphery have to exist to 

prevent autoimmune reactions by those potentially self-reactive T cells. Only recently 

it became clear that DCs which are essential for the induction of adaptive immune 

responses are also key players not only for the maintenance of central tolerance but 

also for the maintenance of peripheral tolerance. 

Dendritic cells (DCs) are the most important antigen presenting cells in the immune 

system. In non-lymphoid organs DCs act as sentinels and take up and process 

antigens from the periphery (skin, airways, intestine, interstitial spaces of various 

organs). Antigen loaded DCs migrate to the lymphoid tissues and interact with 

antigen-specific T cells. Besides the uptake of soluble antigens, DCs are also able to 

ingest apoptotic cells, tumor cells, or infected cells to present their antigen. The 

induction of peripheral tolerance or immunity depends on the DC differentiation state. 

In the steady-state immature DCs express low levels of CD80, CD86 or MHC class II 

molecules on their cell surface. Recognition of the MHC peptide complex by a T cell 

in the steady-state causes deletion or anergy of self reactive T cells after an initial 

phase of proliferation. This process is resulting in peripheral tolerance (Dudziak et al., 

2007, Dudziak, 2009). 

In contrast, during a microbial or viral infection antigen uptake is paralleled by DC 

activation and maturation. In this process pattern-recognition receptors (PRRs) 

expressed on DCs, such as Toll-like receptors (TLRs) and proteins of the C-type 

lectin receptor family, recognize conserved parts of pathogens such as lipids, sugar 

93

esidues, or nucleic acids. This stimulation causes upregulation of cell surface 

activation markers (e.g. CD80, CD83, and CD86) and expression of cytokines and 

chemokines such as TNFα, IL6, IL12 or Rantes 9 (Dudziak et al., 2005, Dudziak, 

2009). Under these circumstances T cells that recognize bacterial or viral antigens 

become activated, clonally expand and develop into memory T cells (Dudziak et al., 

2007; Dudziak, 2009). 

Production of antigen carrying recombinant antibodies against different 

Dendritic cell subpopulations 

Diana Dudziak, Gordon Heidkamp, Nathalie Eissing, Kirsten Ehrenspeck 

At least 6 different subtypes of murine DCs can be distinguished based on their 

localization and expression of cell surface molecules (Fig. 1, Dudziak, 2009). We are 

only just beginning to understand why such a great variety of DC subtypes has 

evolved and what their precise role is in the maintenance of peripheral tolerance or in 

the induction of immunity. The two main subpopulations of conventional DCs in the 

spleen can be distinguished by the expression of CD11c and CD8 as CD11c + CD8 - 

(myeloid) and CD11c + CD8 + (lymphoid) DCs (Dudziak, 2009). We found that these 

DC subpopulations express different endocytosis receptors and C-type lectin 

receptors on their cell surface, e.g. CD11c + CD8 - DCs express DCIR2 whereas 

CD11c + CD8 + DCs express DEC205 (CD205) (Fig. 1, Dudziak et al., 2007; Soares et 

al., 2007; Yamazaki et al., 2008; Do et al., 2010, O’Kamphorst et al., 2010; Loschko 

et al., 2011(1); Loschko et al., 2011(2)). We could show that DCIR2 and DEC205 

were rapidly internalized after interaction with antibodies against these endocytosis 

receptors (Dudziak et al., 2007). In addition to the internalization of the receptors we 

found that also the bound antibodies were internalized into the DCs. These findings 

let us to the idea of producing recombinant endocytosis receptor antibodies that are 

genetically fused with an antigen of choice. 

94 

Figure 1. Murine splenic 

DCs. A) Overview of 

different murine DC subpopulations 

and their localization. 

B) Immunofluorescence 

of murine spleen 

showing B cells (B220), 

CD11c + CD8 - (DCIR2/ 

33D1 + ) in the bridging area 

and red pulp and 

CD11c + CD8 + (DEC205 + ) 

DCs in the T cell area. C) 

FACS analysis of CD11c + 

DCs in murine spleen and 

the expression of CD8, 

DEC205 and DCIR2/33D1. 

(Dudziak et al., 2009)

We cloned the variable regions of the DEC205 and the 33D1 rat antibodies (the latter 

we identified by microarray analysis and expression cloning to be DCIR2) and fused 

them by overlap PCR to the constant regions of the heavy and light chain of murine 

IgG1. The Fc region of the murine IgG1 was mutated and therefore binding to Fc 

receptors is inhibited. In the heavy chain we inserted the Ovalbumin antigen (and for 

some experiments Hen egg lysozyme) as model antigen of choice. The light and 

Ovalbumin carrying heavy chains of DEC205 and 33D1 antibodies were transiently 

transfected into 293T cells and recombinant antibody containing supernatants were 

enriched with protein G. We found that the recombinant antibodies DEC205-Ova and 

33D1/DCIR2-Ova were binding to the DC subsets CD11c + CD8 + or CD11c + CD8 - , 

respectively, in vivo and in vitro, were internalized and thus were therefore ready for 

antigen targeting experiments (Dudziak et al., 2007). Currently we are producing new 

antibodies for future targeting of other DC subpopulations in vivo. 

Differential Antigen processing and presentation by Dendritic cell 

subpopulations 

Diana Dudziak, Anna Baranska, Gordon Heidkamp, Kirsten Ehrenspeck 

To analyze if DC subpopulations can be targeted with our recombinant Ovalbumin 

carrying antibodies we injected C57BL/6 mice with 33D1-Ova and DEC205-Ova 

antibodies to analyze how the different DC subpopulations uptake, process and 

present the antigens to T cells. In our experiments we are using transgenic mice that 

contain T cells that express either a T cell receptor that recognizes Ovalbumin 

peptide when it is presented as peptide MHCI complex or transgenic mice whose T 

cell receptor recognizes Ovalbumin antigen when it is presented as peptide MHCII 

complex on the surface of antigen presenting DCs. Therefore, both cytotoxic and T 

helper cell responses can be analyzed in vivo. First, we performed in vivo antigen 

targeting and in vitro T cell stimulation experiments with transgenic Ovalbumin 

specific T cells. After antigen targeting of only 10μg DEC205-Ova, or 33D1/DCIR2- 

Ova (normally, uncoupled Ovalbumin-protein needs to be injected as 1000-5000 

μg/mouse to receive T cell responses) we found, that delivering Ovalbumin antigens 

with DEC205-Ova to CD11c + CD8 + DCs induces a strong CD8 T cell response and 

antigen targeting with 33D1/DCIR2-Ova to CD11c + CD8 - DCs induced a more 

prominent CD4 T cell response in vitro. 

Further, we analyzed how T cells respond to antigen loaded DC subpopulations in 

vivo. Within 3 days we found that CD4 or CD8 transgenic T cells showed a 

proliferative response after interaction with antigen loaded DCs in vivo. We found that 

with less than 30 ng of the recombinant antibodies we were able to induce a CD8 T 

cell response when DEC205-Ova antibodies were targeted to CD11c + CD8 + DCs, 

whereas when we targeted 33D1-Ova to CD11c + CD8 - DCs in vivo we were able to 

induce a strong CD4 T cell response in vivo. By microarray analysis and the use of 

transgenic mice that express human DEC205 on all DC subpopulations in vivo we 

were able to demonstrate that the DC subpopulations were different in antigen 

processing and presentation and that the differences were not due to the receptors 

we have had targeted in vivo. Thus, we can conclude that CD11c + CD8 + DCs excel in 

antigen presentation as peptide MHCI complexes whereas CD11c + CD8 - DCs excel in 

antigen presentation on MHCII in the steady state. 

95

Targeting antigens under immunizing conditions induced a strong prolonged 

proliferative response in either CD4 transgenic T cells (targeting CD11c + CD8 - with 

33D1-Ova) or CD8 transgenic T cells (targeting of CD11c + CD8 + DCs with DEC205- 

Ova). On the other hand when we targeted antigens under tolerizing conditions 

transgenic T cells started to proliferate till day 3, but were deleted afterwards. Left T 

cells clearly were of regulatory T cell phenotype (Yamazaki et al., 2008). We further 

could show by 2-Photon microscopy that T cells that were interacting with DCs that 

presented the antigen under tolerizing conditions or presented the antigen under 

immunizing conditions were behaving very similar in dependency on interaction time, 

velocity and activation markers expressed on the T cells (Lindquist et al., 2004; 

Shakhar et al., 2005). Regarding the induced immune responses we induced under 

immunizing conditions with anti CD40 antibody, as gold standard activation stimulus, 

we found that targeting antigens to CD11c + CD8 - DCs predominantly induced a TH2 T 

cell response when we analyzed Balb/c mice but on the other hand a TH1 T cell 

response when CD11c + CD8 + DCs were targeted (Soares et al., 2007). 

Altogether, our data indicate that targeting of CD11c + CD8 + DCs by anti- 

DEC205 antibody seems to be an efficient way to induce cross presentation in the 

classical MHCI pathway. In contrast, CD11c + CD8 - DCs preferentially processed and 

presented antigens in the traditional MHCII pathway. Currentlc we are investigating 

the antigen processing machineries when DCs were activated with TLR ligands. 

Analysis of early T cell tolerance and T cell immunity 

Diana Dudziak, Kirsten Neubert 

We and others could show that antigen targeted DCs elicited a peripheral tolerance 

in antigen-specific T cells under non-inflammatory conditions. In contrast, delivery of 

recombinant antigen-carrying antibodies together with a stimulatory anti CD40 

antibody established an antigen-specific immune response. Because of severe side 

effects such as systemic inflammation and the non-specific activation of all immune 

cells that express CD40 on their cell surface the usage of systemic anti CD40 

antibody cannot be a feasible costimulatory strategy for human therapy. 

As TLR stimulation is an approach already used in clinical trials we believe that TLR 

stimulation in combination with our antibody targeting strategy might be an alternative 

approach to induce protective immune responses in vivo. TLRs are very well 

characterized class of pattern-recognition-receptors (PRRs) in mammalian species. 

The TLRs are differentially expressed on various cells of the immune system 

including DCs. TLRs 1, 2, 4, 5, and 6 are expressed on the cell surface and seem to 

have evolved to recognize bacterial products. In contrast TLRs 3, 7, 8, and 9 are 

expressed intracellularly in endosomes and lysosomes and recognize nucleic acids. 

Several pathogen-associated molecules such as double stranded RNA (poly(I:C) = 

pIC), bacterial or viral double stranded DNA (CpG), bacterial lipopolysaccharides 

(LPS), lipoproteins (Pam3Cys = Pam3), lipoteichoide acids, zymosan, or flagellin 

have been identified to bind to TLR receptors resulting in a strong inflammatory and 

protective immune response. 

96

To determine if TLR agonists are able to support the initiation of an immune response 

in combination of in vivo antigen targeting of C-type lectin receptors we are analyzing 

the early T cell activation and proliferation after interaction of the T cells with antigen 

loaded DC subsets in vivo to identify 1) if T cells can respond to TLR ligands similar 

to anti CD40 stimulating antibody, 2) to analyze different pathways of TLR stimulation 

(MyD88 dependent versus MyD88 independent), 3) to identify which kind of T cell 

subset emerged after antigen targeting and TLR stimulation, and 4) to analyze what 

type of antibody response can be induced. 

To investigate if T cells can respond to antigen loaded and TLR ligand activated DCs 

in vivo, we perform T cell transfer experiments in antibody targeted mice. To target 

DC subsets in vivo we use the recombinant chimeric antibody DEC205, which 

recognizes predominantly CD11c + CD8 + splenic DCs and the 33D1 antibody which 

binds uniquely to the molecule DCIR2 on CD11c + CD8 - splenic DCs. For the 

phenotypical and functional analysis of the emerged T cells it is planned to reisolate 

CD4 + or CD8 + transgenic T cells after activation with TLR-ligands and targeting 

antibody in vivo. By CBA-cytokine bead array, FACS-analysis, Western blots and 

microarray analyses we will be able to identify important transcription factors, 

cytokines, and genes involved in establishing immunity or tolerance in T cells. With 

these data we will be able to identify which stimulus needs to be given to induce a 

strong and long lasting immune response in vivo. 

Analysis of cell migration in steady state und immunity in lymphoid 

organs 

Anna Baranska; Christina Weiss 

In this project we focus on the role of DC migration for the induction of efficient and 

life long memory T cell responses. Therefore, we inject mice with different TLR 

ligands or anti CD40 antibody to analyze the expression of activation markers by 

FACS-analysis, DC migration by confocal microscopy, and the produced cytokines by 

CBA-assay at different time points after activation. We found that the C-type lectin 

receptor DCIR2 was not influenced by the DC activation, whereas DEC205 was up 

regulated by diverse TLR ligands. In addition, we found that the upregulation of 

MHCII and the activation markers CD80 and CD86 were different dependent on the 

TLR stimulus as well as the DC subpopulation (Baranska et al., in preparation). We 

further established a 6 color confocal microscopy analysis to identify where the 

different DC subsets localize after activation. Our next steps will be the simultaneous 

targeting of antigens to either CD11c + CD8 - or CD11c + CD8 + DCs under stimulating or 

tolerizing conditions to analyze when and were DCs interact with T cells in vivo. 

These data will help to understand the important role of DCs in initiation of an 

immune response. 

New molecules for Antigen targeting in vivo 

Targeting inhibitory and activating Fc-receptors in vivo 

Christian Lehmann 

Fc-receptors are widely accepted as key mediators of specific, antibody-dependent 

reactions of the immune system against pathogens (Biburger et al., 2011, 

97

Baerenwald et al., 2011; Nimmerjahn et al., 2010). In the last couple of years it 

became clear, that antibodies can also down-regulate immune-responses by acting 

on inhibitory Fc-receptors like Fc-gamma-RIIb (which contains an inhibitory signaling 

motif, ITIM). The Fc-gamma-RIIB is an Fc-receptor expressed on B cells, and 

therefore gives a negative feed-back to the antibody producing B cells. On the other 

hand all other Fc-receptors e.g. Fc-gamma RIV (which interacts with the common 

gamma chain and therefore induces an activating signal, ITAM), activate the cells 

after binding antibodies. As we are interested to understand how the immune system 

maintains peripheral tolerance and induces immunity, we would like to study the role 

of intrinsic signaling properties of the targeted receptors. As Fc receptors have a 

strong endocytosis capacity, and as they are expressed on a variety of antigen 

presenting cells, Fc receptors seem to be ideal molecules for targeting antigens in 

vivo. Targeting these molecules can model reactions to antigens bound by 

antibodies. In our studies we concentrate on the activating Fc-gamma-RIV and the 

inhibitory Fc-gamma-RIIb. We already know from literature that monocytes, but not B 

cells express Fc-gamma-RIV, whereas Fc-gamma-RIIb is expressed on both cell 

types. 

For addressing these questions we have cloned the variable regions of the antibodies 

directed against the two mentioned Fc-gamma receptors as mouse IgG1 isotype. 

Since the constant region is mutated these antibodies are only able to bind the Fc 

receptors via their variable regions and not via their Fc part. In addition, we 

genetically fused the model antigen Ovalbumin to the heavy chains, which has been 

widely used for investigating specific CD4 and CD8 T cell responses in vivo (Dudziak 

et al., 2007, Soares et al., 2007; Do et al., 2010, O’Kamphorst et al., 2010). We are 

currently producing these antibodies in HEK293T cells by transient transfection. 

Afterwards they will be used to compare T cell responses after targeting different 

cells (e.g. CD11c + CD8 + DCs, CD11c + CD8 - DCs, B cells, monocytes) and receptors for 

the induction of CD4 and CD8 T cells responses. In future, we are planning to use 

our antibodies against the inhibitory Fc-gamma-RIIb to protect mice prone to diabetes 

from the disease. 

Functional characterization of human Dendritic cell 


Characterization of Dendritic cell subpopulations in human tissues 

Gordon Heidkamp; Nathalie Eissing, Lukas Heger; Simone Beck, Yang Jiao 

As everything we know about immune responses is restricted to the murine system, 

we are investigating the function of DC subpopulations in human lymphoid organs. In 

order to obtain single cell suspensions, a method to efficiently process human splenic 

tissue needed to be established (Heidkamp et al., manuscript in preparation). 

Extensive multi-colour FACS analysis was used to identify Dendritic cell 

subpopulations. Here, Dendritic cells in the human spleen, tonsils, thymus, bone 

marrow, blood and cord blood was analyzed according to the already described 

expression profiles of blood DCs. So far, two major human blood DC lineages are 

defined: myeloid DCs and plasmacytoid DCs. The latter is characterized by the coexpression 

of BDCA-2, BDCA-4, CD123 and HLA-DR, whereas myeloid DCs can be 

further subdivided into mDC type 1 (BDCA-1 + , CD14 + , CD11c hi ) and mDC type 2 

98

(BDCA-3 + , CD14 + , CD11c lo ). These subpopulations have now been characterized for 

the expression profil of their cell surface receptors, by their RNA-expression profile in 

genearray analyses and by the localization of the DC subpopulations in the tissue on 

the tissues mentioned above with the focus on antigen targeting receptors and 

antigen processing machinery in the human DC subpopulations. 

Antigen targeting of human DCs with anti C-type lectin antibodies 

Gordon Heidkamp, Nathalie Eissing, Kirsten Ehrenspeck, 

As 33D1 antibody targeting induced a strong CD4 T cell response in mice and 

antigen targeting by DEC205 antibody induced a strong CD8 T cell response in vivo 

we wanted to investigate the role of the human counterpart of DCIR2 (33D1-antigen) 

and DEC205 in peripheral blood and human organs. As the human DEC205 antibody 

already existed, we concentrated on the production of an antibody against human 

DCIR. ClecSF6/DCIR/LLIR is the counterpart of the murine DCIR2. The antibodies 

were first tested by ELISA, FACS, immunofluorescence, Western blot, and 

immunoprecipitations if they bind specifically to the C-type lectin hDCIR (Heidkamp et 

al., 2010). Later we have chosen the clone 15E12 which showed the best binding 

specificities. We cloned the variable regions of the antibody and produced 

recombinant hDCIR antibodies in the tissue culture. In addition, our recombinant 

antibodies contained the Hemagglutinin antigen of Influenza virus as model antigen. 

With the recombinant HA-conjugated hDCIR-HA and hDEC205-HA antibodies we 

were able to target human monocyte derived DCs in the tissue culture and to induce 

a restimulation of Influenza-specific CD4 and CD8 T cells (Heidkamp et al., 

manuscript submitted). New antibodies are currently produced against newly 

identified endocytic receptors specifically expressed in human tissue DCs for future 

antigen targeting as therapeutic option. 

