Download W. Drobnik, E. Orso, W. Diederich, G. Schmitz

O1
THE ZINC FINGER PROTEIN 202 (ZNF202) IS A TRANSCRIPTIONAL
REPRESSOR OF ABCA1 AND ABCG1 GENE EXPRESSION IN MACROPHAGES
AND MODULATES CELLULAR LIPID EFFLUX
T Langmann, S. Heimerl, M. Porsch-Öczürümez, H. Borsukova, W. E. Kaminski, W. Drobnik,
C. Honer, C. Schumacher, G. Schmitz
Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, 93042 Regensburg,
Germany (E-mail: [email protected])
The zinc finger gene 202 (ZNF202) located within a hypoalphalipoproteinemia susceptibility locus on
chromosome 11q23 is a transcriptional repressor of various genes involved in lipid metabolism. To provide
further evidence for a functional linkage between ZNF202 and hypoalphalipoproteinemia, we investigated the
effect of ZNF202 expression on ABCA1 and ABCG1. ABCA1 is a key regulator of the plasma HDL pool size
whereas ABCG1 is another mediator of cellular cholesterol and phospholipid efflux in human macrophages. We
demonstrate here that the full length ZNF202m1 isoform binds to GnT repeats within the promoters of ABCA1
(-229/-210) and ABCG1 (-572/-552). ZNF202m1 expression in HepG2 cells dose-dependently repressed the
promoter activities of ABCA1 and ABCG1. This transcriptional effect required the presence of the
SCAN-domain in ZNF202 and the functional integrity of a TATA box at position -24 of ABCA1, whereas the
presence of GnT binding motifs was nonessential. The state of ZNF202 SCAN domain oligomerization affected
the ability of the adjacent ZNF202 KRAB domain to recruit the transcriptional corepressor KAP1.
Overexpression of ZNF202m1 in RAW264.7 macrophages prevented the induction of ABCA1 gene expression
by 20(S)-OH-cholesterol and 9-cis retinoic acid further substantiating the interference of ZNF202 in critical
elements of transcriptional activation. Finally, HDL3 and apoA-I mediated lipid efflux was significantly reduced
in RAW264.7 cells stably expressing ZNF202m1. In conclusion, we have identified ABCA1 and ABCG1 as
target genes for ZNF202 mediated repression in macrophages and thus provide evidence for a functional linkage
between ZNF202 and hypoalphalipoproteinemia.
O2 CHITOTRIOSIDASE ACTIVITY IN MODELS OF LYSOSOMAL STORAGE
AND MACROPHAGE STIMULATION
T. Korolenko, O. Falameyeva, S. Djanaeva, G. Paul1, R. Wevers2, and J.W. Nowicky3
Institute of Physiology, RAMS, Novosibirsk, Russia; 1Novosibirsk Diagnostic Center, Novosibirsk, Russia;
2
University of Nijmegen, Nijmegen, Holland; 3Nowicky Pharma, Vienna, Austria (E-mail:
[email protected])
Chitotriosidase (Ch), endo -glucosaminidase, belonging to the protein family of chitinases, is strikingly
elevated in plasma of patients with Gaucher disease type 1, possibly as a result of massive production by storage
cells - lipid-loaded macrophages (Mph), called Gaucher cells. Plasma Ch was suggested to originate from
activated Mph; Ch mRNA is specifically expressed by phagocytes (Guo et al., 1995). The aim: to study the
origin of elevated serum Ch activity in Gaucher disease, using different models of Mph stimulation and
lysosomal storage syndromes. Serum Ch activity was determined fluorometrically (Guo et al., 1995). Models of
Mph stimulation induced by zymosan (100 mg/kg) and by carboxymethylated (13)-;-D-glucan (CMG),
chitoCMG (produced by Chem. Inst., Bratislava, Slovakia) both in a dose of 25 mg/kg were used (Korolenko et
al., 2000). Triton WR 1339-induced lysosomal storage was followed by liver Mph overloading with
non-digested lipids and simultaneously by Mph stimulation (increased rate of carbon particle phagocytosis in
vivo). As was shown earlier (Guo et al., 1995), in our investigation Ch activity was elevated more than 500-fold
in serum of Gaucher type 1 patient and about twice in serum of patients with MPS I, II. In experimental study
Mph stimulation (zymosan, CMG, chitoCMG, Triton WR 1339) was followed by 2-2.5 fold increase of serum
Ch activity in CBA mice or Wistar rats. Similar results were obtained in the groups with different glucans
studied - ChitoCMG and CMG (in the same dose of 25 mg/kg, i.p). Mph depression (”negative” control)
induced by gadolinium chloride in vivo was characterized by decreased serum Ch activity both in mice and rats.
