Supplementum 163 - Swiss Medical Weekly

41 S SWISS MED WKLY 2008;138(Suppl 163) · www.smw.ch
Poster Viewing
HGF receptor, c-met, is restricted to the peak phase of EAE disease
and is mainly expressed by CD11b+ macrophages/microglia within
demyelinated lesions in the spinal cord. In addition, a small
percentage of CD11c+ dendritic cells (DCs) and NG2+ OPCs were
found to express c-met, whereas CD4+ T-cells were completely
c-met negative. Flowcytometric analysis confirmed these
observations. The timing of c-met expression, with expression
limited to the peak phase of disease, and the robust expression by
macrophages indicates a potential pro-inflammatory role for the
HGF-c-met pathway in EAE.
Subsequent in vitro experiments, using bone marrow-derived
macrophages (BMM), showed that quiescent macrophages do not
express c-met but that c-met mRNA and protein expression can be
induced by treatment with TNF-alpha or LPS. Furthermore,
experiments with soluble TNF receptor and monoclonal anti-TNFalpha
antibodies demonstrated that LPS-induced c-met expression is
mediated via autocrine TNF-alpha signalling.
Although the function of c-met in macrophages remains unclear our
data suggests it plays no role in classical macrophage functions such
as phagocytosis, cytokine secretion and NO production. Currently,
EAE experiments with macrophage-specific c-met knockout mice as
well as gene chip arrays with HGF-treated BMM are being performed
to elucidate the role of c-met in macrophages in EAE.
P180
The role of the pro-apoptotic Bcl-2 family member Bim
in GM-CSF-regulated neutrophil apoptosis
N. Andina, H-U. Simon.
Pharmakologisches Institut (Bern, CH)
Background: Neutrophils are constantly produced in large numbers
in the bone marrow. After maturation, they enter into the circulation
and undergo apoptosis. However, under inflammatory conditions,
neutrophil apoptosis is delayed due to survival factor exposure, and,
consequently, cell numbers increase. An important pro-inflammatory
cytokine involved in the regulation of neutrophil survival/activation is
granulocyte-macrophage colony stimulating factor (GM-CSF).
Although GM-CSF mediates anti-apoptotic effects in neutrophils, it
does not prevent apoptosis, and the survival effect is both timedependent
and limited. We therefore hypothesised that GM-CSF, in
spite of mediating anti-apoptotic effects, allows the activation of proapoptotic
pathways.
Methods: Global gene expression was assessed using
oligonucleotide microarrays. For protein detection cell lysates were
prepared and Bim protein expression was assessed using
immunoblotting. Cell survival was determined by flow cytometric
analysis of ethidium bromide uptake.
Results: Analyzing global gene expression in untreated and GM-CSF
– stimulated human peripheral blood neutrophils revealed that GM-
CSF induced the expression of pro-apoptotic Bcl-2 family member
Bim. To verify these findings, we performed immunoblotting analysis
and observed increased Bim protein levels in GM-CSF – stimulated
blood neutrophils compared to freshly isolated or cultured control
blood neutrophils. Bim upregulation was dependent on de novo
transcription and translation as it was blocked by both cycloheximide
(translation inhibitor) and actinomycin D (transcription inhibitor).
Pharmacological inhibition of PI3K (LY294002) blocked GM-CSF –
mediated survival and, surprisingly, decreased Bim expression in
neutrophils. The functional role of Bim was investigated using Bim
deficient mouse neutrophils. Lack of Bim expression reduced
spontaneous neutrophil death. Moreover, in the absence of Bim, both
GM-CSF and IL-3 demonstrated much higher efficacy to block
neutrophil apoptosis.
Conclusions: These data demonstrate a functional role for Bim in the
regulation of neutrophil apoptosis and suggest that GM-CSF and
other neutrophil hematopoietins initiate a pro-apoptotic counter
regulation that involves up-regulation of Bim.