2011/2012 



Heidkamp GF, Eissing N, Heger L, Schmitt V, Neubert K, Baranska A, Bozzacco F, 

Trumpfheller C, Zebroski H, Schuler G, Nimmerjahn F, Nussenzweig MC, , Dudziak 

D. Differential and directed antigen presentation after targeting of DEC205 and DCIR 

on human dendritic cells. manuscript submitted 

Biburger M, Albert H, Woigk M, Dudziak D, Mack M, Ravetch JV, Nimmerjahn F. 

Monocyte subsets involved in immunoglobulin G mediated effector functions in vivo. 

Immunity. 2011 Dec 23;35(6):932-44 

Loschko J, Heink S, Hackl D, Dudziak D, Reindl W, Korn T, Krug AB. Antigen 

Targeting to Plasmacytoid Dendritic Cells via Siglec-H Inhibits Th Cell-Dependent 

Autoimmunity. J Immunol. 2011 Dec 15;187(12):6346-56. 

Baerenwaldt A, Lux A, Danzer H, Spriewald BM, Ullrich E, Heidkamp G, Dudziak D, 

Nimmerjahn F. Fcγ receptor IIB (FcγRIIB) maintains humoral tolerance in the human 

immune system in vivo. Proc Natl Acad Sci U S A. 2011 Nov 15;108(46):18772- 

18777. 

99

Loschko J, Hackl D, Dudziak D, Reindl W, Krug A. Tuning T cell responses by 

antigen targeting to plasmacytoid dendritic cells in vivo. J Immunol. 2011 Jun 

15;186(12):6718-25 

2010 

Nimmerjahn F, Lux A, Albert H, Woigk M, Lehmann C, Dudziak D, Smith P, Ravetch 

JV. FcγRIV deletion reveals its central role in mediating IgG2a and IgG2b activity in 

vivo. Proc Natl Acad Sci U S A. 2010 Nov 9;107(45):19396-401. 

Kamphorst, AO, Guermonprez P, Dudziak D, Nussenzweig MC. Route of antigen 

uptake differentially impacts presentation by dendritic cells and activated monocytes. 

J Immunol. 2010 Sep 15;185(6):3426-35. Epub 2010 Aug 20. 

Do Y, Koh H, Park CG, Dudziak D, Seo P, Mehandru S, Choi JH, Cheong C, Park S, 

Perlin DS, Powell BS, Steinman RM. Targeting of LcrV virulence protein from 

Yersinia pestis to dendritic cells protects mice against pneumonic plague. Eur J 

Immunol. 2010 Oct;40(10):2791-6. 

Heidkamp GF, Neubert K, Haertel E, Nimmerjahn F, Nussenzweig MC, Dudziak D*. 

Efficient generation of a monoclonal antibody against the C-type Lectin receptor 

DCIR by targeting murine dendritic cells. Immunol Lett. 2010 Aug 16;132(1-2):69-78. 

Epub 2010 Jun 8. 

2009-2011/2012 

Review Articles 

Ullrich E, Bosch J, Aigner M, Voelkl S, Kroeger I, Hoffmann P, Kreutz M, Dudziak D, 

Gerbitz A. Advances in cellular therapy: 6th international symposium on the clinical 

use of cellular products, March 24 and 25, 2011, Erlangen, Germany. Cancer 

Immunol Immunother. 2011 Dec 27. 

Ullrich E, Bosch J, Aigner M, Völkl S, Dudziak D, Spriewald B, Schuler G, Andreesen 

R, Mackensen R. Advances in Cellular Therapy: 5th International Symposium on the 

Clinical Use of Cellular Products, March 19 and 20, 2009, Nürnberg, Germany. 

Cancer Immunology Immunotherapy. 2010. 

Dudziak D, Dendritic cells as Master regulators of T cell responses. BioForum 

Europe. 2009. 

100


Doctoral theses (Biology, Dr. rer. nat.) 

Gordon Heidkamp (Dipl. Biol.) 

Characterization of human Dendritic cell subpopulations 

Anna Baranska (Dipl. Biol.) 

Antigen targeting and antigen processing in maturing Dendritic cell subpopulations 

Christian Lehmann (Dipl. Chem.) 

Antigen targeting to inhibitory and activating Fc-receptors in vivo 

Doctoral theses (Molecular Medicine, Dr. rer. nat.) 

Lukas Heger 

Chemotactic characterization of human DC subpopulations in lymphoid and nonlymphoid 

tissues 

Doctoral theses (Medicine, Dr. med.) 

Yang Jiao 

Characterization of pDC subpopulations in human tissues 

Doctoral theses (Veterinarian Medicine, Dr. med. vet.) 

Nathalie Eissing (maiden name: Nathalie Bleny) 

Characterization of Dendritic cell subpopulations and their expression of C-type lectin 

receptors in human tissues by immunofluorescence microscopy 

Kirsten Ehrenspeck 

Characterization of stem cells in human tissues and their differentiation into Dendritic 

cells 

101

Experimental Immunology and Immunotherapy 

(Medical Department III) until March 2010 

Head: Falk Nimmerjahn, Dr. rer. nat. 

Associate Professor of Experimental Immunology and 

Immunotherapy (until March 2010) 

Since April 2010: 

Professor and Chairman of the Institute of Genetics at 

the Department of Biology 

Address: Chair of Genetics 

Erwin-Rommelstr. 5 


Telefone: + 49 (9131) 85 25050 

Fax: + 49 (9131) 85 28526 

E-mail: fnimmerj@biologie.uni-erlangen.de 

Markus Biburger, Dr. rer. nat. 

Anne Bärenwaldt (PhD 

received in 2011) 

Susanne Aschermann 

(PhD received in 2012) 

Head 

Falk Nimmerjahn, Prof. Dr. rer. nat. 

Professor of Genetics 


Doctoral Students 

Technicians 

102 

Anja Lux, Dr. rer. nat. 

Inessa Schwab 

Birgit Lehmann 

Sybille Böhm


Heike Albert Melissa Woigk 

Heike Danzer 

The research of our laboratory focuses on understanding the mechanisms that underlie the 

different activities of immunoglobulin G (IgG) antibodies in vivo. IgG antibodies are the 

primary mediators of protective humoral immunity against pathogens and have been used 

therapeutically for over a century. They were first used as antitoxins for the treatment of 

infectious diseases in the pre-antibiotic era. Today, hyperimmune sera from human donors 

recovering from infection with specific viruses, such as hepatitis B, cytomegalovirus, and 

varicella zoster, are used to provide protective immunity to susceptible populations. 

Moreover, tumor specific antibodies have been successfully used in human cancer therapy 

and have been included in standard therapeutic regimens in lymphoma and breast cancer. 

Besides these protective activities, IgG autoantibodies are the principal mediators of 

autoimmune diseases such as immune thrombocytopenia (ITP), autoimmune haemolytic 

anemia (AHA), and systemic lupus erythematosus (SLE). In addition to this pro-inflammatory 

activity antibodies also are known to have an anti-inflammatory activity. If infused at high 

doses, IgG can effectively suppress autoimmune mediated inflammation (IVIG therapy). 

Recent evidence suggests that both the pro- and anti-inflammatory activity of IgG is 

regulated by the sugar side chain that is attached to the CH2-domain of all IgG subclasses. 

Subtle variations in the composition of this sugar moiety will either enhance or decrease the 

pro-inflammatory activity. Using a variety of in vivo model systems of autoimmune diseases 

and syngeneic tumor models we are trying to delineate which effector mechanisms the 

different IgG subclasses depend on and which effector cell populations are involved in this 

process. 

Focus I: Effector cells responsible for IgG activity (Biburger, 

Aschermann, Lehmann, Schwab) 

Immunoglobulins (Ig) are important mediators of the immunological defence against 

pathogens, however, autoantigen-specific antibodies may elicit autoimmunediseases. 

Due to these double-edged effects, immunoglobulin-mediated responses 

are strictly regulated. This is achieved by the existence of both activating and 

inhibitory receptors recognizing these antibodies (Fc receptors). Such Fc receptors 

are present in different combinations on the cell surface of various cell types and are 

characterized by different affinities for the respective IgG subclasses. In spite of the 

physiological importance of the interactions between immunoglobulins and their 

103

activating or inhibitory Fc receptors, there are still many unresolved questions. In this 

project we aim to understand how antibody-dependent cell-mediated cytotoxicity 

(ADCC) reactions work in vivo using a model of autoantibody mediated 

thrombocytopenia (ITP). During the last funding period we were able to show that a 

certain activating Fc-receptor, FcγRIV, which is crucial for autoantibody mediated 

platelet depletion is selectively expressed on tissue macrophages, neutrophils and on 

resident monocytes. By using a variety of cell depletion strategies we could 

demonstrate that neither neutrophils nor macrophages but resident monocytes were 

responsible for IgG dependent platelet removal (Figure 1). 

Figure 1: (A) Quantification of cell numbers of Ly6C hi and Ly6C lo monocytes in mice that were either 

left untreated, injected with PBS or with 10μl of clodronate liposomes to deplete the Ly6C lo monocyte 

subset. (B) Shown are liver sections stained with an F4/80 antibody specific for liver resident 

macrophages two hours after injection of 10µl clodronate liposomes or PBS. (C) Blood neutrophil 

counts in mice left either untreated or injected with PBS or 10µl of clodronate liposomes two hours 

after injection. (D) Platelet depletion activity of the 6A6-IgG2a antibody in mice either left untreated or 

pretreated with PBS or clodronate liposomes 2 hours before antibody injection. (E) Platelet depleting 

activity of the 6A6-IgG2a antibody in the indicated mouse strains twenty four hours after pretreatment 

with diphtheria toxin (+DT) or PBS (-DT). (F) Residual platelet count in C57BL/6 and Fcgr1 -/- Fcgr4 -/- 

mice four hours after injection of the 6A6-IgG2a anti-platelet antibody. * indicates p< 0.05. 

104

In addition to these passive models of autoimmunity we are also investigating more 

complex spontaneous mouse models of chronic autoimmune diseases. Specifically 

we are trying to elucidate which parts of the immune system contribute to the severe 

systemic autoimmune disease in Scurfy mice, which have a mutation in the 

transcription factor Foxp3, the master regulator of CD4+ CD25+ regulatory T cell 

development and function. As a member of the forkhead/winged-helix family of 

transcriptional regulators Foxp3 is essential for normal immune homeostasis. Mice 

hemizygous for the X-linked (Xsf/Y) mutation (scurfy mice) suffer from a fatal 

lymphoproliferative disorder caused by an impaired development of regulatory T cells 

(CD4+ CD25+ Foxp3+). Mice develop multi-organ lymphocytic infiltrates, resulting in 

lethal autoimmunity 3-6 weeks after birth. In our studies during the last years, we 

could identify autoantibody specificities associated with arthritis as well as murine 

lupus, which is the prototype of immune complex (IC)-mediated autoimmune disease. 

In addition, renal function was severely impaired. Interestingly, depletion of B cells 

either with a monoclonal antibody or by crossing Scurfy mice to B cell deficient 

mouse strains resulted in a prolonged survival of mice, suggesting that B cells and 

the respective autoantibodies play a critical role in the systemic inflammatory 

response. 

Focus II: Humanized mouse models to understand human IgG 

activity and pathways of humoral tolerance (Lux, Bärenwaldt, 

Böhm) 

Mice and humans differ in several aspects of IgG and FcγR biology. Therefore, one 

aim of our group is to establish in vivo models that allow studying the mechanism of 

human IgG activity and checkpoints of humoral tolerance in the setting of a human 

immune system in vivo. Humanized mice are generated by reconstitution of 

immunodeficient, irradiated mice (e.g. NOD/SCID/gamma-chain -/- ) with human CD34 + 

hematopoetic stem cells (HSC) purified from umbilical cord blood. Following 

reconstitution, the development of the human immune system is confirmed by flow 

cytometry and immunofluorescence staining. Human immune cells such as B, T, NK 

and dendritic cells are present in peripheral blood, spleen and bone marrow and 

human haematopoetic stem cells are able to persist in the bone marrow for months 

after reconstitution ensuring an ongoing human hematopoesis. As a model for human 

IgG activity we study B cell depletion in the peripheral blood, spleen, lymph nodes 

and bone marrow by IgG subclass variants of the CD20 specific antibody rituximab. 

In the second major project we investigated how impaired FcγRIIB alleles associated 

with the development of SLE in humans are involved in the maintenance of humoral 

tolerance. For this, humanized mouse colonies were generated that carried either the 

functionally normal FcγRIIB-232I allele or the functionally impaired FcγRIIB-232T 

allele. By following the development of the human immune system in these mouse 

cohorts we were able to show that mice carrying a human immune system with 

impaired FcγRIIB function showed a more rapid production of serum immunoglobulin 

105

M and G levels. This was mirrored by a higher number of late B cell developmental 

stages including memory B cells and plasma cells, consistent with previous data 

obtained in classical mouse models. More importantly, however, we could also show 

that human B cells in mice with impaired human FcγRIIB function started to produce 

autoantibodies (Figure 2). These findings establish an important function of human 

FcγRIIB as a late checkpoint in the human humoral immune system, preventing the 

expansion of autoantibody producing B cells. 

Figure 2: Development of autoantibodies in humanized mice. (A-B) The levels of serum IgM and 

serum IgG were analyzed in humanized mice carrying the different FcγRIIB allelic variants at 4 and 6 

months of age. (C-F) Detection of IgM autoantibody responses to double stranded DNA (anti-DNA), 

glucose 6-phosphate-isomerase (anti-GPI), rheumatoid factor (RF) and cyclic citrullinated peptides 

(anti-CCP) in humanized mice at 16 and 24 weeks of age. The level of autoantibody production is 

shown as arbitrary units (AU) for anti-DNA, -GPI, and –RF responses and as optical density (OD) for 

anti-CCP responses. Statistical significance was calculated with the Mann-Whitney U test. *P < 0.05. 

Data points of mice that received human hematopoetic stem cells from the same donor are depicted in 

the same color. 

106



2012 

Biburger, M., and Nimmerjahn, F. (2012). Low level of FcgammaRIII expression on 

murine natural killer cells. Immunol Lett 143, 53-59. 

Bohm, S., Schwab, I., Lux, A., and Nimmerjahn, F. (2012). The role of sialic acid as a 

modulator of the anti-inflammatory activity of IgG. Semin Immunopathol. 

Harre, U., Georgess, D., Bang, H., Bozec, A., Axmann, R., Ossipova, E., Jakobsson, 

P.J., Baum, W., Nimmerjahn, F., Szarka, E., et al. (2012). Induction of 

osteoclastogenesis and bone loss by human autoantibodies against citrullinated 

vimentin. J Clin Invest 122, 1791-1802. 

Kasperkiewicz, M., Nimmerjahn, F., Wende, S., Hirose, M., Iwata, H., Jonkman, M.F., 

Samavedam, U., Gupta, Y., Moller, S., Rentz, E., et al. (2012). Genetic identification 

and functional validation of FcgammaRIV as key molecule in autoantibody-induced 

tissue injury. J Pathol. 

Lehmann, B., Schwab, I., Bohm, S., Lux, A., Biburger, M., and Nimmerjahn, F. 

(2012). FcgammaRIIB: a modulator of cell activation and humoral tolerance. Expert 

Rev Clin Immunol 8, 243-254. 

Moldt, B., Shibata-Koyama, M., Rakasz, E.G., Schultz, N., Kanda, Y., Dunlop, D.C., 

Finstad, S.L., Jin, C., Landucci, G., Alpert, M.D., et al. (2012). A Nonfucosylated 

Variant of the anti-HIV-1 Monoclonal Antibody b12 Has Enhanced FcgammaRIIIa- 

Mediated Antiviral Activity In Vitro but Does Not Improve Protection against Mucosal 

SHIV Challenge in Macaques. J Virol 86, 6189-6196. 

Ruiz-Heiland, G., Horn, A., Zerr, P., Hofstetter, W., Baum, W., Stock, M., Distler, J.H., 

Nimmerjahn, F., Schett, G., and Zwerina, J. (2012). Blockade of the hedgehog 

pathway inhibits osteophyte formation in arthritis. Ann Rheum Dis 71, 400-407. 

Schwab, I., Biburger, M., Kronke, G., Schett, G., and Nimmerjahn, F. (2012). IVIgmediated 

amelioration of ITP in mice is dependent on sialic acid and SIGNR1. Eur J 

Immunol 42, 826-830. 

Uderhardt, S., Herrmann, M., Oskolkova, O.V., Aschermann, S., Bicker, W., Ipseiz, 

N., Sarter, K., Frey, B., Rothe, T., Voll, R., et al. (2012). 12/15-lipoxygenase 

107

orchestrates the clearance of apoptotic cells and maintains immunologic tolerance. 

Immunity 36, 834-846. 

2011 

Baerenwaldt, A., Lux, A., Danzer, H., Spriewald, B.M., Ullrich, E., Heidkamp, G., 

Dudziak, D., and Nimmerjahn, F. (2011). Fcgamma receptor IIB (FcgammaRIIB) 

maintains humoral tolerance in the human immune system in vivo. Proc Natl Acad 

Sci U S A 108, 18772-18777. 

Biburger, M., Aschermann, S., Schwab, I., Lux, A., Albert, H., Danzer, H., Woigk, M., 

Dudziak, D., and Nimmerjahn, F. (2011). Monocyte subsets responsible for 

immunoglobulin G-dependent effector functions in vivo. Immunity 35, 932-944. 

Lux, A., and Nimmerjahn, F. (2011). Impact of differential glycosylation on IgG 

activity. Adv Exp Med Biol 780, 113-124. 

Nimmerjahn, F., and Lunemann, J.D. (2011). Expression and function of the 

inhibitory Fcgamma-receptor in CIDP. J Peripher Nerv Syst 16 Suppl 1, 41-44. 

Nimmerjahn, F., and Ravetch, J.V. (2011). FcgammaRs in health and disease. Curr 

Top Microbiol Immunol 350, 105-125. 

Santiago-Raber, M.L., Baudino, L., Alvarez, M., van Rooijen, N., Nimmerjahn, F., and 

Izui, S. (2011). TLR7/9-mediated monocytosis and maturation of Gr-1(hi) 

inflammatory monocytes towards Gr-1(lo) resting monocytes implicated in murine 

lupus. J Autoimmun 37, 171-179. 

2010 

Anthony, R.M., and Nimmerjahn, F. (2010). The role of differential IgG glycosylation 

in the interaction of antibodies with FcgammaRs in vivo. Curr Opin Organ Transplant. 

Aschermann, S., Lux, A., Baerenwaldt, A., Biburger, M., and Nimmerjahn, F. (2010). 

The other side of immunoglobulin G: suppressor of inflammation. Clin Exp Immunol 

160, 161-167. 