Effective antitumor treatment by macrophage stimulator CMG (25 mg/kg) in combination with cyclophosphane
restored up to the normal values decreased serum Ch activity in CBA mice with LS lymphosarcoma. Obviously
during Mph stimulation secreted into serum form of Ch was elevated, but in all experiments it was not possible
to reproduce so drastical increase of Ch activity as in Gaucher type 1 disease. One can conclude that serum Ch
activity may serve as a valuable parameter for monitoring the efficacy of Mph stimulation in vivo.
Acknowledgements: We are very grateful to Dr. R. Wevers (Nijmegen, Holland) for support and kind gift of
spectrofluorometer; to Dr. B. Czartoryska (Poland) for kind help.
References:
Guo Y et al (1995) J Inher. Metab. Dis. 18: 717-722
Korolenko T et al (2000) Proc. 3rd World Meeting APV/APGI, Berlin, p.343-345
O3
TRANSCRIPTIONAL REGULATION OF HUMAN CARTILAGE 39 KDA
GLYCOPROTEIN, A MARKER GENE FOR LATE STAGES OF MACROPHAGE
DIFFERENTIATION
Michael Rehli, Hans-Helmut Niller*, Christoph Ammon, Lucia Schwarzfischer, Reinhard
Andreesen, Stefan W. Krause
Department of Hematology and Oncology, and *Institute of Medical Microbiology, Universitiy Hospital,
Regensburg, Germany
(E-mail: [email protected])
The human cartilage 39 kD glycoprotein (HCgp-39) is a tissue-restricted chitin-binding lectin and member of the
family 18 glycosyl hydrolases. It is strongly expressed in mature macrophages and serves as a specific marker
for late stages of macrophage differentiation in vitro. To understand the molecular mechanisms underlying the
tissue-restricted and maturation-associated expression of its gene we studied the regulation of the proximal
HCgp-39 promoter. Deletion analysis of reporter constructs localizes a region directing high levels of
macrophage-specific reporter gene expression to approximately 300 bp proximal to the major transcriptional
start site. The sequence contains consensus binding sites for several known factors, including AML-1, C/EBP
and Sp1 family members, and several motifs that represent potential binding sites for Ets family transcription
factors. In macrophages, but not in monocytes, corresponding sequences were protected in footprinting assays
with DMS in vivo. Specific binding of PU.1, Sp1, Sp3, USF, AML-1 and CEBP proteins was detectable in
EMSA. The importance of C/EBP, Sp1, USF and Ets binding sites was further demonstrated by transient
transfection studies using deleted or specifically mutated constructs of the proximal HCgp-39 promoter.
Co-transfection studies in Drosophila S2 and HeLa cells indicate cooperative effects between various
promoter-binding transcription factors. Although the proximal promoter specifically directs expression in a
macrophage cell line, bisulfite sequencing of the proximal promoter in a wide range of cell types indicates that
additional regulatory levels, e.g. local CpG methylation and chromatin structure are involved in the
maturation-associated and tissue-restricted regulation of the CHI3L1 gene.
O4
PAMIDRONATE AND ZOLEDRONATE INHIBIT THE GENERATION OF
OSTEOCLASTS BY INTERFERING WITH THE MEVALONATE PATHWAY
S. Brosch, L. Schuster, R. Gruber, M. Shehata, P. Pietschmann, M. Peterlik
Department of Pathophysiology, University of Vienna
(E-mail: [email protected])
Bisphosphonates are widely used in the treatment of bone diseases such as osteoporosis, Paget´s disease and
tumor hypercalcaemia. In order to investigate the mechanism of action of aminobisphosponates we studied the
influence of pamidronate and zoledronate on the generation of osteoclasts derived from hematopoetic precurser
cells. Osteoclasts were generated in vitro from murine bone marrow cells in the presence of 10 -8 M 1,25
(OH)2-vitamin D3. Both bisphosphonates led to a significant reduction of osteoclastogenesis. Pamidronate had
its effective concentration at 10-5 M, zoledronate with an effective concentration of 10-7 M clearly was more
potent. In addition a resorption pit assay where osteoclasts were generated on bovine bone slices was performed.
