Supplementum 163 - Swiss Medical Weekly

43 S SWISS MED WKLY 2008;138(Suppl 163) · www.smw.ch
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P187
Cathepsin D initiates neutrophil apoptosis and its inhibition
blocks the resolution of inflammation
S. Conus1 , R. Perozzo2 , T. Reinheckel3 , C. Peters3 , L. Scapozza2 ,
S. Yousefi1 , H-U. Simon1 .
1 University of Berne (Berne, CH); 2 University of Geneva (Geneva,
CH); 3 University of Freiburg (Freiburg, D)
Background: Although the lysosomal cathepsins have often been
considered as intracellular proteases able to mediate caspaseindependent
death, there is evidence that they act in concert with
caspases in apoptotic cell death. In particular, the cysteine protease
cathepsin B and the aspartic protease cathepsin D have been
reported to be involved in apoptosis regulation. Since neutrophils
rapidly undergo apoptosis following phagocytosis of bacteria, we
hypothesized that azurophilic granules, in which cathepsins are
located and intracellular bacterial killing occurs, might be able to
somehow trigger the normal apoptotic program in these cells.
Methods: To resolve the issue of whether cathepsins are involved in
neutrophil apoptosis pathways, we specifically inactivated cathepsin
B and D, respectively, by both genetic and pharmacological means.
Neutrophil death was assessed by uptake of ethidium bromide and
flow cytometric analysis. To determine whether cell death was
apoptosis, redistribution of phosphatidylserine in the presence of
propidium iodide and oligonucleosomal DNA fragmentation were
measured by flow cytometry. Immunofluorescence analysis of freshly
isolated or cultured neutrophils was performed to follow timedependently
the release of cathepsin D from azurophilic granules into
cytosol. Subsequent activation of caspase-8 and caspase-3 by
cathepsin D was analyzed either by cell-free assay followed by
western blot analysis or by enzymatic assay.
Results: We report here a new pro-apoptotic pathway in which
cathepsin D directly activates caspase-8. Cathepsin D is released
from azurophilic granules in neutrophils in a caspase-independent
manner. Under inflammatory conditions, however, the translocation of
cathepsin D in the cytosol is blocked. Pharmacological or genetic
inhibition of cathepsin D resulted in delayed caspase activation and
reduced neutrophil apoptosis. Cathepsin D deficiency or lack of its
translocation in the cytosol prolongs innate immune responses in
experimental bacterial infection and in septic shock.
Conclusion: Thus, we identified a new function of azurophilic
granules, which regulate, besides their role in bacterial defense
mechanisms, the life-span of neutrophils and therefore the duration of
innate immune responses through the release of cathepsin D.
P188
Acute physical exercise affects natural killer cell numbers
and function
P.V. Valli 1 , A-L. Millard1 , N.J. Müller1 , M.K.J. Schneider1 ,
J.D. Seebach2 .
1 University Hospital (Zurich, CH);
2 University Hospital (Geneva, CH)
Introduction: Psychological and chronic physical stress increase the
number of natural killer (NK) cells in the circulation, but decrease NK
cell cytotoxicity. Since the influence of acute physical exercise (APE)
on PBMC and NK cells remains unclear, we analyzed kinetics,
phenotype and NK cell function before and immediately after short
intensive exercise.
Material and methods: Heparinized peripheral blood (50 ml) was
obtained from 5 healthy volunteers immediately before and after
running up and down 6 flights of stairs. The phenotype of PBMC and
NK cells, including the NK activating receptors NKp30, NKp44,
NKp46, and NKG2D, was analyzed by flow cytometry. The
percentage of IFN-g secreting NK cells was determined by a capture
assay after 4 and 24 h, total IFN-g release by ELISA after 48 h of
incubation with either low (50 U/ml) or high (500 U/ml) doses of IL-2.
Results: APE led to an increased number of PBMC (x 1.2 to 1.5) and
NK cells (x 2.1 to 6.0) in all 5 donors. A 40% decrease in the
percentage of CD56bright NK cells and a slightly decreased
expression of NKG2D and NKp46 (preliminary results) was noted after
APE. In contrast, APE did not influence the basal percentage of IFN-g
secreting NK cells. After 4 h of IL-2 activation we found a 2-3 fold
increase in the percentage of IFN-g secreting NK cells (n = 3),
whereas a reduction of 60–70% (±15%) was detected after 24h
(n = 5). After 48 h, IFN-g release was reduced to 50% (low dose IL-2)
and 34% (high dose IL-2).
