Supplementum 163 - Swiss Medical Weekly

45 S SWISS MED WKLY 2008;138(Suppl 163) · www.smw.ch
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Material and methods: Human monocyte-derived immature DC were
stimulated with different TLR-2 and 4 ligands (hyaluronic acid [HA],
LPS or lipoteichoic acid [LTA]) under normoxia. Furthermore, we
incubated DC under hypoxic conditions (1.5% oxygen) with or
without stimulation with LPS. HIF-1a expression was examined by
western blot at 2h, 4h, 6h, 8h, 12h and 24h after TLR stimulation. DC
were analyzed for the expression of the co-stimulatory molecules and
maturation markers CD80 and CD86 by flow cytometry (FACScan,
B&D). Mitochondrial respiration of digitinon-permeabilized cells was
determined using a High Resolution Oxygraph (Oroboros Instruments,
Innsbruck, Austria) and DatLab 4.2 software for data acquisition and
analysis.
Results: All tested TLR agonists stimulated the expression of HIF-1a
in a time-dependent manner. Interestingly, TLR induced HIF-1a
expression levels in normoxia were even higher than in hypoxia. HA,
LPS and LTA led to DC maturation, as shown by the up-regulation of
CD80 and CD86 expression. LPS also increased complex IIdependent
mitochondrial respiration of DC.
Conclusions and Outlook: The current data demonstrate that HIF-1a
expression in DC is induced under normoxic conditions via TLR-2
and -4 agonists in a time-dependent manner. Furthermore, LPS
treatment leads to an increased complex II-dependent mitochondrial
respiration. In further investigations we will examine if a HIF-1a
knock-down in human DCs by siRNA leads to an impaired
immunostimulatory capacity measured by the ability to activate
T cells and if TLR ligation leads to a HIF-1a-dependent release of
cytokines and growth factors and modification of mitochondrial
respiration.
P195
Crosstalks between neutrophils and dendritic cells during
leishmania major infection
M. Charmoy1 , S. Agten1 , P. Launois1 , G. Milon2 , F. Tacchini-Cottier1 .
1 Université de Lausanne (Epalinges, CH); 2 Institut Pasteur (Paris, F)
Neutrophils are key components of the inflammatory response and
contribute to the development of pathogen-specific immune
response. Neutrophils are recruited within hours of an infection with
Leishmania major (L. major). C57BL/6 mice are resistant to infection
with L. major developing a protective Th1 response, while BALB/c
mice, developing a Th2 response, are susceptible to infection and do
not control parasite replication nor healing of lesions. Dendritic cells
play a crucial role in initiating and regulating the T cell immune
response. Uptake of L. major by dendritic cells results in their
activation and IL-12 secretion, a cytokine which is critically involved
in the Th1 induced killing of the parasite. In this report, we
investigated in vitro and in vivo, the crosstalks between neutrophils
and dendritic cells following L. major infection in C57BL/6 and
BALB/c mice. The secretion of dendritic cells attracting chemokines
by neutrophils was first investigated. C57BL/6 neutrophils transcribed
and secreted significantly higher levels of CCL3, CCL4 and CCL5
chemokines in response to L. major compared to BALB/c neutrophils.
In vitro migration assay showed that the higher secretion of dendritic
cells attracting chemokines by C57BL/6 neutrophils correlated with a
higher migration of dendritic cells towards C57BL/6 neutrophils
supernatant as compared to that of BALB/c neutrophils. Following
s.c. injection of L. major, 24 hours after infection, a significantly higher
amount of dendritic cells were found to be transiently recruited to the
site of injection in resistant C57BL/6 mice compared to susceptible
BALB/c mice. Depletion of neutrophils prior to infection led to
inhibition of the migration of dendritic cells to the site of infection,
suggesting that neutrophils are essential for early cellular recruitment.
Altogether, these results indicate that the differential release of
dendritic cells attracting chemokines by neutrophils following
infection with L. major induces the migration of dendritic cells to the
site of infection and thus may influence L. major-specific immune
response.