In this set of experiments we found a clear reduction of the number of resorption pits formed in the presence of
the aminobisphosphonates. Since it has be suggested that bisphosphonates inhibit the synthesis of several
intermediates of the mevalonate pathway; this possibility was addressed in further experiments. Both in bone
marrow cultures and in the co-culture of bone marrow cells with murine osteoblasts the addition of mevalonic
acid (10-3 M) antagonized, at least in part, the effect of pamidronate and zoledronate on the generation of
osteoclasts. In co-cultures treated with zoledronate the mevalonate pathway intermediate geranylgeraniol (10 -5
M) also partly prevented the inhibition of osteoclastogenesis. This could indicate, that zoledronate acts on the
geranylgeranylation of proteins and therefore membrane structures or the fusion of preosteoclasts are disturbed.
The intermediates squalene or farnesol did not prevent pamidronate or zoledronate induced inhibition of
osteoclast generation. Our results demonstrate that the aminobisphosphonates pamidronate and zoledronate
decrease the generation of osteoclasts in vitro. This effect is mediated, at least in part, by interfering with
intermediates of the mevalonate pathway
O5 PLATELET - RELEASED SUPERNATANTS STIMULATE THE FORMATION
OF OSTEOCLAST - LIKE CELLS TROUGH A PROSTAGLANDIN / RANK-L DEPENDENT MECHANISM
Reinhard Gruber, Florian Karreth, Michael B. Fischer*, and Georg Watzek
School of Dentistry, Department of Oral Surgery, *Department of Transfusion Medicine University of Vienna,
Vienna, Austria (E-mail: [email protected])
Platelets are activated at fracture sites or upon the insertion of implants as a consequence of vascular disruption.
The regeneration of injured tissue requires bone remodeling and therefore the resorbing activity of osteoclasts.
To determine the role of platelets in osteoclastogenesis, murine bone marrow cells were cultured in the presence
of supernatants released from thrombin - activated platelets for 7 days. Histochemical analysis indicated the
presence of bone resorbing, tartrate-resistant acid phosphatase (TRAP) positive, multinucleated cells, termed
osteoclast-like cells (OCL). Transcripts that are characteristically expressed in OCL were increased in these
cultures, as determined by semiquantitative reverse transcription (RT)-PCR. The addition of Indomethacin and
NS398, both at 10-7 M, completely inhibited OCL formation and decreased Prostaglandin (PG) E2 levels.
Platelet - released supernatants (PRS) stimulated the expression of receptor activator of NF-kB-ligand
(RANK-L) 2-fold, whereas mRNA levels of osteoprotegerin (OPG) were marginally decreased.
The formation of OCL was prevented by recombinant OPG, and partially suppressed through a recombinant
Interleukin-1 receptor antagonist (IL-1ra). Our results indicate that PRS stimulate cyclooxygenase (COX)-2 dependent PGE2 production in bone marrow cells. PGE2 can in turn increase the RANK-L / OPG ratio, resulting
in the formation of OCL. The data support the assumption that platelets can contribute to bone remodeling and
therefore to the regeneration of injured mineralized tissue.
O6 RETINOIC ACID MODULATES DENDRITIC CELL ANTIGEN EXPRESSION
AND FUNCTION
Marina Kreutz, Katharina Menninger, Jana Fritsche and Reinhard Andreesen
Dept.
of
Hematology
and
Oncology,
(E-mail:[email protected])
University
of
Regensburg,
Germany
Objective: Dendritic cells (DC) play a central role in the regulation of a cellular immune response.
Differentiation and terminal maturation of DC determines the outcome of antigen presentation to T-cells:
immune activation or tolerance induction. Retinoic acid (RA) has multiple effects on the differentiation of
myeloid hematopoietic cells and inhibits the differentiation of blood monocytes into macrophages. The aim of
this study was to investigate the role of all-trans RA and 9cis RA for the differentiation of blood monocytes into
DC.
Methods/Results: Monocytes were cultured for 7 days either in the presence or absence of RA, respectively and
antigen expression was determined by flow cytometry. The upregulation of the DC marker CD1a during
monocyte to DC differentiation was suppressed by both RA isoforms. However, the typical
monocyte/macrophage antigen CD14 was completely downregulated with or without the addition of RA.