Baerenwaldt, A., Biburger, M., and Nimmerjahn, F. (2010). Mechanisms of action of 

intravenous immunoglobulins. Expert Rev Clin Immunol 6, 425-434. 

Comabella, M., Montalban, X., Kakalacheva, K., Osman, D., Nimmerjahn, F., Tintore, 

M., and Lunemann, J.D. (2010). B cell expression of the inhibitory Fc gamma 

receptor is unchanged in early MS. J Neuroimmunol 223, 135-137. 

Heidkamp, G.F., Neubert, K., Haertel, E., Nimmerjahn, F., Nussenzweig, M.C., and 

Dudziak, D. (2010). Efficient generation of a monoclonal antibody against the human 

108

C-type lectin receptor DCIR by targeting murine dendritic cells. Immunol Lett 132, 69- 

78. 

Lux, A., Aschermann, S., Biburger, M., and Nimmerjahn, F. (2010). The pro and antiinflammatory 

activities of immunoglobulin G. Ann Rheum Dis 69 Suppl 1, i92-96. 

Najjar, I., Deglesne, P.A., Schischmanoff, P.O., Fabre, E.E., Boisson-Dupuis, S., 

Nimmerjahn, F., Bornkamm, G.W., Dusanter-Fourt, I., and Fagard, R. (2010). 

STAT1-dependent IgG cell-surface expression in a human B cell line derived from a 

STAT1-deficient patient. J Leukoc Biol 87, 1145-1152. 

Nimmerjahn, F., Lux, A., Albert, H., Woigk, M., Lehmann, C., Dudziak, D., Smith, P., 

and Ravetch, J.V. (2010). FcgammaRIV deletion reveals its central role for IgG2a 

and IgG2b activity in vivo. Proc Natl Acad Sci U S A 107, 19396-19401. 

Nimmerjahn, F., and Ravetch, J.V. (2010). Antibody-mediated modulation of immune 

responses. Immunol Rev 236, 265-275. 

Ritter, M., Gross, O., Kays, S., Ruland, J., Nimmerjahn, F., Saijo, S., Tschopp, J., 

Layland, L.E., and Prazeres da Costa, C. (2010). Schistosoma mansoni triggers 

Dectin-2, which activates the Nlrp3 inflammasome and alters adaptive immune 

responses. Proc Natl Acad Sci U S A 107, 20459-20464. 

Schoenen, H., Bodendorfer, B., Hitchens, K., Manzanero, S., Werninghaus, K., 

Nimmerjahn, F., Agger, E.M., Stenger, S., Andersen, P., Ruland, J., et al. (2010). 

Cutting edge: Mincle is essential for recognition and adjuvanticity of the 

mycobacterial cord factor and its synthetic analog trehalose-dibehenate. J Immunol 

184, 2756-2760. 

Tackenberg, B., Nimmerjahn, F., and Lunemann, J.D. (2010). Mechanisms of IVIG 

efficacy in chronic inflammatory demyelinating polyneuropathy. J Clin Immunol 30 

Suppl 1, S65-69. 

Zaiss, M.M., Frey, B., Hess, A., Zwerina, J., Luther, J., Nimmerjahn, F., Engelke, K., 

Kollias, G., Hunig, T., Schett, G., et al. (2010). Regulatory T cells protect from local 

and systemic bone destruction in arthritis. J Immunol 184, 7238-7246. 

Zaiss, M.M., Sarter, K., Hess, A., Engelke, K., Bohm, C., Nimmerjahn, F., Voll, R., 

Schett, G., and David, J.P. (2010). Increased bone density and resistance to 

ovariectomy-induced bone loss in FoxP3-transgenic mice based on impaired 

osteoclast differentiation. Arthritis Rheum 62, 2328-2338. 

2009 

109

Grevers, L.C., van Lent, P.L., Koenders, M.I., Walgreen, B., Sloetjes, A.W., 

Nimmerjahn, F., Sjef Verbeek, J., and van den Berg, W.B. (2009). Different 

amplifying mechanisms of interleukin-17 and interferon-gamma in Fcgamma 

receptor-mediated cartilage destruction in murine immune complex-mediated 

arthritis. Arthritis Rheum 60, 396-407. 

Guo, S., Muhlfeld, A.S., Wietecha, T.A., Peutz-Kootstra, C.J., Kowalewska, J., Yi, K., 

Spencer, M., Pichaiwong, W., Nimmerjahn, F., Hudkins, K.L., et al. (2009). Deletion 

of activating Fcgamma receptors does not confer protection in murine 

cryoglobulinemia-associated membranoproliferative glomerulonephritis. Am J Pathol 

175, 107-118. 

Santiago-Raber, M.L., Amano, H., Amano, E., Baudino, L., Otani, M., Lin, Q., 

Nimmerjahn, F., Verbeek, J.S., Ravetch, J.V., Takasaki, Y., et al. (2009). Fcgamma 

receptor-dependent expansion of a hyperactive monocyte subset in lupus-prone 

mice. Arthritis Rheum 60, 2408-2417. 

Syed, S.N., Konrad, S., Wiege, K., Nieswandt, B., Nimmerjahn, F., Schmidt, R.E., 

and Gessner, J.E. (2009). Both FcgammaRIV and FcgammaRIII are essential 

receptors mediating type II and type III autoimmune responses via FcRgamma-LATdependent 

generation of C5a. Eur J Immunol 39, 3343-3356. 

Tackenberg, B., Jelcic, I., Baerenwaldt, A., Oertel, W.H., Sommer, N., Nimmerjahn, 

F., and Lunemann, J.D. (2009). Impaired inhibitory Fcgamma receptor IIB expression 

on B cells in chronic inflammatory demyelinating polyneuropathy. Proc Natl Acad Sci 

U S A 106, 4788-4792. 

Werninghaus, K., Babiak, A., Gross, O., Holscher, C., Dietrich, H., Agger, E.M., 

Mages, J., Mocsai, A., Schoenen, H., Finger, K., et al. (2009). Adjuvanticity of a 

synthetic cord factor analogue for subunit Mycobacterium tuberculosis vaccination 

requires FcRgamma-Syk-Card9-dependent innate immune activation. J Exp Med 

206, 89-97. 

Current Research Funding 

2007-2012 Bayerisches Genomforschungsnetzwerk 

2009-2012 DFG Forschergruppe 832: Regulatoren der humoralen Immunität 

2008-2012 SFB 643: Strategien zur zellulären Immunintervention 

2010-2013 SPP 1468: Immunobone 

110

2011-2015 GK1660: Schlüsselsignale der adaptiven Immunität 

2011-2013 Bill and Melinda Gates Foundation 


Doctoral theses 

Anja Lux (PhD in 2011) 

Anne Bärenwaldt (PhD in 2011) 

Susanne Aschermann (PhD in 2012) 

Inessa Schwab 

Birgit Lehmann 

Sybille Böhm 

111

Clinical Research Group 

(Reinhard Voll, Med.3) 

Head: Reinhard Voll, Dr. med. 

Professor of Rheumatology and Clinical Immunology 

(Since Dec. 2010 director of the Division Rheumatology 

and Clinical Immunology, University Medical Center 

Freiburg) 

Address: Clinical Research Group 

Glückstr. 6 


Telefone: + 49 (9131) 85 39303 

Fax: + 49 (9131) 85 39311 

E-mail: reinhard.voll@uniklinik-freiburg.de 

Head 

Reinhard Voll, Prof. Dr. med. 

Professor of Rheumatolgy and Clinical Immunology 

Vilma Urbonaviciute, Dr. rer. nat.* 

Silke Meister, Dr. rer. nat. * 




112 

Bettina Sehnert, Dr. rer. nat. *

Charlotte Starke 


Sandy Pohle 



Eva Gückel° 


Daniela Graef * 



Technicians 


The clinical research group is mainly interested in the etiology and pathogenesis of 

autoimmune diseases, especially systemic lupus erythematosus and rheumatoid 

arthritis. Based on the insights into the pathogenesis, new treatment strategies are 

going to be developed, initially in mouse models and, eventually, transferred to 

clinics. Targeting plasma cells for the treatment of autoantibody-mediated diseases 

and selective inhibition of the transcription factor NF-κB within activated endothelial 

cells represent such novel treatment strategies. 

113

From the immunopathogenesis of systemic lupus erythematosus to 

new treatment strategies 

Autoantibodies against nuclear antigens, especially double stranded (ds) DNA and 

nucleosomes represent a hallmark of SLE. However, the mechanisms involved in 

breaking the immunological tolerance against these normally poorly immunogenic 

nuclear components are not understood yet. We have shown previously that impaired 

phagocytosis of apoptotic cells with consecutive release of nuclear antigens may 

critically contribute to the immune pathogenesis of SLE. The architectural 

chromosomal protein and alarmin high mobility group box protein 1 (HMGB1) is 

tightly attached to the chromatin of apoptotic cells. Our results demonstrate that 

HMGB1 remains bound to nucleosomes released from late apoptotic cells in vitro. In 

addition, HMGB1-nucleosome complexes were also detected in the blood of SLE 

patients. Biochemically purified HMGB1-containing nucleosomes from apoptotic cells 

induced secretion of proinflammatory cytokines from monocytes/macrophages and 

maturation as well as activation of dendrtic cells. Immunizaton of non-autoimmune 

mice with nucleosomes from apoptotic cells, but not with those from living cells, 

induced autoantibodies to dsDNA. Further investigations are focused on the 

characterization of receptors for HMGB1-nucleosome complexes such as TLR2 and 

their roles in the pathogenesis of SLE. In addition, we are investigating if TLR2 and 

HMGB1 are suitable therapeutic targets in SLE. 

Refractory disease courses of SLE might be caused by long-lived plasma cells 

producing pathogenic autoantibodies. Hence, the elimination of long-lived plasma 

cells appears to represent an important treatment goal. However, long-lived plasma 

cells are resistant toward conventional therapies. We found that proteasome 

inhibitors can efficiently eliminate plasma cells including long-lived ones in murine 

SLE models. 

Recently, we described that long-lived plasma cells can be also found within the 

inflamed kidneys of diseased NZB/W F1 lupus mice. Interestingly, the frequency of 

autoreactive antibody-producing cells in the kidneys was markedly higher than in 

bone marrow and spleen (Starke et al., 2011). Currently we try to characterize these 

pathogenic plasma cells within kidneys and explore ways of their efficient elimination. 

Selective inhibition of NF-kB in activated endothelium as 

treatment strategy in immune-mediated diseases 

One research focus of our group is the investigation of the role of the transcription 

factor NF-κB in the pathogenesis of inflammatory diseases and the exploration of 

new strategies for therapeutic NF-κB inhibition. NF-κB represents a master switch of 

the inflammatory and immune responses. Most pro-inflammatory cytokines, tissue 

degrading enzymes, and several adhesion molecules are expressed in an NF-κB- 

114

dependent manner. Due to its central position in the inflammatory process 

interference with NF-κB activation represents a promising target for the treatment of 

inflammatory and autoimmune diseases. However, NF-κB plays also an important 

role in function and survival of several cell types, tissues, and organs. Therefore, we 

explore also the physiological function of NF-κB in lymphocyte development and the 

consequences of NF-κB inhibition for developing and mature lymphocytes. 

In collaboration with Stefan Dübel (Braunschweig), Harald Burkhardt (Frankfurt), 

Alexander Steinkasserer (Erlangen), Falk Nimmerjahn (Erlangen), Georg Schett 

(Erlangen) we developed a strategy for cell-type specific interference with NF-κB 

activation. Prototypic “sneaking ligand constructs” (SLC) were designed to target 

selectively activated endothelial cells via E selectin-binding peptides. The second 

domain of the fusion protein contains an endosomal release sequence, which 

translocates the IKK2 inhibiting NEMO-binding peptide into the cytoplasm. We could 

demonstrate that SLC1 efficiently ameliorates immune-mediated diseases such as 

murine arthritis models and experimental autoimmune encephalomyelitis (Sehnert et 

al. submitted). 

2011/12 



Hainz N, Thomas S, Neubert K, Meister S, Benz K, Rauh M, Daniel C, Wiesener M, Voll RE, 

Amann K. The Proteasome Inhibitor Bortezomib Prevents Lupus Nephritis in the NZB/W F1 

Mouse Model by Preservation of Glomerular and Tubulointerstitial Architecture. Nephron 

Exp Nephrol. 2012;120(2):e47-58. Epub 2012 Jan 26. 

Pullerits R, Urbonaviciute V, Voll RE, Forsblad-D'Elia H, Carlsten H. Serum Levels of 

HMGB1 in Postmenopausal Patients with Rheumatoid Arthritis: Associations with 

Proinflammatory Cytokines, Acute-phase Reactants, and Clinical Disease Characteristics. J 

Rheumatol. 2011 Jul;38(7):1523-5. 

Starke C, Frey S, Wellmann U, Urbonaviciute V,Herrmann M, Amann K, Schett G, Winkler , 

Voll RE High frequency of autoantibody-secreting cells and long-lived plasma cells within 

inflamed kidneys of NZB/W F1 lupus mice. Eur J 2011 Jul;41(7):2107-12 

Gückel E, Frey S, Zaiss MM, Schett G, Ghosh S, Voll RE. Cell-Intrinsic NF-kappaB Activation 

Is Critical for the Development of Natural Regulatory T Cells in Mice. PLoS One 

2011;6(5):e20003. Epub 2011 May 18 

Polzer K, Neubert K, Meister S, Frey B, Baum W, Distler JH, Gückel E, Schett G, Voll RE, 

Zwerina J.Proteasome inhibition aggravates tumor necrosis factor-mediated bone resorption 

in a mouse model of inflammatory arthritis.Arthritis Rheum. 2011 Mar;63(3):670-80. 

Gomez AM, Vrolix K, Martínez-Martínez P, Molenaar PC, Phernambucq M, van der Esch E, 

Duimel H, Verheyen F, Voll RE, Manz RA, De Baets MH, Losen M. Proteasome inhibition 

115

with bortezomib depletes plasma cells and autoantibodies in experimental autoimmune 

myasthenia gravis. J Immunol. 2011 Feb 15;186(4):2503-13. 

Mosca M, Govoni M, Tomietto P, Aringer M, Boumpas D, Cervera R, Conti F, D'Cruz D, 

Doria A, De La Fuente D, Galeazzi M, Houssiau F, Huizinga TW, Khamashta MA, Ines L, 

Duarte C, Couto M, Meroni P, Montecucco C, Norkuviene E, Riemekasten G, Rios V, 

Schneider M, Shoenfeld Y, Steup-Beekman GM, Szmyrka-Kaczmarek M, Tani C, Tincani A, 

Tzioufas AG, Voll R, Bencivelli W, Salaffi F, Bombardieri S. The development of a simple 

questionnaire to screen patients with SLE for the presence of neuropsychiatric symptoms in 

routine clinical practice. Lupus. 2011;20(5):485-92. 

2010 

Lang VR, Mielenz D, Neubert K, Böhm C, Schett G, Jäck HM, Voll RE, Meister S. The early 

marginal zone B cell-initiated T-independent type 2 response resists the proteasome inhibitor 

bortezomib. J Immunol. 2010 Nov 1;185(9):5637-47. 

Jellusova J, Düber S, Gückel E, Binder CJ, Weiss S, Voll R, Nitschke L.: Siglec-g regulates 

B1 cell survival and selection. J Immunol. 2010 Sep, 15;185(6):3277-84. 

Weber CK, Haslbeck M, Englbrecht M, Sehnert B, Mielenz D, Graef D, Distler JH, Mueller 

RB, Burkhardt H, Schett G, Voll RE, Fürnrohr BG. Antibodies to the endoplasmic reticulumresident 

chaperones calnexin, BiP and Grp94 in patients with rheumatoid arthritis and 

systemic lupus erythematosus. Rheumatology (Oxford). 2010 Aug 27. [Epub ahead of print] 

van Bavel CC, Dieker JW, Kroeze Y, Tamboer WP, Voll R, Muller S, Berden JH, van der 

Vlag J. Apoptosis-induced histone H3 methylation is targeted by autoantibodies in systemic 

lupus erythematosus. Ann Rheum Dis. 2010 Aug 10. [Epubahead of print] 

Meister S, Frey B, Lang VR, Gaipl US, Schett G, Schlötzer-Schrehardt U, Voll RE. Calcium 

channel blocker verapamil enhances endoplasmic reticulum stress and cell death induced by 

proteasome inhibition in myeloma cells. Neoplasia. 2010, Jul;12(7):550-61. 

Zaiss MM, Sarter K, Hess A, Engelke K, Böhm C, Nimmerjahn F, Voll R, Schett G, David JP. 

Increased bone density and resistance to ovariectomy-induced bone loss in FoxP3transgenic 

mice based on impaired osteoclast differentiation. Arthritis Rheum. 2010 

Aug;62(8):2328-38. 

Fürnrohr BG, Wach S, Kelly JA, Haslbeck M, Weber CK, Stach CM, Hueber AJ, Graef D, 

Spriewald BM, Manger K, Herrmann M, Kaufman KM, Frank SG, Goodmon E, James JA, 

Schett G, Winkler TH, Harley JB, Voll RE. Polymorphisms in the Hsp70 gene locus are 

genetically associated with systemic lupus erythematosus. Ann Rheum Dis. 2010 May 24. 

[Epub ahead of print] 

Vogelbacher R, Meister S, Gückel E, Starke C, Wittmann S, Stief A, Voll R, Daniel C, Hugo 

C. Bortezomib and sirolimus inhibit the chronic active antibody-mediated rejection in 

experimental renal transplantation in the rat. Nephrol Dial Transplant. 2010 Apr 28. [Epub 


Hakkim A, Fürnrohr BG, Amann K, Laube B, Abed UA, Brinkmann V, Herrmann M, Voll RE, 

Zychlinsky A. Impairment of neutrophil extracellular trap degradation is associated with lupus 

nephritis. Proc Natl Acad Sci U S A. 2010 May, 25;107(21):9813-8. Epub 2010 May 3. 

Kruse K, Janko C, Urbonaviciute V, Mierke CT, Winkler TH, Voll RE, Schett G, Muñoz LE, 

Herrmann M. Inefficient clearance of dying cells in patients with SLE: anti-dsDNA 

autoantibodies, MFG-E8, HMGB-1 and other players. Apoptosis. 2010, Sep;15(9):1098- 

113. 

116

Rödel F, Frey B, Capalbo G, Gaipl U, Keilholz L, Voll R, Hildebrandt G, Rödel, C. 