P181
TGFb receptor II gene deletion in leukocytes exacerbates
experimental autoimmune encephalomyelitis
U. Malipiero1 , F. Ackermann1 , T. Suter1 , W. Reith2 , A. Fontana1 .
1 Universitätsspital Zürich (Zürich, CH); 2 Centre Medical Universitaire
(Genève, CH)
The TGFb family of proteins are secreted molecules with potent
immunoregulatory properties. All three isoforms, TGFb -1, -2 or -3,
signal via the same heteromeric receptor complex, consisting of a
ligand binding TGFb receptor type II (TRII) and a TGFb receptor type
I. TGFb -1 secreted by macrophages and monocytes can regulate
many activities of these immune cells such as expression of MHCII,
chemotaxis, production of cytokines and their receptors, production
of reactive oxygen species and nitric oxide. Activation and infiltration
of macrophages is seen in various disease models including
experimental autoimmune encephalomyelitis (EAE). The current study
investigates the role of TGFb on the macrophage compartment in
inflammatory and autoimmune disease models. In order to interrupt
the TGFb-signaling pathway in macrophages, we delete the TRII by
breeding floxed TRII mice with mice expressing Cre recombinase
under the macrophage-specific mouse lysozymeM promoter. The
LPS-induced production of TNFa is not inhibited by TGFb in Creexpressing
macrophages. Macrophages of wildtype animals show a
50–80% reduction of TNFa secretion in the presence of TGFb. The
results show clearly that the TGFb-signaling pathway in macrophages
is not functional. TGFb macrophage receptor knockout mice
immunized with MOG35-55 to develop EAE show a significantly
higher disease score in the late phase of the disease compared to
control animals. Mononuclear cells isolated from the CNS of EAE
mice are currently tested for cytokine production.
P182
Activation of NK cells in drug hypersensitivity
A. Zawodniak1 , B. Gerber1 , T. Kawabata2 , W. Pichler1 .
1 University Berne (Berne, CH); 2 Pfizer (Groton, USA)
Introduction: Cytotoxic cells are critically involved in different forms
of drug hypersensitivity. We have recently shown that drug-specific
cytotoxic T cells can be detected in peripheral blood of drug-allergic
patients. The role of natural killer (NK) cells, as an important
component of innate immunity, in drug induced cytotoxicity has not
been investigated so far.
Methods: To asses whether specific drug stimulation can induce
activation and degranulation of NK cell in peripheral blood of drug
allergic patients, peripheral blood mononuclear cells (PBMC) of drug
allergic patients and healthy subjects were isolated from blood and
frozen. After thawing, PBMC were incubated with medium alone
(negative control), with the culprit drug and superantigen SEB
(positive controls). Following stimulation, three parameters were
evaluated: a) cell surface expression of CD69 as a stimulation marker
and b) CD107a as a degranulation marker by FACS, and c)
measurement of granzymeB production by ELISPOT.
Results: Stimulation of PBMC from allergic patients with the specific
drug lead to the upregulation of CD69 and CD107a on the surface of
NK cells. Delayed upregulation of CD107a (the earliest after 24h) on
NK cells from freshly isolated PBMC correlated with the kinetics of
CD107a expression on CD4 and CD8 T cells. Interestingly, the
percentage of NK cells upregulating CD69 and CD107a after
stimulation with the drug was 10–20 times higher than the percentage
of activated T cells, and was thus a more robust marker for drug
sensitization than the activation of T cells itself. Within total PBMC,
the number of cells releasing granzymeB was much lower and
corresponded better to the number of CD107a-positive T cells,
however. Restimulation after a 7d culture with the drug and antigen
presenting cells caused immediate (5h) degranulation of T cells but
not of NK cells.
Conclusions: Activation of peripheral blood cells of drug allergic
patient by drugs leads to activation of NK cells. This activation is
probably due the cytokines released by drug specific T cells and is
not able to cause a granzymeB-dependent cytolytic activity of NK
cells. Since the reaction of NK cells is stronger than the reaction of
specific T cells, monitoring of NK cell activation may be
advantageous in in vitro testing of drug hypersensitivity.
P183
Towards improvement of structural and functional basis
of TCR avidity for tumour antigen
D.A. Schmid1 , V. Zoete2 , M. B. Irving1 , M. André1 , P. Reichenbach3 ,
V. Posevitz4 , R. Gomez5 , T. Schumacher5 , P. Romero4 , D. Speiser4 ,
O. Michielin2 , N. Rufer1 .
1 Centre Pluridisciplinaire d’Oncologie (Epalinges, CH);
2 Swiss Institute of Bioinformatics (Lausanne, CH);
3 ISREC (Epalinges, CH); 4 Ludwig Institute for Cancer Research
(Lausanne, CH); 5 The Netherlands Cancer Institute (Amsterdam, NL)
Background: CD8+ T lymphocytes recognize and destroy virusinfected
or tumor cells. However, the tumor-specific cytotolytic
T lymphocytes often fail to mediate a clinically effective immune
response. A major reason might be the relative lack of high avidity