Furthermore RA-cultivated DC showed an upregulation of CD86, HLA-DR and CD40 expression. Functional
analysis of DC with the mixed lymphocyte reaction (MLR) revealed a decreased ability of RA-cultivated DC to
induce an allogeneic T-cell response. Next we analyzed whether RA also influences the terminal maturation of
DC. DC were cultured without RA up to day 5 and then TNF-alpha or LPS, respectively, was added in the
absence or presence of RA. All-trans RA led to a weak suppression of the maturation marker CD83 and also
partially inhibited further upregulation of CD40, CD86 and HLA-DR. 9cis RA had no effect on the terminal
maturation of DC.
Summary/Conclusion: The vitamin A derivatives all-trans RA and 9cis RA both modulate DC differentiation.
RA-conditioned DC show a high expression of costimulatory antigens but fail to express CD1a. Antigen
presentation (MLR) is decreased by RA-cultivated DC. Therefore RA seems to induce a special phenotype of
DC with immunosuppressive properties. Further experiments will clarify whether RA can suppress not only an
allogeneic but antigen-specific T-cell activation, e.g. mediate a tolerogenic immune response.
O7
PHENOTYPIC AND FUNCTIONAL CHARACTERISTICS OF ADULT
LANGERHANS CELLS UPON IN VITRO CULTURE
A. Elbe-Bürger, S. Olt, S. Chang-Rodriguez, G. Stingl
DIAID, Department of Dermatology, VIRCC, Vienna, Austria
(E-mail: [email protected])
In epidermal sheets of adult mice, Langerhans cells (LC) exhibit a pronounced anti-MHC class II reactivity.
Dendritic leukocytes of neonatal epidermis (NDL), in contrast, express only modest amounts of these proteins.
To determine the differentiation and maturation potential of these cells, epidermal cell suspensions from
newborn and adult whole body skin were highly purified for CD45+, MHC class II+ cells. Subsequently, these
populations were cultured for three days in RPMI medium supplemented with FCS and GM-CSF. During
culture, adult LC became highly dendritic and formed numerous homotypic clusters, while NDL remained round
to polygonal and created only few small-sized clusters. After culture, both cell types were CD3- and exhibited
similar intensity in surface expression of CD45, F4/80, and CD11b. NDL, however, expressed slightly more
MHC class I, less CD40, considerably less MHC class II, CD54, CD80 and CD86, and were CD25- and CD205-.
Functionally, cultured NDL were significantly weaker stimulators of naive, allogeneic CD4+ and CD8+ T cells
than adult LC. Furthermore, freshly isolated NDL were twice as efficient as adult LC in the uptake of
FITC-conjugated ovalbumin, but were not able to present ovalbumin to antigen-specific T cell hybridomas.
In summary, we have shown that CD45+/CD3- NDL do not acquire the morphology and phenotype typical of in
vitro cultured adult LC, and are greatly impaired in their capacity to activate antigen-specific T cell proliferative
responses. The finding that certain dendritic cells of neonatal mice do apparently not possess the molecular
machinery needed for the initiation of productive T cell responses should facilitate the search for molecules
involved and engaged in T cell activation.
O8
BACTERIAL METABOLITE INTERFERENCE WITH MATURATION OF
HUMAN MONOCYTE-DERIVED DENDRITIC CELLS
Marcus D. Saemann,* Ornella Parolini,* Georg A. Böhmig,Ý Peter Kelemen,* Josef Neumüller,ý
Walter H. Hörl,Ý Christos Diakos,* Karl Stuhlmeier,§ and Gerhard J. Zlabinger*
*Institute of Immunology, and ÝDepartment of Internal Medicine III, and ýInstitute of Histology, University of
Vienna, Austria; and §Ludwig Boltzmann Institute for Rheumatology, Vienna, Austria
(E-mail:
[email protected])
Dendritic cells (DC), the most potent APC, are central to antimicrobial immunity. Because of evolutionary
pressure it is reasonable that pathogens have evolved strategies to subvert also this host defense mechanism. In
the present study we describe a novel way of bacterial interference with DC maturation.
The bacterial metabolite n-butyrate, which occurs physiologically in high concentrations in the gastrointestinal
tract and has well-known antiinflammatory effects, is able to prevent LPS-induced maturation of DC resulting in
a reduced capability to stimulate T cells. In particular, n-butyrate prevents homotypic DC clustering, inhibits
IL-12 while sparing IL-10 production and, at the molecular level, blocks NF-kB translocation.
These results demonstrate efficient targeting of DC function by a bacterial metabolite, which might explain the
particular type of immune responsiveness in the presence of this bacterial agent as exemplified in the
gastrointestinal tract.