Discontinuous induction of X-linked inhibitor of apoptosis in EA.hy.926 endothelial cells is 

linked to NF-kappaB activation and mediates the anti-inflammatory properties of low-dose 

ionising-radiation. Radiother Oncol. 2010 Feb 17. [Epub ahead of print] 

2009 

Rödel F, Keilholz L, Herrmann M, Weiss C, Frey B, Voll R, Gaipl U, Rödel C. Activator 

protein 1 shows a biphasic induction and transcriptional activity after low dose X-irradiation in 

EA.hy.926 endothelial cells. Autoimmunity. 2009, May;42(4):343-5. 

Krönke G, Katzenbeisser J, Uderhardt S, Zaiss MM, Scholtysek C, Schabbauer G, Zarbock 

A, Koenders MI, Axmann R, Zwerina J, Baenckler HW, van den Berg W, Voll RE, Kühn H, 

Joosten LA, Schett G. 12/15-lipoxygenase counteracts inflammation and tissue damage in 

arthritis. J Immunol. 2009 Sep 1;183(5):3383-9. 

Rödel F, Keilholz L, Herrmann M, Weiss C, Frey B, Voll R, Gaipl U, Rödel C. Activator 

protein 1 shows a biphasic induction and transcriptional activity after low dose X-irradiation in 

EA.hy.926 endothelial cells. Autoimmunity. 2009 May;42(4):343-5. 

Urbonaviciute V, Meister S, Fürnrohr BG, Frey B, Gückel E, Schett G, Herrmann M, Voll RE. 

Oxidation of the alarmin high-mobility group box 1 protein (HMGB1) during apoptosis. 


Muñoz LE, Janko C, Grossmayer GE, Frey B, Voll RE, Kern P, Kalden JR, Schett G, Fietkau 

R, Herrmann M, Gaipl US. Remnants of secondarily necrotic cells fuel inflammation in 

systemic lupus erythematosus. Arthritis Rheum. 2009 Jun;60(6):1733-42. 

Raaz D, Herrmann M, Ekici AB, Klinghammer L, Lausen B, Voll RE, Leusen JH, van de 

Winkel JG, Daniel WG, Reis A, Garlichs CD. FcgammaRIIa genotype is associated with 

acute coronary syndromes as first manifestation of coronary artery disease. 

Atherosclerosis. 2009 Jan 21 [Epub ahead of print]. 


Urbonaviciute V, Voll RE: HMGB1 represents a potential marker of disease activity and novel 

therapeutic target in SLE. J Intern Med. 2011 Jul 27. doi: 10.1111/j.1365- 

2796.2011.02432.x. [Epub ahead of print] 

Feuchtenberger M, Voll RE, Kneitz C.: Impfungen in der Rheumatologie. Z Rheumatol. 2010 

Nov;69(9):803-812. 

Nikolova KA, Mihaylova NM, Voynova EN, Tchorbanov AI, Voll RE, Vassilev TL. Selective 

silencing of autoreactive B lymphocytes-Following the Nature's way. Autoimmun Rev. 2010 

Sep;9(11):775-9. Epub 2010 Jul 1. 

Weiss EM, Frey B, Rödel F, Herrmann M, Schlücker E, Voll RE, Fietkau R, Gaipl US.: Ex 

vivo- and in vivo-induced dead tumor cells as modulators of antitumor responses. Ann N Y 

Acad Sci. 2010 Oct;1209:109-17. 

Muñoz LE, Lauber K, Schiller M, Manfredi AA, Schett G, Voll RE, Herrmann M.[The role of 

incomplete clearance of apoptotic cells in the etiology and pathogenesis of SLE]. Z 

Rheumatol. 2010 Mar;69(2):152, 154-6. 

117

Voll R, Hiepe F. [Depletion of plasma cells - a novel strategy in the therapy of systemic lupus 

erythematosus in mice and man.]. Z Rheumatol. 2009, Mar;68(2):150-3. 



Eva Gückel 

Die Rolle des Transkriptionsfaktors NF-kB für die Entwicklung und Funktion natürlich 

vorkommender regulatorischer T-Zellen 

Charlotte Starkte 

Charakterisierung von Antikörper-sezernierenden Zellen in der entzündeten Niere im 

NZB/W-F1 Lupusmausmodell. 


Sandy Pohle 

Influence of dietary salt intake on the immune response and inflammatory diseases 

Doctoral theses (Medicine) 

Veronika Lang 

Resistenz der von Marginalzonen B- Zellen initiierten frühen Phase der T-Zellunabhängigen 

Immunantwort Typ 2 gegenüber dem Proteasominhibitor Bortezomib 

Ramona Peukert 

Proteasominhibition zur Therapie des SLE 

Christian Weber 

ER-Chaperone als Autoantigene bei der chronischen Polyarthritis 

118

DEPARTMENT OF ORTHOPAEDIC TRAUMA 

SURGERY 

(Molecular Cartilage Research) 

Head: Prof. Dr. med. F.F. Hennig 

(Head of Department of Orthopaedic Trauma Surgery) 

Priv.-Doz. Dr. med. K. Gelse 

(Leader of Research Group) 

Address: Department of Orthopaedic Trauma Surgery, Molecular 

Cartilage Research Group) 

Glückstr. 6 


Telefone: + 49 (9131) 85 42121 

Fax: + 49 (9131) 85 33300 

E-mail: kolja.gelse@uk-erlangen.de 

Patricia Klinger, Dr. rer. nat. 

Head 

Friedrich F. Hennig, Prof. Dr. med. 

Professor of Orthopaedic Trauma Surgery 

Leader of Research Group 

Kolja Gelse, Priv.-Doz. Dr. med. 


119

Matthias Koch 

Franziska Cipa 

Melanie Pflügner 



Simon Obier 

Technicians 

Our group analyses molecular mechanisms that control cartilage and bone biology 

focussing on the mechanisms of chondrogenesis and terminal chondrocyte 

differentiation. The investigations further include the role of hypoxia and epigenetic 

mechanisms on the differentiation of chondroprogenitor cells and mesenchymal stem 

cells. 

Role of HIF-1α for the chondrogenic phenotype 

The aim of this project was to investigate the chondrogenic potential of growth factorstimulated 

periosteal cells with respect to the activity of Hypoxia-inducible Factor 1a 

(HIF-1a). In this project, scaffold-bound autologous periosteal cells, which had been 

activated by Insulin-like Growth Factor 1 (IGF-1) or Bone Morphogenetic Protein 2 

(BMP-2) gene transfer using both adeno-associated virus (AAV) and adenoviral (Ad) 

vectors, were applied to chondral lesions in the knee joints of miniature pigs. Six 

weeks after transplantation, the repair tissues were investigated for collagen type I 

and type II content as well as for HIF-1a expression. The functional role of 

phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) and 

mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) 

signaling on BMP-2/IGF-1-induced HIF-1a expression was assessed in vitro by 

employing specific inhibitors. 

Unstimulated periosteal cells formed a fibrous extracellular matrix in the superficial 

zone and a fibrocartilaginous matrix in deep zones of the repair tissue. This zonal 

difference was reflected by the absence of HIF-1a staining in superficial areas, but 

moderate HIF-1a expression in deep zones. In contrast, Ad/AAVBMP-2-stimulated 

periosteal cells, and to a lesser degree Ad/AAVIGF-1-infected cells, adopted a 

120

chondrocyte-like phenotype with strong intracellular HIF-1a staining throughout all 

zones of the repair tissue and formed a hyaline-like matrix. In vitro, BMP-2 and IGF-1 

supplementation increased HIF-1a protein levels in periosteal cells, which was based 

on posttranscriptional mechanisms rather than de novo mRNA synthesis, involving 

predominantly the MEK/ERK pathway. The data of this project indicated that 

chondrogenesis by chondrogenic precursor cells is facilitated in deeper hypoxic 

zones of cartilage repair tissue and is stimulated by growth factors which enhance 

HIF-1a activity. 

Fig. 1. 

O2 

Toluidine 

blue 

Hypoxia 

HIF-1α 

Hypoxia facilitates 

chondrogenesis. 

Mesenchymal stem 

cells overexpressing 

BMP-2 were 

transplanted into 

cartilage defects in 

the knee joints of a 

minipig model. 

Chondrogenesis was 

facilitated in deep 

hypoxic tissue layers. 

The role of anti-angiogenic factors for stabilizing the 

chondrocyte phenotype 

This study investigated the effect of the anti-angiogenic factors Thrombospondin-1 

(TSP-1) and Chondromodulin-I (Chm-I) on the formation of cartilage repair tissue in 

combination with stimulation by osteogenic protein-1 (OP-1). In miniature pigs, 

articular cartilage lesions in the femoral trochlea were treated by the microfracture 

technique and either received no further treatment (MFX), or were treated by 

additional application of recombinant osteogenic protein-1 (MFX+OP1), recombinant 

TSP-1 (MFX+TSP1), or a combination of both proteins (MFX+TSP1+OP1). Since 

Chm-I is not available as a recombinant protein, we applied self-complementary 

Adeno-Associated-Virus (AAV) vectors carrying the Chm-I cDNA to achieve 

overexpression of Chm-I within the defects. Six and 26 weeks after surgery, the 

repair tissue and the degree of endochondral ossification were assessed by 

histochemical and immunohistochemical methods detecting collagen types I, II, X, 

TSP-1 and CD31. Microfracture treatment merely induced the formation of inferior 

fibrocartilaginous repair tissue. OP-1 stimulated chondrogenesis, but also induced 

chondrocyte hypertrophy, characterized by synthesis of collagen type X, and 

excessive bone formation. Application of TSP-1 inhibited inadvertant endochondral 

ossification, but failed to induce chondrogenesis. In contrast, the simultaneous 

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application of both TSP-1 and OP-1 induced and maintained a permanent, nonhypertrophic 

chondrocyte-like phenotype within cartilage repair tissue. Similar 

favourable results were observed following Chm-I gene transfer. Overexpression of 

Chm-I stabilized the chondrocyte phenotype within the repair tissue and prevented 

excessive endochondral ossification. Gene expression analyses in vitro revealed the 

cell cycle inhibitor p21 cip1/waf1 as one target upregulated by AAVChm-I. The data of 

this study demonstrate that treatment by a combination of OP-1 and TSP-1, as well 

as overexpression of Chm-I stabilizes the chondrocyte phenotype by supporting 

chondrogenesis but inhibiting chondrocyte hypertrophy and endochondral 

ossification. 

Molecular differentiation between transient osteophytic and 

permanent articular cartilage 

The aim of this project to identify the molecular differences between the transient and 

permanent chondrocyte phenotype in osteophytic and articular cartilage. Total RNA 

was isolated from the cartilaginous layer of osteophytes and from intact articular 

cartilage from knee joints of 15 adult human donors and subjected to cDNA 

microarray analysis. The differential expression of relevant genes between these two 

cartilaginous tissues was additionally validated by quantitative RT-PCR and by 

immunohistochemistry. Among 47,000 screened transcripts, 600 transcripts were 

differentially expressed between osteophytic and articular chondrocytes. Osteophytic 

chondrocytes were characterized by increased expression of genes involved in the 

endochondral ossification process (BGLAP, BMP8B, COL1A2, SOST, GADD45ß, 

Runx2), and genes encoding tissue remodelling enzymes (MMP-9, -13, HAS1). 

Articular chondrocytes expressed increased transcript levels of antagonists and 

inhibitors of the BMP- and Wnt-signalling pathways (GREM1, FRZP, WISP3), as well 

as factors that inhibit terminal chondrocyte differentiation and endochondral bone 

formation (PTHLH, SOX9, STC2, S100A1, S100B). Immunohistochemistry of tissue 

sections for GREM1 and BGLAP, the two most prominent differentially expressed 

genes, confirmed selective detection of GREM1 in articular chondrocytes and that of 

BGLAP in osteophytic chondrocytes and bone. 

As a conclusion, osteophytic and articular chondrocytes significantly differ in their 

gene expression pattern. In articular cartilage, a prominent expression of antagonists 

inhibiting the BMP- and Wnt-pathway may serve to lock and stabilize the permanent 

chondrocyte phenotype and thus prevent their terminal differentiation. In contrast, 

osteophytic chondrocytes express genes with roles in the endochondral ossification 

process, which may account for their transient phenotype. 

Epigenetic mechanisms for stabilizing the chondrocyte 

phenotype 

This study investigates the differences in epigenetic mechanisms between transient 

and permanent cartilage. We performed a Methylation Array which demonstrated 

distinct differences in the epigenetic pattern of articular chondrocytes and 

chondrocytic differentiated mesenchymal stem cells. The promotor region of FoxS1 

was one of the most striking regions with a high degree of methylation in articular 

chondrocytes. The hypothesis of this study is that an epigenetic suppression of 

122

FoxS1 increases the transcriptional activity of Foxo3a which stabilizes the postmitotic 

chondrocyte phenotype. Currently, we further analyse the promotor region of Foxs1 

by Pyrosequencing and the role of DNA-methyltransferases. The aim of this project is 

to evaluate the methylation status as tool for screening the potency of chondrogenic 

differentiation of mesenchymal stem cells. 



Gelse K, Ekici, AB, Cipa F, Swoboda B, Carl HD, Olk A, Hennig FF, Klinger P. 

Molecular differentiation between osteophytic and articular cartilage – clues for a 

transient and permanent chondrocyte phenotype. Osteoarthritis Cartilage. 

2012;20(2) :162-71. 

Gelse K, Klinger P, Koch M, Surmann-Schmitt C, von der Mark K, Swoboda B, 

Hennig FF, Gusinde J. Thrombospondin-1 prevents excessive ossification in cartilage 

repair tissue induced by osteogenic protein-1. Tissue Eng Part A. 2011 Aug;17(15- 

16):2101-12. 

Klinger P, Schietke RE, Warnecke C, Swoboda B, Wiesener M, Hennig FF, Gelse K. 

Deletion of the oxygen-dependent degradation domain results in impaired 

transcriptional activity of hypoxia-inducible factors. Transcription. 2011 Nov 

1;2(6):269-75. 

Klinger P, Surmann-Schmitt C, Brem M, Swoboda B, Distler JH, Carl HD, von der 

Mark K, Hennig FF, Gelse K. Chondromodulin 1 stabilizes the chondrocyte 

phenotype and inhibits endochondral ossification of porcine cartilage repair tissue. 

Arthritis Rheum. 2011 Sep;63(9):2721-31. 

Gelse K, Olk A, Eichhorn S, Swoboda B, Schoene M, Raum K. Quantitative 

ultrasound biomicroscopy for the analysis of healthy and repair cartilage tissue. Eur 

Cell Mater. 2010;19:58-71. 

Blanke M, Carl HD, Klinger P, Swoboda B, Hennig F, Gelse K. Transplanted 

chondrocytes inhibit endochondral ossification within cartilage repair tissue. Calcif 

Tissue Int. 2009;85(5):421-33. 

Gelse K, Brem M, Klinger P, Hess A, Swoboda B, Hennig F, Olk A. Paracrine effect 

of transplanted rib chondrocyte spheroids supports formation of secondary cartilage 

repair tissue. J Orthop Res. 2009;27(9):1216-25. 

123


Gelse K, Beyer C. The prostaglandin E(2) system: a toolbox for skeletal repair? 

Arthritis Rheum. 2011 Apr;63(4):871-3. 

Gelse K, Schneider H. In vivo evaluation of gene transfer into mesenchymal cells (in 

view of cartilage repair). Methods Mol Biol. 2011;737:391-405. 

Linke R, Gelse K, Schuch F. The indication for radiosynoviorthesis. From the 

perspective of the nuclear medicine expert, rheumatic orthopedist and internist. Z 

Rheumatol. 2011 Jan;70(1):34-44. 

Gelse K, Swoboda B. Zell- und Gentherapie – eine kurative Therapieoption bei 

Arthrose? Der Internist 2009;50:73. 

Gelse K, Swoboda B. Therapie der Arthrose. Der Allgemeinarzt 2009;5:36-40. 



Simon Obier 

Die Rolle des Transkriptionsfaktors HIF-1a für die Integrität des murinen 

Gelenkknorpels 

Matthias Koch 

Die Rolle von Thrombospondin-1 für die Stabilität des Knorpelzellphänotyps 

Franziska Cipa 

Molekularer Vergleich von „transienten“ Osteophytenknorpel und „permanenten“ 

Gelenkknorpel 

124

DEPARTMENT OF PLASTIC AND HAND SURGERY 

(Tissue Engineering) 

Dr. Subha N. Rath 

Leonie Strobel 

Daniel Hiller 

Head: Raymund E. Horch, Prof. Dr. med. 

Vice chairman: Ulrich Kneser, PD Dr. med. 

Amelie Balzer 

Jennifer Covi 

Address: Department of Hand and Plastic Surgery 

Krankenhausstraße 12 


E-mail: ulrich.kneser@uk-erlangen.de 


Dr. Andreas Brandl 


Quan Yuan 

Johannes Hilgert 

Gregor Bührer 

Doctoral Students (Veterinary Medicine) 

125

Stefan Fleischer 

Technicians 

Marina Milde 


126 

Ilse Arnold


Since the late 1980s, Tissue Engineering has been one of the key technologies in 

regenerative medicine. Regarding bone tissue, an approach based on scaffolds as 

carriers for patient’s own cells and suitable growth factors has been proposed. Our 

group focuses on engineering bioartificial bone tissues and vascularisation models. 

The combination of smart biomaterials, osteoinductive factors and osteogenic cells 

lead to new perspectives in bone regeneration therapy. Endothelial cell (EC) culture, 

osteoblast (OB) culture and marrow stromal cell (MSC) culture are utilised in vitro. 

Furthermore, we use in vivo subcutaneous models and vascularisation models (e.g. 

arteriovenous loop model) in rats (in cooperation with Franz-Penzoldt-Zentrum). 

Bone Tissue Engineering 

Bone defects and impaired bone healing represent substantial challenges in 

reconstructive surgery. By bone tissue engineering it may be possible to heal 

complex bone defects in the future. Our group works on the application of novel 

biomaterials, innovative cell culture models and co-cultures. 

Induction of bone formation in biphasic calcium phosphate ceramics 

Subha N. Rath, Leonie Strobel, Ulrich Kneser 

In cooperation with the Institute of Materials Science (Prof. P. Greil), Erlangen 

Novel biphasic calcium phosphate (BCP) matrices were generated by 3D printing and 

high porosity was achieved by starch consolidation (Department of Materials Science 

(Glass and Ceramics), FAU Erlangen). This project aimed to characterise the porous 

BCP scaffold’s properties and the interaction of osteogenic cells and growth factors 

under in vitro and in vivo conditions. 