Conclusions: In summary, APE had a profound effect on circulating
NK cell numbers (2–6 fold increase) and function (decreased
percentage of IFN-g secreting NK cells after a 24h and decreased
IFN-g accumulation after 48h of IL-2 activation). Therefore, if running
is used to increase the numbers of available human NK cells for ex
vivo experiments, one should be aware of the functional changes
induced by APE and collection conditions should be as uniform as
possible to exclude potential interferences on the experimental
results.
P189
CD8 T cell differentiation and TCR repertoire dynamics in
Epstein-Barr and cytomegalovirus infected individuals
E.M. Iancu1 , M. Bruyninx2 , P. Corthesy1 , P. Baumgaertner2 , E.
Devevre2 , P. Romero2 , D. Speiser2 , N. Rufer1 .
1 Multidisciplinary Oncology Center (Lausanne, CH); 2 Ludwig
Institute for Cancer Research (Lausanne, CH)
Protective immune responses against viral infections involve the
selection and generation of differentiated antigen-specific CD8 T
lymphocytes with potent effector functions, restricted clonal diversity
and increased avidity of T cell receptors (TCR). Our objective is to
identify correlates of immune protection in humans by analyzing the
highly efficient responses to chronic viral infections. We analyzed the
immune responses against chronic Epstein-Barr (EBV) and
cytomegalovirus (CMV) infections in healthy individuals by
determining the phenotype and repertoire characteristics of viral
specific T cells. We found that EBV-specific CD8 T lymphocytes
consist primarily of early-differentiated effector memory cells
(EM/CD28+), while CMV-specific T lymphocytes are mostly
composed of differentiated effector cells (EM and EMRA/CD28-).
Although the proportions of these distinct T cell subsets were
different among EBV- and CMV-specific T cells, ex-vivo analysis of
several functionally relevant proteins (CD27, CD57, CD127, granzyme
B, perforin) revealed that they follow the same pathway of T cell
differentiation, from EM/CD28+ to EMRA/CD28-. Ex-vivo TCR
repertoire analysis showed that the EBV specific responses are
dominated by several T cell clonotypes bearing the BV2 and BV4
chains, while CMV responses are highly restricted to few clonotypes,
but with BV chains that can vary from donor to donor (BV13, BV14,
BV15). In order to determine the frequency of each clonotype and
study the dynamics of dominance along the pathway of
differentiation, we generated EBV and CMV specific T cell clones
from the least differentiated and from the most differentiated subsets.
In vitro TCR repertoire analysis showed that while certain clonotypes
were selected, other clonotypes were lost with differentiation and
were most dominant in the early-differentiated compartment. We are
currently investigating factors such as TCR affinity that may be
involved in the selection of particular clonotypes towards
differentiation. We conclude that immune responses to chronic
infections are unique to each virus, although they consist of antigen
primed and functionally active effector memory T cells. Altogether,
combined analysis of T cell differentiation and TCR repertoire will
provide novel insights in the dynamics of T cell responses, and the
identification of correlates of immune protection will be crucial for the
development of therapeutic vaccination against chronic viral
infections and cancer.
P190
Eosinophils release mitochondrial DNA to trap and kill bacteria
S. Yousefi, D. Simon, E. Kozlowski, I. Schmid, H-U. Simon.
University of Berne (Berne, CH)
Although eosinophils are considered as being useful in defense
mechanisms against parasites, their exact function(s) in innate
immunity, however, remains unclear. The aim of this study was to
better understand the role of eosinophils infiltrating the
gastrointestinal tract. We show here that lipopolysaccharide (LPS)
from gram-negative bacteria activates interlukin (IL)-5 primed
eosinophils to release mitochondrial DNA and granule proteins in a
reactive oxygen species (ROS) dependent manner, but independent
from eosinophil death. The mitochondrial DNA and the granule
proteins from extracellular structures able to bind and kill bacteria
both in vitro and under inflammatory conditions in vivo. Therefore,
eosinophils contribute to innate immune responses by fighting
against bacteria in the extracellular space using mitochondrial DNA.
This newly identified function of eosinophils might represent an
important defense mechanism to protect the host from uncontrolled
invasion of bacteria in inflammatory bowel diseases associated with
epithelial cell damage.