O9 TISSUE-SPECIFIC CPG DEMETHYLATION OF THE MCP-4 PROMOTER IN
DENDRITIC CELLS
Sven Heinz, Michael Rehli, Harald Wagner, Reinhard Andreesen, Stefan W. Krause
Department of Hematology and Oncology, University Hospital, Regensburg, Germany
(E-mail: [email protected])
Dendritic cells (DC) play a key role in initiating antigen-specific T-cell responses. In order to define new DC
markers, we compared mRNA expression profiles of immature DC versus monocytes and macrophages using
RDA. We found the expression of the CC-chemokine MCP-4 to be strongly and specifically up-regulated during
DC differentiation, indicating that DC may be a major source of MCP-4 in vivo. Due to these results we became
interested in the regulation of MCP-4 gene transcription. The MCP-4 promoter has been described before and
contains binding sites for NF-AT, C/EBP-b, STAT1 and oct-1 as well as several CpG motifs. Since CpG
methylation of regions with a low CpG density has been correlated with tissue-specific repression, we started out
by characterizing the methylation status of the MCP-4 promoter during DC differentiation by bisulfite-induced
cytosine deamination. All CpGs of the proximal promoter were found to be methylated in monocytes and
macrophages which both do not express MCP-4. However, during differentiation into DC the three CpGs
adjacent to the transcription start site become demethylated concomitant with a strong increase in MCP-4 gene
transcription. EMSA with oligonucleotides encompassing the proximal CpGs indicate that methylation of the
CpG upstream of the TATA box interferes with the binding of a yet unknown nuclear factor present in both
macrophages and DC. Further studies will be aimed at identifying this factor and characterizing its effect on
MCP-4 promoter activity in transient transfection assays.
O10
CROSSLINKING OF LEUKOSIALIN (CD43) WITH MONOCLONAL
ANTIBODY 6F5 INDUCES APOPTOSIS IN DENDRITIC CELLS
Nicole Selenko, Otto Majdic, *Luzia Deszcs, #Erwin Tschachler, Johannes Stoeckl and Walter
Knapp
Institute of Immunology, *Institute of Medical Biochemistry, Division of Biocemistry,
Immunodermatology, University of Vienna Medical School, Vienna, Austria
[email protected])
#
Department of
(E-mail:
Dendritic cells (DC) play a central role in the induction and regulation of immune responses. This capacity has to
be controlled in order to avoid overwhelming immune reactions. One of these potential control mechanisms is
programmed cell death of DC. Here we report that a monoclonal antibody (mAb) against CD43 generated in our
laboratory induces apoptosis in DC. CD43 is broadly expressed on peripheral blood leukocytes. Yet, we
observed that CD43 triggered apoptosis is specific for DC, since engagement of CD43 on monocytes,
lymphocytes, granulocytes or cell lines did not result in apoptosis. CD43 induced DC death is rapid and efficient
and occurs in immature as well as mature monocyte-derived DC, which is in contrast to apoptosis observed after
HLA-DR triggering. Interestingly, also peripheral blood DC (LIN-, HLA-DR+) were driven into apoptosis upon
CD43 triggering with 6F5mAb. Cross-linking of the CD43 epitope was found to be mandatory, since
Fab-fragments of 6F5 failed to induce apoptosis. DC apoptosis appears to be specific for the epitope recognized
by the 6F5 mAb, since an antibody against a different epitope showed no such effects. We conclude from these
data that the mAb 6F5 recognizes an epitope of CD43 which represents a potential target structure via which DC
can be selectively eliminated.
O11 ROLE OF NEUROPEPTIDE -MELANOCYTE STIMULATING HORMONE
UPON DENDRITIC CELL MIGRATION
E. Becher*, M. Rezvani#, M. Eblenkamp*, L. Tietze*, R. S. Bhardwaj#
*Institute for Pathology, RWTH-Aachen, Pauwelsstrasse 30, 52074 Aachen #IZKF BioMAT., RWTH Aachen,
52074 Aachen, Germany (E-mail: [email protected])
Objective: Dendritic cells are a unique population characterized by the capacity to activate naive T-cells in an
antigen specific way. The POMC-neuropeptide -Melanocyte stimulating hormone (-MSH) is known for its
potent immunomodulatory effects. Especially monocytes seem to be target cells for -MSH, as monocytes are
shown to express specific receptors and stimulation with -MSH leads to an upregulation of the production of
antiinflammatory cytokine IL-10. The aim of our study was to investigate, wether human monocyte derived
dendritic cells, which are known to have binding sites for -MSH, are functionally influenced by -MSH.