Bioreactors are advised for improved cell survival, as they are able to provide a 

controlled flow through the scaffold. Osteogenic cells (rat primary osteoblasts (OB) 

and bone marrow stromal cells (MSCs)) were isolated, cultured and seeded onto 3D 

scaffolds. Samples in static culture without osteoinduction, static culture with 

osteoinduction and dynamic culture with osteoinduction were compared over 6 

weeks. Analyses showed that a continuous flow bioreactor not only preserves the 

number of osteogenic cells, but also keeps their differentiation ability in balance 

providing a suitable cell-seeded scaffold product for applications in regenerative 

medicine (Rath, Strobel et al. 2012). 

Furthermore, these very BCP scaffolds were evaluated in vivo. Five differently 

treated constructs were implanted subcutaneously in syngeneic male Lewis rats: 

127

plain BCP constructs, constructs pre-treated with BMP-2, seeded with osteoblasts 

(OB), seeded with OB and BMP-2 and constructs with OB that were pre-cultivated in 

a flow bioreactor for 6 weeks. Specimens were explanted and subjected to histology 

and molecular biological analysis after 2, 4 and 6 weeks. 

All explanted constructs were invaded by fibrovascular tissue. Samples with both OB 

and BMP-2 demonstrated higher osteogenic gene expression and significantly 

increased bone formation compared to all other groups. Pre-cultivation in the flow 

bioreactor induced bone formation comparable with the freshly seeded group. In 

summary, the combination of OB and BMP-2 enhanced bone formation in the novel 

ceramic scaffolds (Strobel, Rath et al. 2012). 

This work was supported by the German Research Foundation (DFG). 

Rath SN, Strobel LA, Meier AK. 2012, Osteoinduction of osteoblasts and bone marrow stromal cells in 

3D BCP scaffolds: the effect of bioreactor on cell survival and differentiation, Journal of 

Cellular and Molecular Medicine; 2012 Feb 3. doi: 10.1111/j.1582-4934.2012.01545.x. 

Strobel LA, Rath SN, Maier AK. 2012, Induction of Bone Formation in Biphasic Calcium Phosphate 

Scaffolds by Bone Morphogenetic Protein-2 and Primary Osteoblasts Journal of Tissue 

Engineering and Regenerative Medicine; (accepted) 

Haematoxylin-eosin (HE) staining of an explanted biphasic calcium phosphate (BCP) scaffold at 6 

weeks after subcutaneous implantation. The scaffold was seeded with primary osteoblasts previous to 

implantation. Newly formed bone areas (arrows) are visible in the pores of the BCP construct. 

Human marrow stromal cells (hMSCs) on innovative bioglass scaffolds* 

Subha N. Rath, Daniel Hiller, Ulrich Kneser 

In cooperation with the Institute of Biomaterials (Prof. A. Boccaccini), Erlangen 

The aim of this study is to analyse hMSCs response to new bioactive copper-doped 

glass scaffolds. 45S5 bioglass scaffolds containing different concentrations of copper 

are provided by the Institute of Biomaterials. First, these scaffolds were evaluated 

regarding copper and bioactive ion release in vitro. For this purpose, we observed 2dimensional 

cell culture in vicinity to bioactive glass. The constructs proved non-toxic 

and provided continuous release of copper (depending on the initial copperconcentration). 

Furthermore, scaffolds are seeded with hMSCs and evaluated under 

128

different culture conditions. This will provide the basis for further in vivo studies with 

bioactive glass materials. 

Bioglass scaffolds in the arteriovenous (AV) loop model* 

Amelie Balzer, Gregor Bührer, Andreas Arkudas 

Generation of axially vascularised bone tissue engineering constructs in vivo is 

performed using an arteriovenous (AV) loop model. After bioactive glass scaffolds 

proved biocompatible in vitro, scaffolds are evaluated in the AV loop model in rats. 

Different scaffold materials, osteogenic cells and bioactive molecules such as bone 

morphogenetic protein-2 (BMP-2) can be incorporated in the matrix that is inserted in 

the AV-loop (in cooperation with Franz-Penzoldt-Zentrum, Erlangen). 

Co-cultures of human marrow stromal cells (hMSCs) and endothelial cells 

(hECs) in vitro* 

Subha N. Rath, Andreas Brandl, Oliver Bleiziffer, Ulrich Kneser 

Previous reports have shown that hMSCs can be osteo-differentiated into boneforming 

cells and thus can be used for bone tissue engineering. However, when 

applied in vivo in larger dimensions, the lack of development of neovascularization 

into the defect substantially limits their survival. It was also reported that hECs when 

cultured in vitro forms endothelial tube like structures, which vascularizes upon in 

vivo implantation to host blood vessels to supply blood relatively quicker than without 

hECs. 

Therefore, the combined co-culture of hMSCs and hECs can be attempted for a bone 

tissue engineered product which can later be vascularized upon in vivo implantation. 

A perfect scaffold material to study the effect is in bioactive glass, which not only 

have osteoconductive property, but also have vascularization inducing ability. To 

study this effect, our group developes co-culture models to investigate the 

interactions and influences of these cells upon each other and the effect of bio-active 

glass in inducing VEGF from hMSCs. 

*These projects are supported by the Emerging Fields Initiative, FAU Erlangen. 

Neovascularization of bioartificial tissues 

Neovascularization of tissues and organs is based upon two distinct processes: de 

novo formation of blood vessels via the assembly of progenitor cells (vasculogenesis) 

and expansion of pre-existing vascular networks by endothelial cell sprouting 

(angiogenesis). The latter one is the main mechanism in postnatal life. Evidence exist 

that bone marrow-derived progenitor cells can contribute to the formation of new 

vessels by incorporation into sites of active angiogenesis (Rüger et al.; 2008). The 

discovery of putative endothelial progenitor cells (EPC) gave rise to a new era of 

vascular biology to use their highly promising multipotent character for therapeutic 

angiogenesis equally with regard to tissue engineering where they could be used as 

129

a source to establish a reliable perfusion of bioartificial tissues and organs. Bone 

marrow-derived progenitors are known to be heterogenous. Therefore it is absolutely 

necessary to clearly characterize isolated populations from bone marrow that are 

designated to be EPCs, with regard to their morphology and performance in 

endothelial-specific functional assays in vitro prior to using them in in vivo studies. 

Effect of rat primary EPCs on neovascularization using the (AV) loop model* 

Andreas Brandl, Quan Yuan, Oliver Bleiziffer 

Previous studies used a mouse endothelial progenitor cell (EPC) line (T17b) to study 

vascularization in a highly standardarized (AV) loop model in rat. One aim of this 

project is to isolate and characterize a bone marrow-derived EPC-population from rat 

to obtain an allogenic cell population to study angiogenesis in this well-established in 

vivo system. 

First results show that rat bone marrow-derived mononuclear cells can be 

differentiated into E(P)Cs in vitro. MNCs (mononuclear cells) from rat bone marrow 

were isolated using density gradient centrifugation and a differential adhesion 

method as described in Kähler et al., 2007. Cells were followed up concerning their 

morphology for several weeks and they exhibit the typical “cobblestone”-shape after 

about two to three weeks in culture (Fig.1). 

a) b) 

Fig.1: Mononuclear cells from rat bone marrow ( a) day of isolation) can be differentiated 

into endothelial cells ( b) 3 weeks post isolation) in vitro using special differentiation 

medium. 

The cells were also characterized with regard to their surface marker expression 

using flow cytometric analyses and their ability to form tube-like structures on 

Matrigel. The flow cytometry analyses showed that the cultivated cells gain 

endothelial-specific surface marker expression like CD31, VEGFR-2 and CD146 in 

culture. Four weeks old EPC are able to form tube-like structures and sprouts on the 

130

specialized extracellular matrix (ECM) Matrigel as potent as pure endothelial cell 

lines (HDMECs and EC52) do (Fig. 2). 

a) b) 

Fig.2: Isolated primary endothelial cells ( a) 4 weeks after isolation) perform just as pure 

commercially available cell lines ( b) HDMECs) in sprouting experiments on the 

specialized extracellular matrix (ECM) Matrigel. 

Future experiments will investigate the ability of these primary endothelial cells to 

efficiently support or promote neovascularization in bioartificial tissues using the wellestablished 

AV-loop model. 

Distribution and Impact of Hypoxia on blood vessel formation in a developing 

three-dimensional vascular network in the AV-Loop HIF investigated by 

detection of hypoxia-inducible factor 1 alpha (HIF-1 α) 

Hypoxia inducible factor (HIF) is a transcriptional factor that is the master regulator of 

oxygen homeostasis and plays an essential role in angiogenesis. In normoxic 

conditions, HIF could be degraded by prolyl hydroxylases (PHDs). Suppression of 

PHD activity increases endogenous HIF levels, which is probably able to promote the 

angiogenesis. Dimethyloxallyl Glycine (DMOG) is a cell permeable, competitive 

inhibitor of prolyl hydroxylase domain-containing proteins (PHDs and HIF-PHs). 

Future experiments will apply DMOG in Lewis rats with AV-loop systemically; detect 

the angiogenesis-related target genes such as HIF-1a, HIF-2a, VEGFA, eNOS, Flt-1, 

VE-Cadherin, KDR and vWF at transcription level in the loop at certain time points, 

and evaluate the outgrowth of the capillary network with morphology methods. 

* These projects are supported by the Else-Kröner-Fresenius-Stiftung. 

131


Rath SN, Strobel LA, Meier AK. 2012, Osteoinduction of osteoblasts and bone marrow stromal cells in 

3D BCP scaffolds: the effect of bioreactor on cell survival and differentiation, Journal of 

Cellular and Molecular Medicine; 2012 Feb 3. doi: 10.1111/j.1582-4934.2012.01545.x. 

Strobel LA, Rath SN, Maier AK. 2012, Induction of Bone Formation in Biphasic Calcium Phosphate 

Scaffolds by Bone Morphogenetic Protein-2 and Primary Osteoblasts Journal of Tissue 

Engineering and Regenerative Medicine; (accepted) 

Bleiziffer O, Horch RE, Hammon M, Arkudas A, Naschberger E, Rath S, Pryymachuk G, Beier JP, 

Hatzopoulos AK, Stürzl M, Kneser U, T17b murine embryonal endothelial progenitor cells can 

be induced towards both proliferation and differentiation in a fibrin matrix. J Cell Mol Med. 

2009; 13: 926-35. 

Bleiziffer O, Hammon M, Naschberger E, et al. Endothelial Progenitor Cells are integrated in newly 

formed capillaries and alter adjacent fibrovascular tissue after subcutaneous implantation in a 

fibrin matrix. J Cell Mol Med 2010; doi: 10.1111/j.1582-4934.2010.01247.x. 

Arkudas A, Tjiawi J, Saumweber A, Beier JP, Polykandriotis E, Bleiziffer O, Horch RE, Kneser U. 

Evaluation of blood vessel ingrowth in fibrin gel subject to type and concentration of growth 

factors. J Cell Mol Med. 2009; 13: 2864-74 

Arkudas A, Pryymachuk G, Hoereth T, Beier JP, Polykandriotis E, Bleiziffer O, Horch RE, Kneser U. 

Dose-finding study of fibrin gel-immobilized vascular endothelial growth factor 165 and basic 

fibroblast growth factor in the arteriovenous loop rat model. Tissue Eng Part A. 2009; 15: 

2501-11. 

Fiegel HC, Pryymachuk G, Rath S, Bleiziffer O, Beier JP, Bruns H, Kluth D, Metzger R, Horch RE, Till 

H, Kneser U. Foetal hepatocyte transplantation in a vascularized AV-Loop transplantation 

model in the rat. J Cell Mol Med. 2010; 14: 267-74. 

Arkudas A, Beier JP, Pryymachuk G, Hoereth T, Bleiziffer O, Polykandriotis E, Hess A, Gulle H, Horch 

RE, Kneser U. Automatic quantitative micro-computed tomography evaluation of angiogenesis 

in an axially vascularized tissue-engineered bone construct. Tissue Eng Part C Methods. 

2010; 16: 1503-14. 

132

AG Krönke 

(Department of Internal Medicine 3) 

Principal Investigator: Gerhard Krönke, Dr. med. 

Address: AG Krönke 

Glückstr. 6 


Telefone: + 49 (9131) 85 43012 

E-mail: gerhard.kroenke@uk-erlangen.de 

Homepage: www..medizin3.ukerlangen.de/e1846/e741/e614/index_ger.html 

Carina Scholtysek 

Tobias Rothe 

StefanUderhardt° 

Arnd Kleyer 

Susanne Weig 

°MD received 

Cornelia Stoll 

Martin Steffen 


Natacha Ipseiz 

Réne Pfeifle 

Students (Medicine) 

Tobias Fillep 

Technicians 

Isabell Schmidt 

133 

Edith Bottesch


Our group is interested in common mechanisms regulating the initiation and 

resolution of the inflammatory and adaptive immune response. Our aim is to 

elucidate the pathogenesis of inflammatory and autoimmune diseases and the 

consequences of chronic inflammation on tissue homeostasis. To answer these 

questions, we focus on the role and function of cells of the monocytic lineage such as 

macrophages, dendritic cells and osteoclasts as well as on the contribution of lipid 

mediators and lipid-activated nuclear receptors to inflammation, immunity and 

regulation of tissue homeostasis. 

Regulation of the immune response by lipid mediators 

Lipids such as fatty acids, phospholipids and triglycerides are not only an integral 

component of cellular membranes and essential for energy homeostasis, but are also 

involved in the regulation of inflammation and the adaptive immune response. 

Multiple enzymes including different cyclooxygenases and lipoxygenases metabolize 

lipids such as free and esterified polyunsaturated fatty acids and thereby generate 

bioactive lipid mediators including prostaglandins, leukotriens and lipoxins as well as 

less well characterized species of distinct phospholipid oxidation products. Such lipid 

mediators act via multiple mechanisms, which include both activation and inhibition of 

different cell-surface receptors and of nuclear receptors that are involved in the 

modulation of the innate and adaptive immune response. 

Enzymatic lipid oxidation orchestrates the resolution of inflammation 

Gerhard Krönke 

Inflammation is the physiologic response to infection and tissue damage, 

respectively. The inflammatory response, however, has to be tightly regulated and 

controlled, since overwhelming and prolonged inflammation would exacerbate tissue 

damage and prevent the healing process. Typically, different mechanisms cooperate 

to initiate the resolution of inflammation. Interestingly, different lipid oxidation 

products such as lipoxin A4 have been recognized as major factors driving this 

resolving process. 

To study the role of lipoxin A4 during the pathogenesis of chronic inflammatory 

diseases, we are analyzing mice with a targeted deletion in the gene for 12/15lipoxygenase 

(12/15-LO) as the major enzyme involved in the generation of this proresolving 

mediator. These mice display a defect in the resolution of inflammation as 

well as a dramatic exacerbation of the inflammatory response as observed in two 

models of inflammatory arthritis (Fig. 1; Krönke et al., 2009). Likewise, 12/15-LO 

deficiency results in increased inflammation-associated tissue damage, in an altered 

healing response and overwhelming post-inflammatory fibrosis (Krönke et al., 2012). 

Interestingly additional data indicate that 12/15-LO is also involved in the 

pathogenesis of acute lung injury (Zarbock et al., 2009) as well as in the process of 

osteoclast differentiation (Krönke et al., 2009). 

134

Fig. 1 Experimental Arthritis in TNF-transgenic (TNFtg) mice exacerbates in the absence of 12/15- 

LO. (A) survival, (B) weight gain, (C) histological analysis of paws, (D) mRNA expression in joints and 

(E) clinical score of arthritis in TNFtg mice in the presence and absence of 12/15-LO. 

12/15-lipoxygenase orchestrates the clearance of apoptotic cells 

Stefan Uderhardt 

During the inflammatory response and the resolution of inflammation, the immune 

system is confronted with both pathogens and self-antigens in the form of necrotic 

and apoptotic cells (AC). Uptake and processing of AC by inflammatory monocytes 

and dendritic cells would eventually result in an immune response to self antigens. 

Therefore, the non-inflammatory removal and clearance of AC is essential to 

maintain immunologic tolerance. To study the clearance of AC in an inflammatory 

setting, we performed phagocytosis assays with monocytes that were isolated from 

murine peritonitis. Here we observed that, during inflammation, uptake of ACs is 

confined to a population of 12/15-LO-expressing, alternatively activated resident 

macrophages (resMΦ), which actively block uptake of ACs into freshly recruited 

inflammatory Ly6C high monocytes in a 12/15-LO-dependent manner (Fig. 2). On a 

mechanistic basis, we could identify novel 12/15-LO-derived oxidation-products of 

phosphatidylethanolamine, which are exposed on the plasma membranes of resMΦ. 

Thereby these cells generate a sink for distinct soluble receptors for ACs such as 

milk fat globule-EGF factor 8, which are essential for the uptake of ACs into 

inflammatory monocytes. Loss of 12/15-LO-activity, in turn, results in an aberrant 

phagocytosis of ACs by inflammatory monocytes, subsequent antigen-presentation of 

AC-derived antigens, and a lupus-like autoimmune disease (Fig. 2; Uderhardt et al., 

2012). These data indicate that enzymatic phospholipid oxidation products are 

135

essential regulatory mediators during the clearance of AC and in the maintenance of 

self-tolerance. 

Fig. 2 12/15-LO orchestrates the clearance of apoptotic cells. (A and B) Macrophages isolated from 

a thioglycollate-induced peritonitis of WT and 12/15-LO-deficient mice were incubated with apoptotic 

cells (AC; green) and analysed by immunofluorescence. Alternatively activated resident macrophages, 

which ingest most of the AC in WT mice, were identified by their expression of 12/15-LO (blue in A) or 

Tim4 (red in B). (C) FACS-based analysis of the uptake of CFSE-labelled AC by Ly6Chigh 

inflammatory monocytes during a thioglycollate-induced peritonitis in vivo. (D and E) 12/15-LOdeficient 

mice develop a systemic autoimmune disease with signs of glomerulonephritis and different 

classes of autoantibodies. 

Oxidized phospholipids control the maturation of dendritic cells 

Tobias Rothe 

The analysis of murine bone marrow-derived dendritic cells (DC) revealed a high 

expression of 12/15-LO in these cells. Likewise we could identify different 12/15-LOderived 

phospholipid oxidation products in these cells. Our data demonstrates that 

this enzyme and its oxidation products critically modulate the maturation status and 

gene expression profile of DC. Absence of 12/15-LO results in an enhanced DC 

maturation, whereas addition of 12/!5-LO-specific phospholipid oxidation products 

interfered with the maturation process of DC. As a consequence, 12/15-LO-dficient 

mice show an altered activation of T-cells and an exacerbation of T-cell-driven 

autoimmune diseases such as experimental autoimmune encephalomyelitis (Rothe 

et al.; in preperation). 

136

Nuclear receptors as modulators of tissue homeostasis 

Nuclear receptors comprise a superfamily of ligand-activated transcription factors and 

regulate multiple homeostatic processes. Different nuclear receptors such as PPARγ 

have been identified as key regulators of fat and glucose metabolism as well as 

crucial factors influencing inflammation and the adaptive immune response. 