P196
Leishmania major-specific B-cells are necessary for Th2 cell
development and susceptibility to L. major LV39 in BALB/c mice
C. Ronet 1 , H. Voigt 1 , H. Himmelrich 1 , M-A. Doucey 1 , Y. Hauyon-La
Torre 1 , M. Revaz-Breton 1 , F. Tacchini-Cottier 1 , C. Bron 1 , J. Louis 2 ,
P. Launois 1 .
1 Université de Lausanne (Epalinges, CH); 2 Institut Pasteur (Paris, F)
B lymphocytes are considered to play a minimal role in host defence
against L. major. In this study, the contribution of B cells to
susceptibility to infection with different strains of L. major was
investigated in BALB/c mice lacking mature B cells due to the
disruption of the IgM transmembrane domain (uMT). Whereas BALB/c
uMT remained susceptible to infection with L. major IR173 and IR75,
they were partially resistant to infection with another strain of L.
major, L. major LV39. Adoptive transfer of naïve B cells into BALB/c
mMT mice prior to infection restored susceptibility to infection with L.
major LV39, demonstrating a role for B cells in susceptibility to
infection with this parasite. The two main functions of B cells are Ig
production and/or Ag presentation to T cells. First, by transfer of
immune serum in BALB/c uMT mice, we demonstrate that specific Ig
did not contribute to disease progression. Thus, the role of APC
function of the transferred B cell on the expression of a susceptible
phenotype in resistant BALB/c uMT mice following adoptive transfer
of B cells was evaluated. Adoptive transfer of B cells that express an
IgM/IgD specific for HEL, an irrelevant antigen, did not restore
disease progression in BALB/c uMT mice infected with L. major LV39.
This was likely due to the inability of HEL Tg B cells to internalize and
present Leishmania antigens to specific T cells. These data suggest
that direct antigen presentation by specific B cells and not Ig effector
functions is involved in susceptibility of BALB/c mice to infection with
L. major LV39.
P197
In vitro drug-induced cytokine release from peripheral blood
mononuclear cells of patients with delayed-type drug
hypersensitivity
A. Beeler 1 , P. Lochmatter 1 , T. Kawabata 2 , W.J. Pichler 1 .
1 Universität Bern (Bern, CH); 2 Pfizer, Inc. (Groton, USA)
The most prevalent hypersensitivity reactions to drugs are T-cell
mediated. The only established in vitro test for detecting T cell
sensitization to a drug is the lymphocyte transformation test (LTT),
which measures the proliferative response of T cells to the drug. LTT
involves the use of radioactivity and is time-consuming; therefore its
practicability is limited. Our goal was to find an alternative in vitro
method to detect drug-sensitized T cells. We analyzed in vitro the
secretion pattern of 17 cytokines and chemokines from peripheral
blood mononuclear cells (PBMCs) of patients with well-documented
drug allergies (MPE and DRESS), in order to identify those cytokines
which are predominantly secreted upon exposure to the relevant
drug. PBMCs of 5 amoxicillin-allergic patients, 5 sulfamethoxazoleallergic
patients and 5 healthy controls were incubated for 3 days with
the drug antigen. Cytokine concentrations were measured in the
supernatants using commercially available 17-plex, multiplex, beadbased
immunoassay kits. Among the 17 cytokines/chemokines
analyzed, IL-5, IL-13 and IFN-gamma secretion in response to the
drug antigens was increased in patients’ PBMCs compared to healthy
controls. No difference in cytokine secretion patterns between
sulfamethoxazole- and amoxicillin-reactive PBMCs could be
observed. The secretion of other cytokines, such as the Th1
cytokines IL-2 and IL-12, and the Th2 cytokines IL-4 and IL-10,
showed a high variability among patients. We conclude that from the
17 cytokines/chemokines analyzed, the measurement of IL-5, IL-13
and IFN-gamma might be a useful tool for in vitro detection of T cell
sensitization to drugs. Secretion of these cytokines is independent of
the drug antigen and the phenotype of the drug reaction.
P198
CD4+ T-cell help improves CD8+ T-cell memory by retained
CD27 expression
M. Matter, C. Claus, A.F. Ochsenbein.
Universität Bern (Bern, CH)
CD4+ T cell help during the priming of CD8+ T lymphocytes imprints
the capacity for optimal secondary expansion upon re-encounter with
antigen. Helped memory CD8+ T rapidly expand in response to a
secondary antigen exposure, even in the absence of T cell help and,
are most efficient in protection against a re-infection. In contrast,
helpless memory CTL can mediate effector function, but secondary
expansion is reduced. How CD4+ T cells instruct CD8+ memory
T cells during priming to undergo efficient secondary expansion has
not been resolved in detail. Here we show that memory CTL after
infection with lymphocytic choriomeningitis virus (LCMV) are
CD27high whereas memory CTL primed in the absence of CD4+
T cell have a reduced expression of CD27. Helpless memory CTL