Methods: Monocytes were isolated from Buffy Coats by ficoll and subsequent percoll gradient centrifugation.
Differentiation was induced by culture for 7 days in the presence of GM-CSF and IL-4 as described. Maturation
state of the cells was defined by FACS-analysis using antibodies against CD1a, CD14, CD83, CD86, HLA-DR.
Melanocortin-receptor (MC) expression on DCs was analysed by FACS using polyclonal antibodies against each
of MC1 to MC5 (Santa Cruz). For migration studies a two chamber system (Transwell, Costar) was employed
and number of migrated cells was determined with a Casy®Counter.
Results: Human monocyte derived DCs show immunreactivity against all five known MC-receptors. Whereas
antigen-presenting capacity in an in-vitro assay (mixed lymphocyte reaction) was not significantly influenced by
-MSH (data not shown), a regulatory role of -MSH for the migratory capacity could be determined. Only in
DCs, which show no expression of CD14 and high expression of CD1a, CD86 and HLA-DR, but low expression
of CD83, -MSH was able to inhibit the migration at a concentration of 10 -10M. Migration of cells, which are in
the maturation process and express CD1a and HLA-DR, but also CD14, was not inhibited by -MSH. In
monocytes, without stimulation with GM-CSF and IL-4, we could detect a rare supporting effect of -MSH
upon the migration of these cells.
Conclusion: -MSH is a differential modulator of monocyte-dendritic cell migration, which acts strictly
dependent on the maturation state of the cells. Immature DCs can be inhibited in their migratory capacity by the
neuropeptid, which might slow down the antigen transport to the lymph node. This might be a further
mechanism of immunomodulation by -MSH and supports the idea of neuroendocrine-immune communication.
O12 TYPE 2 IMMUNE RESPONSES CONTROL THE SYNTHESIS OF PROLINE
IN MACROPHAGES VIA ARGINASE
M. Modolell, M. Hesse*, N. Justies, V. Weber, R. Escher and K. Eichmann
Max-Planck-Institut für Immunbiologie, Freiburg, Germany and *NIH, Schistosomiasis Immunology and
Pathology Unit, Bethesda, USA
(E-mail: [email protected])
Objective: We have previously shown that, dependent on the Type1/2 polarization of the immune response,
murine macrophages metabolize arginine via NOS 2 (Type 1) or arginase (Type 2). While the role of NOS 2 is
well understood, the function of arginase in macrophages remains unclear. Arginase hydrolizes arginine to urea
and ornithine which can be further metabolized to polyamines, glutamate, or proline. Proline is an essential
amino acid for the synthesis of collagen. Macrophages expressing arginase have been implicated in tissue
regeneration upon wound healing and in the fibrotic response of granuloma formation. Both processes require
collagen synthesis. Here we analysed the metabolic fates of arginine and ornithine and their products, to further
assess the role of the arginase-dependent metabolism in macrophages.
Methods: Murine bone marrow-derived macrophages were treated with Th2 cytokines for induction of arginase.
Radioactive arginine or ornithine were employed to quantify the synthesis of proline and RT-PCR to analyze
the expression of the ornithine metabolizing enzymes. Th2-type granulomas were induced in vivo by
Schistosoma mansoni.
Results: Macrophages synthezise proline from arginine. Arginase is the only non-constitutively expressed
enzyme in this pathway. Thus, the process is regulated by the activity of arginase and not by the other enzymes,
as well as by arginine transport into the cell. The pathology, including collagen deposition, of the S. mansoni
induced granulomas correlates with the levels of arginase.
Conclusions: Arginase is necessary for proline synthesis in macrophages. Through arginase expression and
proline synthesis, macrophages can regulate collagen synthesis. In macrophages the most effective inducers of
arginase are Th2 cytokines whereas the Th1 cytokines inhibit this enzyme. As a consequence, collagen
synthesis may be regulated by the Typ1/2 polarization of the immune response. These results may have
important consequences for wound healing and other tissue regeneration or fibrotic processes.