The nuclear receptor NR4A1 mediates anti-inflammatory effects of apoptotic 

cells 

Natacha Ipseiz 

The non-inflammatory clearance of apoptotic cells (AC) is essential to dispose selfantigens 

and to maintain immunologic tolerance. Mechanisms and factors 

contributing to this process are poorly understood, though. We observed a rapid 

induction of the expression of the nuclear receptor NR4A1 in macrophages ingesting 

AC. This induction of NR4A1 was critically involved in the anti-inflammatory effects 

AC exerted on macrophages. Indeed, AC efficiently blocked the inflammatory 

response in WT, but not NR4A1-deficient macrophages. Moreover, we observed a 

critical role of NR4A1 during the modulation of the activity of the pro-inflammatory 

transcription factor NFκB and the expression of different sets of pro-inflammatory 

genes such as IL12 and TNF. In line with these results, NR4A-/- mice display a break 

in self-tolerance in a model of murine lupus erythematodes (Ipseiz et al. in 

preperation). 

The nuclear receptor PPARβ acts as a key regulator of bone homeostasis 

Carina Scholtysek, Edith Bottesch and Arnd Kleyer, 

The nuclear receptor PPARγ acts as a master regulator of adipocyte differentiation. 

In contrast, PPARγ negatively affects Wnt-signalling in osteoblasts and thereby 

interferes with osteoblast differentiation and bone formation. The role of its family 

member PPARβ during bone homeostasis has not been studied, though. To address 

the role of PPARβ during bone homeostasis, we analyzed its role in osteoblasts and 

observed that this nuclear receptor acted in a permissive manner on Wnt-signaling in 

these cells. Activation of PPARβ by specific ligands induced expression of the Wntco-receptor 

LRP5, promoted nuclear accumulation of β-catenin and consequently 

enhanced TCF-driven transcriptional activity. Thereby, PPARβ augmented 

expression of different Wnt-dependent genes, such as osterix and osteoprotegerin 

(opg), in osteoblasts. Consequently, activation of PPARβ in osteoblasts blocked the 

differentiation of bone- resorbing osteoclasts. Mice deficient in PPARβ, displayed 

reduced Wnt-signaling activity and low serum levels of OPG resulting in an increased 

differentiation of osteoclasts and osteopenia. Conversely, pharmacological treatment 

with a PPARβ-specific agonist blocked the formation of osteoclasts in vivo and 

protected mice from ovariectomy-induced bone loss in a therapeutic manner. These 

data reveal a so far unrecognized role for PPARβ in the crosstalk between energy 

metabolism and bone homeostasis and highlights its potential to serve as a target for 

the treatment of osteoporosis (Fig. 3; Scholtysek et al., in revision). 

137

Fig. 3 The nuclear receptor PPARβ acts as key regulator of bone homeostasis. (A) Gene expression 

analysis in primary osteoblasts after stimulation with specific agonists for PPARα, PPARβ or PPARγ in 

the absence or presence of the Wnt agonist Wnt3a. (B) Analysis of the mineralization of primary 

osteoblasts after treatment with the indicated concentrations of the PPARβ agonist GW501516. (C) 

Measurement of the transcriptional activity of a TCF-luciferase reporter after overexpression of PPARβ 

and stimulation with Wnt3a or a vehicle, respectively. (D) In vitro quantification of osteoclast 

differentiation in osteoblast/osteoclast co-cultures after stimulation with the indicated concentration of 

the PPAR β agonist GW501516 and Wnt3a. (E) Micro-CT analysis of the bone structure and (F) 

determination of the mineral apposition in sham-trated mice and mice after ovariectomy (OVX), which 

have been treated with a vehicle or the PPARβ agonist GW501516 over a period of 6 weeks. 


2011/12 

Uderhardt S, Herrmann M, Oskolkova O, Aschermann S, Bicker W, Ipseiz N, Sarter 

K, Frey B, Rothe T, Voll R, Nimmerjahn F, Bochkov VN, Schett G, Krönke G. 12/15- 

Lipoxygenase Orchestrates the Clearance of Apoptotic Cells and Maintains 

Immunologic Tolerance, Immunity (2012), doi:10.1016/j.immuni.2012.03.010. [Epub 


Schwab I, Biburger M, Krönke G, Schett G, Nimmerjahn F. IVIg-mediated 

amelioration of ITP in mice is dependent on sialic acid and SIGNR1. Eur J Immunol. 

2012 Jan 26. doi: 10.1002/eji.201142260. [Epub ahead of print] 

Krönke G, Reich N, Scholtysek C, Akhmetshina A, Uderhardt S, Zerr P, Palumbo K, 

Lang V, Dees C, Distler O, Schett G, Distler JH. The 12/15-lipoxygenase pathway 

138

counteracts fibroblast activation and experimental fibrosis. Ann Rheum Dis. 2012 

Jan 20. 

Dees C, Akhmetshina A, Zerr P, Reich N, Palumbo K, Horn A, Jüngel A, Beyer C, 

Krönke G, Zwerina J, Reiter R, Alenina N, Maroteaux L, Gay S, Schett G, Distler O, 

Distler JH. Platelet-derived serotonin links vascular disease and tissue fibrosis. 

J Exp Med. 2011 May 9;208(5):961-72. 

2010 

Krönke G, Uderhardt S, Kim KY, Zaiss MM, Katzenbeisser J, Schett G, Abo A. R- 

Spondin-1 protects against inflammatory bone damage during murine arthritis by 

modulating the Wnt pathway. Arthritis Rheum. 2010 Aug;62(8):2303-12. 

Uderhardt S, Diarra D, Katzenbeisser J, David JP, Zwerina J, Richards WG, Krönke 

G, Schett G. Blockade of Dickkopf-1 induces fusion of sacroiliac joints. 

Ann Rheum Dis. 2010 Mar;69(3):592-7 

Stach CM, Bäuerle M, Englbrecht M, Krönke G, Engelke K, Manger B, Schett G. 

Periarticular bone structure in rheumatoid arthritis patients and healthy individuals 

assessed by high-resolution computed tomography. 

Arthritis Rheum. 2010 Feb;62(2):330-9. 

2009 

Böhm C, Hayer S, Kilian A, Zaiss MM, Finger S, Hess A, Engelke K, Kollias G, 

Krönke G, Zwerina J, Schett G, David JP. The {alpha}-Isoform of p38 MAPK 

Specifically Regulates Arthritic Bone Loss. 

J Immunol. 2009 Oct 14. [Epub ahead of print] 

Zarbock A, Distasi MR, Smith E, Sanders JM, Krönke G, Harry BL, von Vietinghoff S, 

Buscher K, Nadler JL, Ley K. Improved Survival and Reduced Vascular Permeability 

by Eliminating or Blocking 12/15-Lipoxygenase in Mouse Models of Acute Lung Injury 

(ALI). 

J Immunol. 2009 Oct 1;183(7):4715-22. Epub 2009 Sep 14. 

Schett G, Stolina M, Dwyer D, Zack D, Uderhardt S, Krönke G, Kostenuik P, Feige U. 

Tumor necrosis factor alpha and RANKL blockade cannot halt bony spur formation in 

experimental inflammatory arthritis. 


Axmann R, Böhm C, Krönke G, Zwerina J, Smolen J, Schett G. . Inhibition of 

interleukin-6 receptor directly blocks osteoclast formation in vitro and in vivo. 


Krönke G, Katzenbeisser J, Uderhardt S, Zaiss M, Scholtysek C, Schabbauer G, 

Zarbock A, Koenders MI, Axmann R, Zwerina J, Baenckler HW, van den Berg W, Voll 

RE, Kühn H, Joosten LAB, Georg Schett G. 12/15-lipoxygenase counteracts 

inflammation and tissue damage in arthritis. 

J Immunol. 2009 Sep 1;183(5):3383-9. Epub 2009 Aug 12. 

Krönke G, Uderhardt S, Katzenbeisser J, Schett G. The 12/15-Lipoxygenase- 

Pathway Promotes Osteoclast Development and Differentiation. 


139


Scholtysek C, Krönke G, Schett G. Inflammation-associated changes in bone 

homeostasis. 

Inflamm Allergy Drug Targets. 2012 Jan 20. 

140

DEPARTMENT OF UROLOGY 

Div. Molecular Urology 

Head: Helge Taubert, Dr. rer. nat. 

Professor of Molecular Urology 

Address: Division of Molecular Urology 

Glückstr. 6 


Telefone: + 49 (9131) 85 23373 

Fax: + 49 (9131) 85 23374 

E-mail: helge.taubert@uk-erlangen.de 

Homepage: www.urologie.uk-erlangen.de 

Sven Wach, Dr. rer. nat. 

Head 

Helge Taubert, Prof. Dr. rer. nat. 

Professor of Molecular Urology 



141

Elke Nolte geb. Löprich, 

Dipl. Biologin 

Omar Al-Janabi , Dipl. 

Biologe 

Christine Ellmann 

Kathrin Holzer 

Katrin Weigelt, MTA 



Technicians 


142 

Anne Theil, BTA


Our group analyses molecular mechanisms that control tumor development and 

progression, focusing on microRNA expression and their target genes and gene 

expression that is hypoxia- or stem cell-associated in solid tumors (urologic tumors, 

soft tissue sarcomas). 

Tumor genetic research with focus on identification of biomarkers 

Dr. rer. nat. S. Wach 

The identification and characterization of specific biological properties of the prostate 

carcinoma as well as other malignant tumors like kidney carcinoma is the main focus 

of the biological research projects. By assessing changes in microRNA (miRNA) 

expression profiles it is already possible to distinguish between samples of tumor and 

non-malignant tissue. Furthermore the prognostic value of miRNA expression profiles 

is currently examined. MiRNAs directly regulate the expression of numerous other 

proteins in cells. Therefore, experimental methods for analyzing protein expression 

(Fig.1) are a vital component of our research. 

The complete spectrum of molecular cytogenetic techniques including fluorescence 

in situ DNA and RNA hybridization is established in the laboratory. The quantification 

of micro-RNA and gene expression as well as the determination of gene copy 

numbers using real time PCR approaches is a central part of the experimental 

methods. 

The role of hypoxia and hypoxia-associated signal transduction 

pathways in solid tumors 

Prof. Dr. rer. nat. Helge Taubert 

The lack of oxygen (hypoxia) is a situation seen in many solid tumors. Especially 

locally advanced malignancies rapidly outgrow the blood vessels that supported their 

growth. By this tumor cells are confronted with a lack of oxygen and nutrition. As a 

consequence, more than 70 genes are activated by the HIF-1 transcription factor. 

Signal molecules are produced that stimulate the growth of new blood vessels, 

enzymes are produced that support a survival of cells under hypoxic conditions and 

stem cell-associated genes are expressed. We are mainly interested in the regulation 

of miRNA genes by HIF-1. Because miRNAs themselves regulate numerous target 

genes, it is obvious that hypoxia has a vital influence on any cell. Using cell culture 

models we examine the functional consequences of hypoxia on tumor cells. 

Projects 

Is the detection of members of the urokinase plasminogen activator 

family and of splice variants of survivin suitable as marker for 

143

prognosis and metastases of prostate cancer in the Clinic of 

Urology? 

Rudolf und Irmgard Kleinknecht-Stiftung (Grant No. 01.10.11-30.9.2013) 

Prof. Dr. H. Taubert 

Although prostate cancer belongs is the most frequent tumor in men there are except 

clinical factors no markers that are valid for an evaluation of the individual disease 

course ort he individual prognosis. We will investigate members of the urokinase 

plasminogen activator family and splice variants of the apoptosis inhibitor survivin on 

the mRNA level in prostate cancer retrospectively and prospectively. The molecular 

results will be correlated with the clinical parameters and tested for their relevance for 

the tumor biological behavior (development of relapses and/or metastases) and the 

individual prognosis. We expect to better characterize factors that can improve 

individual prognosis evaluation and help to support future therapy decisions. 

Alterations of the microRNA expression profile of primary prostate 

cancer 

Wilhelm Sander-Stiftung (Grant No. 2007.025.01) 

Prof. Dr. B. Wullich, Prof. Dr. F. Grässer 

MicroRNAs (miRNAs) belong to the family of non-coding RNAs. Their biological role 

is the regulation of gene expression on a post-transcriptional level. In every human 

tumor entity examined so far, there exists a characteristic pattern of deregulated 

miRNAs. Hereby, miRNAs contribute to tumorigenesis by promoting the expression 

of oncogenes or suppressing the tumor suppressor genes. In prostate cancer we 

were able to establish comprehensive and reliable miRNA expression signatures. 

Using these miRNA expression signatures, we were able to distinguish between 

tumor- and normal tissue with high accuracy. Therefore, miRNAs have the potential 

of being valuable biomarkers for the diagnosis of prostate cancer in the future. 

Besides their properties as biomarkers, miRNAs lead the way to the discovery of 

novel tumor-relevant genes because miRNA deregulation is not merely a symptom 

but a driving force in the process of tumorigenesis. We are currently investigating 

several specific target genes of deregulated miRNAs to unravel their intrerplay and 

functional relevance in prostate carcinogenesis. 

MiRNAs in papillary renal cell carcinoma 

ELAN-Funds (Grant No. 09.11.11.1) 

Dr. Sven Wach 

Papillary renal cell carcinomas (pRCCs) represent the second most common entity of 

renal cell carcinomas. According to histopathological criteria, pRCCs can even be 

further subdivided into two distinct subtypes. Recent insights point towards the fact, 

that pRCCs of type 1 represent a low-malignant phenotype whereas type 2 pRCCs 

are high-risk carcinomas associated with a worse clinical outcome. So far, there is 

little insight into the molecular basis of the differentiation pattern or the different 

clinical behavior of these two subtypes of pRCC. Using our established method of 

miRNA expression profiling, we are currently identifying miRNAs that discriminate 

144

etween the two pRCC subtypes. When target genes of these miRNAs will be 

defined, this may lead to a better understanding of the molecular pathways affected 

in both subtypes of pRCC. 

2012 



Wach S, Nolte E, Szczyrba J, Stöhr R, Hartmann A, Orntoft T, Dyrskjøt L, Eltze E, Wieland 

W, Keck B, Ekici AB, Grässer F, Wullich B. MicroRNA profiles of prostate carcinoma 

detected by multiplat- form microRNA screening. Int J Cancer 2012;130(3):611-21 

Greither T, Würl P, Grochola L, Bond G, Bache M, Kappler M, Lautenschläger C, 

Holzhausen HJ, Wach S, Eckert AW, Taubert H. Expression of microRNA 210 associates 

with poor survival and age of tumor onset of soft-tissue sarcoma patients. Int J Cancer 2012 

130:1230-5. 

2011 

Szczyrba J, Nolte E, Wach S, Kremmer E, Stöhr R, Hartmann A, Wieland W, Wullich B, 

Grässer FA. Down-regulation of Sec23A protein by miRNA-375 in prostate carcinoma. Mol 

Cancer Res. 2011;9(6):791-800. 

Keck B, Stoehr R, Wach S, Rogler A, Hofstaedter F, Lehmann J, Montironi R, Sibonye M, 

Fritsche HM, Lopez-Beltran A, Epstein JI, Wullich B, Hartmann A. The plasmacytoid 

carcinoma of the bladder- rare variant of aggressive urothelial carcinoma. Int J Cancer. 

2011;129(2):346-54. 

Eckert AW, Lautner MH, Schütze A, Taubert H, Schubert J, Bilkenroth U. (2011) 

Coexpression of hypoxia-inducible factor-1α and glucose transporter-1 is associated with 

poor prognosis in oral squamous cell carcinoma patients. Histopathology 2011;58:1136-47. 

Kappler M, Taubert H, Eckert AW. Oxygen sensing, homeostasis, and disease. N Engl J 

Med. 2011;365(19):1845-6 (letter/author reply). 

Rot S1, Taubert H1, Bache M, Greither T, Würl P, Eckert AW, Schubert J, Vordermark D, 

Kappler M. A novel splice variant of the stem cell marker LGR5/GPR49 is correlated with the 

risk of tumor-related death in soft-tissue sarcoma patients. BMC Cancer. 2011 Oct 6;11:429. 

(1equally contributed) 

Bache M, Zschornak MP, Passin S, Keßler J, Wichmann H, Kappler M, Paschke R, 

Kaluđerović GN, Kommera H, Taubert H, Vordermark D. Increased betulinic acid induced 

cytotoxicity and radiosensitivity in glioma cells under hypoxic conditions. Radiat Oncol. 2011 

Sep 9;6(1):111. 

Kotzsch M, Magdolen V, Greither T, Kappler M, Bache M, Lautenschlager C, Fussel S, 

Eckert AW, Luther T, Baretton G, Wurl P, Taubert H. Combined mRNA expression levels of 

members of the urokinase plasminogen activator (uPA) system correlate with diseaseassociated 

survival of soft-tissue sarcoma patients. BMC Cancer. 2011 Jun 25;11(1):273. 

[Epub ahead of print] 

145

Grochola LF, Taubert H, Greither T, Bhanot U, Udelnow A, Würl P. Elevated transcript levels 

from the MDM2 P1 promoter and low p53 transcript levels are associated with poor 

prognosis in human pancreatic ductal adenocarcinoma. Pancreas. 2011 Mar;40(2):265-70. 

2010 

Szczyrba J, Loprich E, Wach S, Jung V, Unteregger, G, Barth S, Grobholz R, Wieland W, 

Stohr R, Hartmann A, Wullich B, Grässer F. The MicroRNA Profile of Prostate Carcinoma 

Obtained by Deep Sequencing. Mol Cancer Res. 2010;8(4):529-38. 

Goebell PJ, Keck B, Wach S and Wullich B. [Value of biomarkers in urology]. Urologe A 

2010, 49, 547-559. 

Fürnrohr BG, Wach S, Kelly JA, Haslbeck M, Weber CK., Stach CM, Hueber AJ, Graef, D, 

Spriewald BM, Manger K Herrmann M, Kaufman KM, Frank SG, Goodmon E, James JA, 

Schett G, Winkler TH, Harley JB, Voll RE. Polymorphisms in the Hsp70 gene locus are 

genetically associated with systemic lupus erythematosus. Ann Rheum Dis. 

2010;69(11):1983-9. 

Taubert H, Heidenreich C, Holzhausen HJ, Schulz A, Bache M, Kappler M, Eckert AW, Wurl 

P, Melcher I, Hauptmann K, Hauptmann S, Schaser KD. Expression of survivin detected by 

immunohistochemistry in the cytoplasm and in the nucleus is associated with prognosis of 

leiomyosarcoma and synovial sarcoma patients. BMC Cancer. 2010,10(1):65. 

Taubert H, Würl P, Greither T, Kappler M, Bache M, Lautenschläger C, Füssel S, Meye A, 

Eckert AW, Magdolen V, Kotzsch M. Co-detection of members of the urokinase plasminogen 

activator system in tumor tissue and in serum correlates with a poor prognosis for soft-tissue 

sarcoma patients. Brit J Cancer 2010 Feb 16;102(4):731-7. 

Greither T, Grochola L, Udelnow A, Lautenschläger C, Würl P, Taubert H. Elevated 

expression of microRNAs 155, 203, 210 and 222 in pancreatic tumours associates with 

poorer survival. Int J Cancer. 2010 Jan 1;126(1):73-80. 

Vazquez A, Grochola LF, Bond EE, Levine AJ, Taubert H, Müller TH, Würl P, Bond GL. 

Chemosensitivity Profiles Identify Polymorphisms in the p53 Network Genes 14-3-3{tau} and 

CD44 That Affect Sarcoma Incidence and Survival. Cancer Res. 2010; 70(1):172-80. 

Bache M, Kappler M, Wichmann H, Rot S, Hahnel A, Greither T, Said HM, Kotzsch M, Würl 

P, Taubert H, Vordermark D. (2010) Elevated tumor and serum levels of the hypoxiaassociated 

protein osteopontin are associated with prognosis for soft tissue sarcoma 

patients. BMC Cancer. 10:132. 

Eckert AW, Schubert J, Taubert H. Optimising the therapeutic ratio in head and neck cancer. 

Lancet Oncol. 2010 Jun;11(6):511-2. (letter/author reply) 

Eckert AW, Schutze A, Lautner MH, Taubert H, Schubert J, Bilkenroth U. HIF-1a is a 

prognostic marker in oral squamous cell carcinomas. Int J Biol Markers. 2010 Apr- 

Jun;25(2):87-92. 

Eckert AW, Lautner MHW, Schuetze A, Bolte K, Bache M, Kappler M, Schubert J, Taubert H, 

Bilkenroth U. (2010) Co-expression of Hif1a and CAIX is associated with poor prognosis in 

oral squamous cell carcinoma patients. J Oral Pathol Med. 39(4):313-7. 

Hahnel A, Wichmann H, Kappler M, Kotzsch M, Vordermark D, Taubert H, Bache M. Effects 

of osteopontin inhibition on radiosensitivity of MDA-MB-231 breast cancer cells. Radiat 

Oncol. 2010;5:82. 

Grochola LF, Müller TH, Bond GL, Taubert H, Udelnow A, Würl P. MDM2 SNP309 

Associates With Accelerated Pancreatic Adenocarcinoma Formation. Pancreas. 2010 

Jan;39(1):76-80. 

146

2009 

Hoffman AC, Danenberg KD, Taubert H, Danenberg PV, Wuerl P. A three-gene signature for 

outcome in soft tissue sarcoma. Clin Cancer Res. 2009 15(16):5191-8. 

Bluemke K, Bilkenroth U, Meye A, Fuessel S, Lautenschlaeger C, Goebel S, Melchior A, 

Heynemann H, Fornara P, Taubert H. Detection of circulating tumor cells in peripheral blood 

of patients with renal cell carcinoma correlates with prognosis. Cancer Epidemiol Biomarkers 

Prev 2009; 2009 Aug;18:2190-4. 

Grochola LF, Vazquez A, Bond EE, Würl P, Taubert H, Müller TH, Levine AJ, Bond GL. 

Recent Natural Selection Identifies a Genetic Variant in a Regulatory Subunit of Protein 

Phosphatase 2A that Associates with Altered Cancer Risk and Survival. Clin Cancer Res. 

2009;15(19):6301-8. 

Schmidt H, Taubert H, Lange H, Kriese K, Schmitt WD, Hoffmann S, Bartel F, Hauptmann 

S.Small polydispersed circular DNA contains strains of mobile genetic elements and occurs 

more frequently in permanent cell lines of malignant tumors than in normal lymphocytes. 

Oncol Rep. 2009 Aug;22:393-400. 

Seifert A, Taubert H, Hombach-Klonisch S, Fischer B, Navarrete Santos A.: TCDD mediates 

inhibition of p53 and activation of ERalpha signaling in MCF-7 cells at moderate hypoxic 

conditions. Int J Oncol. 2009;35:417-24. 


147

DEPARTMENT OF INTERNAL MEDICINE 5 

(Haematology / Oncology) 

Head: Andreas Mackensen, Prof. Dr. med. 

Research Cellular Immunoregulation 

Group: Evelyn Ullrich, Prof. Dr. med. 

Address: Department of Internal Medicine 5 

Ulmenweg 6 


Telefone: + 49 (9131) 85 43091 

Fax: + 49 (9131) 85 35958 

E-mail: evelyn.ullrich@uk-erlangen.de 

Homepage: http://www.medizin5.uk-erlangen.de 


Ruth Bauer, Dr. rer. nat. 


Kathrin Meinhardt 

Johanna Rothamer 


Stephanie Krieg 

148


Technicians 

Julia Schneider 

Franziska Ganß 

Our group analyses human and murine natural killer cells in different lymphoid and 

non-lymphoid organs. NK cells are a heterogeneous population of immune cells with 

cytotoxic capacity and multiple immunoregulatory properties. Until recently, studies 

on human NK cells have been mainly focused on NK cells from peripheral blood. The 

project based in the Nikolaus-Fiebiger-Center aims to characterize the genomic, 

phenotypical and functional profile of human NK cell subsets from different organs 

with a special interest in early forms of thymic NK cells in comparison to mature 

peripheral blood NK cells from adults and children. 

Phenotypical, functional and genomic analyses of human NK cell 


Human NK cells can be classified according to the expression of CD56 and CD16 

into immunoregulatory, cytokine producing CD56 high CD16 dim and mature cytotoxic 

CD56 dim CD16 high NK cells. In contrast to murine NK cell subpopulations that have 

been extensively studied in different compartments, studies on human NK cells have 

been mainly focused on NK cells from peripheral blood. Little is known about the 

functional role of human NK cell subpopulations in different lymphoid organs. 

Therefore, a translation of murine studies, mainly performed on splenic NK cells, into 

the human system remains quite difficult. In our first analyses of human NK cell 

subsets from peripheral blood, spleen, bone marrow, and thymus, we observed 

relevant differences concerning the distribution of CD56 high CD16 dim and 

CD56 dim CD16 high NK cells. In addition, the expression of functionally relevant surface 

molecules varies on the NK cell subsets. 

One important observation from our phenotypic analyses was that especially the 

composition of NK cell subpopulations in the thymus differs in particular from those in 

spleen and peripheral blood. For that reason that thymus is already well-known for 

being an organ for immune cell development, we further focused on the 

characterization of thymic NK cells. NK cells from thymus have not only a lower 

CD56-expression but also a different ratio of CD16 high / CD16 dim NK cells compared 

to peripheral blood NK cells (Fig.1). While peripheral blood NK cells from adults have 

a ration of 10 / 1, children have a ratio of only 4 / 1 and those of child thymus have an 

even lower ratio of < 1 (Fig.2). 

149

Fig. 1 FACS analysis of NK cell subpopulations in child PBMC and 

thymic MNC. Two populations can be distinguished according to 

the expression of CD56 and CD16. CD16 dim thymic NK cells have a 

lower CD56-expression compared to peripheral blood NK cells. 

Importantly, the NK cell subset ratio shows an age-dependency that differs in 

peripheral blood and thymus. Especially the number of CD16 dim immunoregulatory 

NK cells increases in thymus, but decreases in peripheral blood with age so that the 

ratio of CD16 high / CD16 dim NK cells increases in total (Fig.2). This process might be 

explained by acquisition of surface molecules that regulate migration, homing and 

differentiation of early forms of NK precursors, immature, immunoregulatory or 

mature NK cells. 

Fig. 2 Ratio of CD16 high / CD16 dim NK cells in peripheral blood and 

thymus of children. This NK subset ratio is lower in thymus and 

showed a negative correlation with increasing donor’s age. 

To get further insights, we performed microarray analyses that will provide us with 

detailed information on the gene expression profile of NK cell populations from 

different organs. In addition to the genomic and phenotypical analysis, we 

investigated the cytokine production and cytotoxicity of these different NK cell 

150

subpopulations. Our data clearly demonstrates that NK cells from thymus as well as 

from peripheral blood of young children produce only low amounts of IFN-γ and need 

additional IL-2 stimulation to develop an antitumor capacity that is still under 

averaged compared to adult NK cells (data not shown). 

In summary, this comparative study of NK cell subsets provides important information 

on the development and function of NK cells that will be of great value for the 

optimization of cellular therapy. 



Ullrich E, Bosch J, Aigner M, Völkl S, Dudziak D, Spriewald B, Schuler G, 

Andreesen R, Mackensen A. Advances in Cellular Therapy: 5th International 

Symposium on the Clinical Use of Cellular Products, March 19 and 20, 2009, 

Nürnberg, Germany. Cancer Immunol. Immunother. 2009; Oct 28. [Epub ahead of 

print] 

Terme M, Ullrich E, Delahaye N, Chaput N, Zitvogel L. NK cell-directed 

therapies: from unexpected results to successful strategies. Nat.Immunol. 

2008;9(5):486-94. 

Ullrich E, Bonmort M, Mignot G, Jacobs B, Bosisio D, Sozzani S, et al. Transpresentation 

of Interleukin-15 dictates IKDC effector functions. J.Immunol. 

2008;180(12):7887-97. 

Mignot G, Ullrich E, Bonmort M, Menard C, Apetoh L, Taieb J, et al. The 

critical role of IL-15 in the antitumor effects mediated by the combination therapy 

imatinib and IL-2. J.Immunol. 2008; 180(10):6477-83. 

Ullrich E, Chaput N, Zitvogel L. Killer dendritic cells and their potential role in 

immunotherapy. Hormone and Metabolic Research 2008;40(2):75-81. 

Ullrich E, Bonmort M, Mignot G, Kroemer G, Zitvogel L. Tumor stress, cell 

death and the ensuing immune response. Cell Death.Differ. 2008;15(1):21-8. 

Ullrich E, Menard C, Flament C, Terme M, Mignot G, Bonmort M, et al. 

Dendritic cells and innate defense against tumor cells. Cytokine Growth Factor Rev. 

2008; 19(1):79-92. 

151

Ullrich E, Bonmort M, Mignot G, Chaput N, Taieb J, Menard C, et al. Therapy- 

Induced Tumor Immunosurveillance Involves IFN-Producing Killer Dendritic Cells. 

Cancer Res. 2007;67(3):851-3. 

Taieb J, Chaput N, Menard C, Apetoh L, Ullrich E, Bonmort M, et al. A novel 

dendritic cell subset involved in tumor immunosurveillance. Nat.Med. 2006;12(2):214- 

9. 



Kathrin Meinhardt 

Isolation and characterization of murine NK cell subpopulations in vitro und in vivo 

Johanna Rothamer 

Investigation of the role of TH17 cells in a murine model of GVHD 


Stephanie Krieg 

Functional, phenotypical und genomic analyses of human NK cells subpopulations 

152

AG Wiesener 

Department of Nephrology and Hypertension 

Group leader: Michael Wiesener, Prof. Dr. med. 

Address: Hypoxia Research Group 

Glückstr. 6 


Telefone: + 49 (9131) 85 36353 

Fax: + 49 (9131) 85 39209 

E-mail: michael.wiesener@uk-erlangen.de 

Karl Knaup, Dr. rer. nat. 

Ruth Schietke, Dr. rer. nat.* 

* period of time reported 

Head 

Michael Wiesener, Prof. Dr. med. 


153 

Thomas Hackenbeck, Dr. rer. nat.

Tilman Jobst-Schwan 

Sina Grupp 


Juliana Monti* 



Stefan Thieme 

Regina Günther 

Technicians 


154 

- 

Johanna Stöckert


The constant availability of molecular oxygen is crucial for function and survival of 

any cell and tissue of organisms of higher order. All mammalian cells investigated to 

date are capable of utilising an important adaptive mechanism, which is strongly 

influenced by oxygen tensions. Hypoxia-Inducible transcription-Factors (HIF) are 

activated by a decline of oxygen supply and stimulate numerous target genes that 

convey adaptation to hypoxia. This system is probably of great importance for the 

growth of solid tumours. Inactivation of the “von Hippel Lindau” tumour suppressor 

gene, which is part of the destruction apparatus of HIF, leads to strong upregulation 

of HIF in clear cell renal carcinoma. Importantly, early HIF activation may play an 

important role in tumorigenesis of VHL associated tumours. These tumours could 

therefore function as a model system to study causes and consequences of 

pronounced HIF expression. 

Our group mainly investigates the role and effect of overexpression of both HIFα 

subunits, HIF-1α and HIF-2α, in tumour tissues, cell culture models, as well as rodent 

models. The role of selected HIF target genes in tumour growth is being analysed. 

Genetically modified mice for HIF overexpression in the kidney have been generated, 

to investi-gate whether HIF plays a role in tumorigenesis. 

The role of HIF in renal epithelial cells and tumorigenesis 

Renal tubular HIF-2α expression requires VHL inactivation and causes fibrosis 

and cysts 

A number of publications have indicated tumorigenic potential of HIF, which may 

primarily concern HIF-2α and particularly for clear cell RCC. We have studied the 

expression patterns of HIFα isoforms in the renal tubules in detail. In three different 

species (mouse, rat and human) microenvironmental stimulation by hypoxia always 

leads to tubular HIF-1α activation, but never to HIF-2α stabilization. Since HIF-2α is 

widely expressed in human RCC and may be functionally more important in terms of 

tumorigenesis, de novo expression of HIF-2α may indicate an oncogenic switch of 

renal tubular cells. In line with this, we have studied two different kidney specific 

knockout models for the tumour suppressor VHL, which shows de novo expression of 

HIF-2α, next to HIF-1α. Thus, the deletion of VHL may enable expression of HIF-2α, 

which may have an oncogenic potential. 

We have therefore established transgenic mice for overexpression of HIF-2α in distal 

tubuli of the kidney, driven by the Ksp-Cadherin promotor (Schietke and Hackenbeck 

et al., PLoSOne 2012). We have not observed any malignant tumors in this model, 

but the transgenic HIF-2α animals develop an interesting phenotype at 12 months of 

age. The kidneys showed obvious morphological differences on the surface in 

155

comparison to the control animals. Control animals displayed a uniform and smooth 

kidney surface, whereas the kidneys of HIF-2 expressing mice had an irregular and 

rough appearance and a significantly reduced kidney weight as judged by kidney to 

body weight ratio. 

Histological analysis of the kidneys from the transgenic mice showed a strong 

increase of fibrotic tissue in comparison to healthy kidneys and an increase of 

fibrosis-associated genes like TGFβ1. We additionally determined plasma 

parameters for renal function. Furthermore, transgenic mice had significantly 

increased creatinine levels confirming impaired renal function (Fig. 1) 

Figure 1: Renal tubular HIF-2α expression induces fibrosis and results in reduced kidney 

function. A. Immunohistochemical analysis of HIF-2α and collagen I in a kidney from a transgenic 

HIF-2 expressing mouse. B. Blinded analysis of renal fibrosis after SiriusRed (total collagen) staining 

C. mRNA expression (RT-PCR) of TGFβ1 from total kidney lysates. D. Kidney function as a 

measurement of serum creatinine of transgenic HIF-2α mice (* p < 0.05). All figures: tmHIF-2α.HA(-): 

transgen negative; tmHIF-2α-HA(+): transgen positive. 

In addition to the fibrotic phenotype, the histological analysis of the tmHIF-2α.HA(+) 

transgenic mice revealed frequent formation of cysts in the renal cortex, which was 

not observed in the control strain (Fig. 2). 

156

Figure 2: Renal cyst development in aged HIF-2α transgenic mice. 12 month and older HIF-2α 

expressing mice develop multiple renal cysts, mainly in the kidney cortex. These partly form around 

glomeruli, where the glomerular tuft can be seen (arrows in A). Other cysts arise from distal tubular 

segments. 

In summary we could demonstrate, that continuous transgenic expression of HIF-2α 

by the Ksp-Cadherin promotor leads to renal fibrosis and insufficiency, next to 

multiple renal cysts. Our data cleary show, that unphysiological activation of HIF 

contributes to renal epithelial dedifferentiation. 

Differential role of HIFalpha subunits 

HIF-1 or HIF-2 induction is sufficient to achieve cell cycle arrest in NIH3T3 

mouse fibroblasts independent from hypoxia 

Hypoxia is a severe stress which induces physiological and molecular adaptations, 

where the latter is dominated by the Hypoxia-inducible transcription Factor (HIF). A 

well described response on cellular level upon exposure to hypoxia is a reversible 

cell cycle arrest, which probably renders the cells more resistant to the difficult 

environment. The individual roles of hypoxia itself and of the isoforms HIF-1α and 

HIF-2α in cell cycle regulation are poorly understood and discussed controversially. 

In order to characterize the isolated effect of both HIFα isoforms on the cell cycle we 

generated tetracycline inducible, HIF-1α and -2α expressing NIH3T3 cells 

(Hackenbeck et al., Cell Cycle 2009). The cDNAs for HIFα were mutated to generate 

stable and active HIF under normoxia. Upon activation of both HIFα subunits, the 

total number of living cells was reduced and long-term stimulation of HIF led to 

complete loss of transgene expression, implicating a strong negative selection 

pressure. Equally, colony forming activity was reduced by activation of both HIFα 

subunits. Cell cycle analyses showed that HIF activation resulted in a prominent cell 

cycle arrest in G1-phase, similarly to the hypoxic effect. Both, HIF-1α and HIF-2α 

157

were able to induce the expression of the cyclin-dependent kinase inhibitor p27 on 

reporter gene and protein level. 

Our study shows that HIF-1 and HIF-2 can individually arrest the cell cycle 

independent from hypoxia. These findings have implications for the resistance of 

tumor cells to the environment and treatment, but also for physiological cells. 

Importantly, recent approaches to stabilize HIFα in normoxia could have deleterious 

effects on proliferating tissues. 