O13 MOLECULAR MECHANISMS REGULATING MEMBRANE TRAFFIC IN
MACROPHAGES. LESSONS FROM THE MICRODOMAIN REGULATING
PROTEIN ABCA1
G. Schmitz, W. Drobnik, E. Orso, H. Borsukova.
Institute of Clinical Chemistry and Laboratory Medicine, University of Regensburg, 93042 Regensburg,
Germany (E-mail: [email protected])
Recently, our laboratory identified ABCA1 as a central regulator of membrane traffic in macrophages and other
cell types. Defects in ABCA1 are responsible for genetic HDL-deficiency syndromes, that are characterized by
impaired apo AI and HDL mediated cholesterol and phospholipid efflux as well as aberrant macrophage
differentiation. These HDL-deficiency syndromes are associated either with splenomegaly or cardiovascular
disease due to aberrant targeting of monocyte/macrophages (Bodzioch et al., Nat Gen 1999, Orso et al. Nat Gen
2000). Thus, ABCA1 beyond regulating HDL maturation of phospholipid-rich pre-beta-HDL precursors to
mature cholesteryl ester rich alpha-HDL is also involved in monocyte-macrophage differentiation and
extravasation. The underlying mechanism involves ABCA1 dependent regulation of plasma membrane
microdomains, that are known as centers of signal transduction. Thus, cholesterol loading of human monocytes
resulted in increased raft staining with confluence of small to large raft structures, while HDL incubation
reversed this effect via an ABCA1 dependent pathway. In addition, cellular sub-fractionation studies revealed
that apo AI induced cholesterol efflux is derived from Lubrol insoluble microdomains, in which ABCA1 is
co-localized with the LPS receptor CD14 and CD55. Moreover, cholesterol loading induced filipodia formation
concomitant with up-regulation of flotillin, a process that is reversed by ABCA1 function. ABCA1 deficient
cells are characterized by abnormal cytoskeletal organization and reduced number and of raft domains, which
may be related to the impaired cellular spreading observed in ABCA1 deficient monocytes. In summary, the data
indicate that ABCA1, as a regulator of cellular membrane traffic, affects the composition of raft domains and
also effects differentiation and extravasation of monocytes.
O14
SIRP-1A EXPRESSION FOLLOWING INFECTION WITH LEISHMANIA
DONOVANI
Alison Jane Bune and Paul Kaye
Immunology Unit, Dept Infectious & Tropical disease, London School of Hygiene & Tropical Medicine, Keppel
Street, London, W2 1PG, UK
(E-mail: [email protected])
Objective: Signal Regulatory Protein-1a (SIRP-1a)/P84 belongs to a novel family of transmembrane
glycoproteins and is expressed on neurones and myeloid cells. A variety of studies have indicated that SIRP-1a
is involved in cell adhesion and cell-cell fusion, and notably, ligation by CD47 has been shown to induce
macrophage fusion. Macrophage fusion is a common feature of granulomatous infections, including both
visceral and cutaneous leishmaniais. However, neither the role, nor regulation of SIRP-1a have previously been
examined in an infectious context.
Methods: To evaluate the regulation of SIRP-1a in granulomatous pathology, mice were infected with the
viscerotropic parasite Leishmania donovani i.v. and tissues isolated at varying times following infection. Liver
cryo-sections were stained with the P84 mAb specific for murine SIRP-1a (Chuang and Lagenaur, Dev. Biol.,
137; 219-232, 1990).
Results: Immunohistochemical analysis demonstrates that Kupffer cells (KC) in naïve mice express minimal or
no SIRP-1a, whereas expression was readily detected as early as 5 hours post-infection (p.i.). Indeed, all
L.donovani-infected KC were SIRP-1a positive, and expression was pronounced in KC undergoing initial cell
fusion. SIRP-1a expression persisted throughout the time course of infection (56 days p.i) principally within
developing granulomas but also in infected KC and infiltrating macrophages, monocytes and neutrophils. The
early induction of SIRP-1a was seen with equal intensity in RAG-/- mice, as well as IFN-g and IL-12 knockout
mice. In contrast injection of latex beads did not induce SIRP-1a expression at 5 hours and suggests specificity in
SIRP-1a induction by L. donovani infection.
Conclusions: This data indicates that T-cells, and the pro-inflammatory cytokines IFN- and IL-12 are not
required for this early induction. Ongoing studies are aimed at defining the mechanisms regulating SIRP-1a
expression in this model.