Regulation of HIF by the mammalian target of rapamycin (mTOR) 

Consequences of genetic mTOR activation on HIF expression 

Stabilisation of the Hypoxia Inducible Transcriptionfactor (HIF) is a global 

phenomenon in most solid tumors. The predominant regulation of HIF is controlled 

via a prolyl hydroxylase (PHD) mediated oxygen-dependent, posttranslational 

degradation. The PI3K/TSC/mTOR pathway is an alternative pathway, which is 

capable of enhancing HIF levels. mTOR itself is negatively regulated via the 

Tuberous Sclerosis Complex (TSC), which consists of and is only functional in 

combination of both subunits (TSC1 and TSC2). In the disease of “Tuberous 

Sclerosis” (TS) patients harbour a heterozygous germline mutation of either the 

TSC1 or TSC2 gene. Due to a somatic “second hit” these individuals develop a 

variety of tumors, including angiomyolipomas of the kidney. Our study aims to 

analyze the impact and the consequences of this genetic mTOR activation on HIF 

accumulation. We are approaching this by pursuing three experimental avenues: 

1. in vitro analysis of the molecular mechanisms with an doxycyclin inducible siTSC2 

HeLa cell line (in cooperation with Dr. Kathrin Thedick, Albert-Ludwigs-University of 

Freiburg) shows that HIF is stabilized quicker, if there is previous mTOR activation to 

hypoxic exposure. (Fig. 3). 

158 

Fig. 3: Immunoblots of doxycyclin inducible 

siTSC2 HeLa cells. TSC2 reduction leads to an 

(A) activation of mTOR by increased rpS6 

phosphorylation at Ser 235/236 and (B) an 

earlier accumulation of HIF-1α under mild 

hypoxia (3% O2). ß-actin is implied as a loading 

control.

Apart from our previously described interplay of the mTOR and the HIF system 

(Knaup et al., Mol Canc Res 2009) this supports the thesis of an oxygen independent 

regulation pathway of HIF by mTOR. 

2. Immunohistochemical analysis of the mTOR and the HIF pathway in kidney samples 

of patients with TS reveals an unphysiological HIF accumulation in the tubules. 

This is probably due to the genetic inactivation of TSC and the resulting subsequent 

activation of mTOR (Fig. 4). Whether this unphysiological HIF expression in the 

healthy kidney directly influences the development and/or the growth of the tumors 

(angiomyolipomas) is subject of ongoing analysis. 

Figure 4: Immunohistohemical analysis of mTOR activation (P-rpS6) and HIF-1α stabilization in both 

angiomyolipoma and adjacent kidney samples from human tuberous sclerosis patients. 

HIF and the HIF target gene HIG2 (data not shown) are strongly expressed in angiomyolipomas. 

This could be due to either regional hypoxia as well as mTOR 

activation, possibly a combination of both scenarios. 

3. We are currently analysing the role of HIF activation in the tumor development and 

growth in a TSC2 knockout mouse model which was supplied to us by the group of 

Prof. T. Noda, Tokyo, Japan (Kobayashi, T. et al, Cancer Res. 1999 Mar 

15;59(6):1206-11). 

159

Figure 5: A. Kidney of a 7 months old TSC2 knockout mouse (supplied to us from the group of Prof. 

T. Noda, Tokyo, Japan), which developed a macroscopically visible tumor on the kidney surface. B. 

Morpholgical evaluation classifies this as being a papillary tumor. C and D. mTOR activation in the 

tumor can be detected by immunohistochemical staining of P-rpS6 (Ser 235/236). 

The knockout mice develop macroscopically visible tumors on the surface of the kidneys 

(Fig. 5, A). These tumors have papillary structures (B) and express high levels 

of phospho-rpS6 (Ser235/236) (C and D), which is a profound marker of mTOR 

activation. Our current data suggests that this genetic mTOR activation can enhance 

accumulation of HIF and therefore support tumor development and growth. We are 

currently analyzing the kidneys and the developing tumors of these mice in this 

context. 

In this ongoing study we hope to gain more insight into the molecular mechanisms of 

the mTOR-HIF pathway. Further, we are aiming to investigate the impact of 

pharmacological mTOR inhibition on tumor development and growth and its 

consequences on HIF expression and functionality in vitro and in vivo. Ongoing 

clinical studies with TS-patients and mTOR inhibitors are showing promising results, 

encouraging us in our effort to dissect the details and mechanisms of the mTOR-HIF 

interplay. 

Identification and characterisation of new HIF target genes 

In order to understand the function and mechanism of action of the HIF transcription 

factors it is important to characterise the spectrum of its target genes. We have 

previously performed affymetrix arrays to identify novel HIF-1α and HIF-2α targets 

(Warnecke et al., Exp. Cell Res. 2008) and selected a number of those for further 

analysis with potential functionality in tumor biology. 

160

The GTPase Rab20 is a HIF target with mitochondrial localization mediating 

apoptosis in hypoxia 

We were able to demonstrate that Rab20, which is a member of the Rab family of 

small GTP-binding proteins, regulating intracellular trafficking and vesicle formation, 

is a new HIF-1 target gene. Rab20 is directly regulated by HIF-1, resulting in rapid 

upregulation of Rab20 mRNA as well as protein under hypoxia. Furthermore, 

exogenous as well as endogenous Rab20 protein colocalizes with mitochondria. 

Knockdown studies revealed that Rab20 is involved in hypoxia induced apoptosis. 

Since mitochondria play a key role in the control of cell death, we suggest that 

regulating mitochondrial homeostasis in hypoxia is a key function of Rab20. 

Furthermore, our study implicates that cellular transport pathways play a role in 

oxygen homeostasis. Hypoxia-induced Rab20 may influence tissue homeostasis and 

repair during and after hypoxic stress. (Hackenbeck et al., Biochim Biophys Acta 

2011). 

The lysyl oxidases LOX and LOXL2 are necessary and sufficient to repress Ecadherin 

in hypoxia: insights into cellular transformation processes mediated 

by HIF-1. 

In our array experiments, the lysyl oxidases Lox and LoxL2 showed a very high level 

of regulation, where LOX has previously been shown to be a HIF-1 target gene (Erler 

et al., Nature 2006). We were able to show that LOXL2 is a direct target as well. Both 

genes are very highly expressed in clear cell RCC and are involved in repression of 

the epithelial marker E-cadherin – a hallmark of the epithelial to mesenchymal 

transformation (EMT). EMT is of great importance for tumour cells to progress into an 

increasingly aggressive phenotype, as well as for diseased tissues into fibrosis. Thus, 

we believe that the lysyl oxidases represent a relevant mechanism in tumour biology, 

possible an effective area for pharmacological intervention (Schietke et al., J Biol 

Chem. 2010). 

Miscellaneous kidney related research projects 

The inhibitor of apoptosis protein surviving accumulates at the apical 

membrane of renal proximal tubules, which colocolises with megalin 

In the past report period we also analysed the inhibitor of apoptosis protein survivin 

and its role in kidney function. Survivin is a bifunctional molecule, which regulates 

cellular division and survival. A paradigm of a restricted oncofetal role for survivin has 

been formulated, where most of the published work implicates a function merely in 

embryonic and malignant tissues. In contrast, we have previously shown that survivin 

161

protein is highly expressed in the adult kidney, particularly in the proximal tubules and 

to a weaker extent in podocytes (Lechler et al., Am J Pathol 2007). 

We were able to confirm and expand our previous work, which identified survivin as 

being strongly present in healthy adult kidneys. Survivin predominantly accumulates 

at the apical membranes of proximal tubules, demonstrated clearly in vivo by 

immunostaining and electron microscopy. The apical localisation at the membrane of 

the proximal tubules reveals a pattern, which resembles that of many absorbed 

proteins. Survivin is a small protein of approximately 14 kDa which could, if 

circulating, be freely filtered by the glomerular apparatus. Many proteins in primary 

urine are internalised by megalin, an endocytosis receptor, of which the expression 

pattern is in principle comparable to the observed survivin pattern. Therefore, we 

generated a hypothesis of an active renal uptake mechanism for survivin, possibly by 

a megalin dependent process. We could clearly demonstrate in vitro, that polarised 

primary renal tubular cells store survivin at the apical membrane, which is true for 

both, endogenous and exogenously added recombinant survivin protein. Finally, 

knockout animals for renal megalin showed accumulation of survivin in their urine. 

We are currently in the process of preparing a manuscript for submission for these 

data (Jobst-Schwan et al.). 

Analyses of molecular causes of different genetic renal diseases 

We have recently been engaged in attempting to clarify the genetic cause of a 

number of hereditary diseases, which lead to end-stage renal disease. A moderate 

number of families are treated in our department, where we have undertaken linkage 

and locus analyses by next generation sequencing. We have identified families with 

medullary cystic kidney disease type 1 (MCKD1), as well as Alport´s disease and 

membranoproliferative glomerulonephritis, as well as so far uncharacterised 

hereditary diseases. These studies are ongoing. We hope to be able to take some of 

these genetic studies on to functional and molecular biology investigations after 

identification of the causative genes. 

2011/12 

Publications (2009-2011/12) 


162

Buettner M, Xu H, Böhme R, Seliger B, Jacobi J, Wiesener M, Benz K, Amann K. 

Predominance of Th2 cells and plasma cells in polyoma virus nephropathy: a role for 

humoral immunity? Hum Pathol. 2012 Mar 8. 

Schietke RE, Hackenbeck T, Tran M, Günther R, Klanke B, Warnecke CL, Knaup 

KX, Shukla D, Rosenberger C, Koesters R, Bachmann S, Betz P, Schley G, Schödel 

J, Willam C, Winkler T, Amann K, Eckardt KU, Maxwell P, Wiesener MS. Renal 

tubular HIF-2α expression requires VHL inactivation and causes fibrosis and cysts. 

PLoS One. 2012;7(1):e31034. 

Hainz N, Thomas S, Neubert K, Meister S, Benz K, Rauh M, Daniel C, Wiesener M, 

Voll RE, Amann K. The Proteasome Inhibitor Bortezomib Prevents Lupus Nephritis in 

the NZB/W F1 Mouse Model by Preservation of Glomerular and Tubulointerstitial 

Architecture. Nephron Exp Nephrol. 2012;120(2):e47-58. 

Klinger P, Schietke RE, Warnecke C, Swoboda B, Wiesener M, Hennig FF, Gelse K. 

Deletion of the oxygen-dependent degradation domain results in impaired 

transcriptional activity of hypoxia-inducible factors. Transcription. 2011 Nov- 

Dec;2(6):269-75. 

Schley G, Klanke B, Schödel J, Forstreuter F, Shukla D, Kurtz A, Amann K, Wiesener 

MS, Rosen S, Eckardt KU, Maxwell PH, Willam C. Hypoxia-inducible transcription 

factors stabilization in the thick ascending limb protects against ischemic acute 

kidney injury. J Am Soc Nephrol. 2011 Nov;22(11):2004-15. 

Hackenbeck T, Huber R, Schietke R, Knaup KX, Monti J, Wu X, Klanke B, Frey B, 

Gaipl U, Wullich B, Ferbus D, Goubin G, Warnecke C, Eckardt KU, Wiesener MS. 

The GTPase RAB20 is a HIF target with mitochondrial localization mediating 

apoptosis in hypoxia. Biochim Biophys Acta. 2011 Jan;1813(1):1-13. 

2010 

Bernhardt WM, Wiesener MS, Scigalla P, Chou J, Schmieder RE, Günzler V, Eckardt 

KU. Inhibition of prolyl hydroxylases increases erythropoietin production in ESRD. J 

Am Soc Nephrol. 2010 Dec;21(12):2151-6. 

Gimm T, Wiese M, Teschemacher B, Deggerich A, Schödel J, Knaup KX, 

Hackenbeck T, Hellerbrand C, Amann K, Wiesener MS, Höning S, Eckardt KU, 

Warnecke C. Hypoxia-inducible protein 2 is a novel lipid droplet protein and a specific 

target gene of hypoxia-inducible factor-1. FASEB J. 2010 Nov;24(11):4443-58. 

Schietke R, Warnecke C, Wacker I, Schödel J, Mole DR, Campean V, Amann K, 

Goppelt-Struebe M, Behrens J, Eckardt KU, Wiesener MS. The lysyl oxidases LOX 

163

and LOXL2 are necessary and sufficient to repress E-cadherin in hypoxia: insights 

into cellular transformation processes mediated by HIF-1. J Biol Chem. 2010 Feb 

26;285(9):6658-69. 

2009 

Morris MR, Hughes DJ, Tian YM, Ricketts CJ, Lau KW, Gentle D, Shuib S, Serrano- 

Fernandez P, Lubinski J, Wiesener MS, Pugh CW, Latif F, Ratcliffe PJ, Maher ER. 

Mutation analysis of hypoxia-inducible factors HIF1A and HIF2A in renal cell 

carcinoma. Anticancer Res. 2009 Nov;29(11):4337-43. 

Bernhardt WM, Gottmann U, Doyon F, Buchholz B, Campean V, Schödel J, 

Reisenbuechler A, Klaus S, Arend M, Flippin L, Willam C, Wiesener MS, Yard B, 

Warnecke C, Eckardt KU. Donor treatment with a PHD-inhibitor activating HIFs 

prevents graft injury and prolongs survival in an allogenic kidney transplant model. 

Proc Natl Acad Sci U S A. 2009 Dec 15;106(50):21276-81. 

Weidemann A, Kerdiles YM, Knaup KX, Rafie CA, Boutin AT, Stockmann C, Takeda 

N, Scadeng M, Shih AY, Haase VH, Simon MC, Kleinfeld D, Johnson RS. The glial 

cell response is an essential component of hypoxia-induced erythropoiesis in mice. J 

Clin Invest. 2009 Nov;119(11):3373-83. 

Tanaka T, Wiesener M, Bernhardt W, Eckardt KU, Warnecke C. The human HIF 

(hypoxia-inducible factor)-3alpha gene is a HIF-1 target gene and may modulate 

hypoxic gene induction. Biochem J. 2009 Oct 23;424(1):143-51. 

Kroening S, Neubauer E, Wessel J, Wiesener M, Goppelt-Struebe M. Hypoxia 

interferes with connective tissue growth factor (CTGF) gene expression in human 

proximal tubular cell lines. Nephrol Dial Transplant. 2009 Nov;24(11):3319-25. 

Schödel J, Klanke B, Weidemann A, Buchholz B, Bernhardt W, Bertog M, Amann K, 

Korbmacher C, Wiesener M, Warnecke C, Kurtz A, Eckardt KU, Willam C. HIF-prolyl 

hydroxylases in the rat kidney: physiologic expression patterns and regulation in 

acute kidney injury. Am J Pathol. 2009 May;174(5):1663-74. 

Hackenbeck T, Knaup KX, Schietke R, Schödel J, Willam C, Wu X, Warnecke C, 

Eckardt KU, Wiesener MS. HIF-1 or HIF-2 induction is sufficient to achieve cell cycle 

arrest in NIH3T3 mouse fibroblasts independent from hypoxia. Cell Cycle. 2009 May 

1;8(9):1386-95. 

164

Wacker I, Sachs M, Knaup K, Wiesener M, Weiske J, Huber O, Akçetin Z, Behrens J. 

Key role for activin B in cellular transformation after loss of the von Hippel-Lindau 

tumor suppressor. Mol Cell Biol. 2009 Apr;29(7):1707-18. 

Knaup KX, Jozefowski K, Schmidt R, Bernhardt WM, Weidemann A, Juergensen JS, 

Warnecke C, Eckardt KU, Wiesener MS. Mutual regulation of hypoxia-inducible factor 

and mammalian target of rapamycin as a function of oxygen availability. Mol Cancer 

Res. 2009 Jan;7(1):88-98. 


Wiesener MS, Maxwell PH, Eckardt KU. Novel insights into the role of the tumor 

suppressor von Hippel Lindau in cellular differentiation, ciliary biology, and cyst 

repression. J Mol Med (Berl). 2009 Sep;87(9):871-7. 



Ruth Schietke (2009) 

Thomas Hackenbeck (2009) 


Stefan Thieme 

Tilman Jobst-Schwan 

Sina Grupp 

Regina Günther 

165

TEACHING ACTIVITIES 

at the Nikolaus-Fiebiger-Center 

LECTURES, SEMINARS AND PRACTICAL COURSES 

Members of the Nikolaus-Fiebiger-Center participate in courses and lectures that are 

currently offered to students enrolled in the Graduate Programs in Biology at the 

School of Basic Sciences II (Naturwissenschaftliche Fakultät II) and in Molecular 

Medicine at the Medical School as well as to medical students. The focus is on basic 

and advanced molecular cell biology (Behrens, von der Mark, Müller) and basic and 

clinical immunology (Jäck). 

166

SEMINARS AND CONFERENCES 

at the Nikolaus-Fiebiger-Center 

Selection of Speakers since January 2009: 

NIKOLAUS-FIEBIGER-SEMINAR 

K. Förstemann, München 

Gene Regulation by Small RNAs- General Principles and Examples from Drosophila 

28.01.2009 

C. Stocking, Hamburg 

Activated FLT3 Tyrosine Receptor Kinase in Acute Lymphoblastic Leukemiea 

20.05.2009 

H. Urlaub, Göttingen 

Quantitative Mass Sepctrometry to Elucidate B Cell Receptor Signaling 

29.07.2009 

K. Toellner, Birmingham, UK 

Imprinting B Cells: Germinal Centres and Plasma Cells – Two sides of the Coin 

21.10.2009 

E. Izaurralde, Tübigen 

Mechansim of miRNA-mediated gene Silencing 

31.03.2010 

T. Nguyen, Paris 

The (pro)renin receptor: important for blood pressure and organ damage or just in 

development? 

23.02.2011 

G. Weidinger, Dresden 

Wnt-signaling in Development and Regeneration 

11.05.2011 

R. Renkawitz-Pohl, Marburg & S. Lindner, Hamburg, D. Mielenz, Erlangen 

Interdisciplinary Minisymposium 

18.05.2011 

R. Obst, München 

Timed antigen presentation and T cell responses 

25.05.2011 

M. Flajnik, Baltimore, USA 

Evolution of the Immune System 

15.06.2011 

167

G. Riemekasten, Berlin 

Systemic sclerosis as prototypic disease for functional autoantibodies against vascular 

receptors 

06.07.2011 

H. Bastians, Göttigen 

Mechanisms underlying Chromosomal Instability in Human Cancer 

12.07.2011 

N. Abdalah 

The interplay of behavior and adult neurogenesis for cognitive and affective disorders 

27.07.2011 

B. Blomberg, Miami, USA 

Molecular mediators of autonomous B cell deficiencies in aging mice and humans 

19.09.2011 

R. Blum, Würzburg 

The sodium channel Nav1.9 regulates activity-dependent axon growth and might serve as a 

target for axon regeneration and maintenance 

05.10.2011 

S. Herzog, Freiburg 

BIOSS – Centre for Biological Signaling Studies 

23.05.2012 

A. Blesch, Heidelberg 

Challenges and Opportunities in Spinal Cord Regeneration 

20.06.2012 

168

Research Report - Nikolaus-Fiebiger-Zentrum für Molekulare Medizin

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