O15
APO AI AND HDL3 INHIBIT SPREADING OF HUMAN MONOCYTES
THROUGH A MECHANISM THAT INVOLVES CHOLESTEROL DEPLETION OF
RAFT DOMAINS AND REGULATION OF CDC42
W. Drobnik, E. Orso, W. Diederich, G. Schmitz
Institute of ClinicalChemistry and Laboratory Medicine, University of Regensburg, 93042 Regensburg,
Germany (E-mail: [email protected])
The objective of the current study was to characterize the influence of HDL on processes related to the vascular
recruitment of human monocytes, which may contribute to the anti-atherogenic properties of these lipoproteins.
We show that HDL3 and apo AI, inhibit the following processes in human monocytes: (1) M-CSF induced cell
spreading (2) M-CSF stimulated expression of surface molecules involved in adhesion, migration and
scavenging and (3) fMLP induced chemotaxis. These processes are obviously modulated by the regulation of
cellular cholesterol pools as indicated by the following findings. In ABCA1 deficient monocytes apo AI had no
influence on the spreading response. In control cells, stimulation of cholesterol efflux by p-cyclodextrin
mimicked the effect of apo AI and HDL3 on spreading and chemotaxis, whereas cholesterol loading with
enzymatically modified LDL (E-LDL) showed the opposite effect. Finally, a similar inverse regulation by
E-LDL and apo AI/HDL3 was also observed in regard to the surface expression of b1- and b2-integrins as well
as the scavenger receptor CD163 and the Fc-IIIaR. CDC42 was identified as a potential downstream target
linking changes in cellular cholesterol content to monocyte spreading and chemotaxis. Thus, CDC42 antisense
markedly reduced spreading and in parallel with their influence on monocyte spreading, HDL3, apo AI and
p-cyclodextrin down-regulated CDC42 expression while E-LDL had the inverse effect. The apo AI induced
decrease of CDC42 protein expression was paralleled by the reduction of active GTP-bound CDC42. In
summary, the data provide insight into mechanisms by which HDL3 and apo AI may regulate the recruitment of
monocytes into the vessel wall.
O16 MACROPHAGE SCAVENGER RECEPTORS IN HOST DEFENSE AGAINST
LISTERIA MONOCYTOGENES INFECTION
Makato Naito,1 Takuro Ishiguro,1 Tatsuhiko Kodama2
1
Second Department of Pathology, Niigata University School of Medicine, Niigata and 2Research Center for
Advanced Science and Technology, University of Tokyo, Tokyo, Japan (E-mail: [email protected])
Objective: Type I and type II macrophage scavenger receptors (SR-AI/II) recognize a variety of polyanions
including chemically modified lipoproteins and bacterial cell wall products. In this study, we examined a role
for SR-AI/II in immunity against listerial infection.
Methods: For the observation of granuloma formation, we injected intravenously 1X104 CFU of listeriolysin-O
(LLO)-producing Listeria to either SR-AI/II-deficient (SR-AI/II-/-) or wild type (SR-AI/II+/+) mice. To test the
involvement of SR-AI/II in the listerial uptake and killing, peritoneal macrophages from SR-AI/II+/+ and
SR-AI/II-/- mice were used. Immunohistochemical, electron microscopical and bacteriological examinations
were performed.
Results: SR-AI/II-deficient mice were more susceptible to infection with LLO-producing Listeria
monocytogenes. After infection, the number and the diameter of granulomas in the liver were larger in
SR-AI/II-/- mice than SR-AI/II+/+ mice. The replication of L. monocytogenes in the liver was more remarkable
in the SR-AI/II-/- mice than SR-AI/II+/+ mice, and macrophages from SR-AI/II-/- mice showed impaired ability
to kill listeria in vitro. However, macrophages from SR-AI/II+/+ and SR-AI/II-/- mice showed similar levels of
listericidal activity against isogenic mutant inactivated of gene encoding LLO. The listerial phagocytic
activities of SR-AI/II-/- macrophages were significantly lower than those of SR-AI/II+/+ macrophages,
indicating that SR-AI/II function as a receptor for Listeria. Electron microscopy revealed that most Listeria had
been eliminated from the lysosomes of SR-AI/II+/+ macrophages in vivo and in vitro. In contrast, L.
monocytogenes rapidly lysed the phagosomal membrane and escaped to cytosol in SR-AI/II-/- macrophages
prior to phagosome-lysosome fusion.
Conclusions: SR-AI/II play a crucial role in host defense against listerial infection not only by functioning as a
receptor but also by mediating listericidal mechanisms through the regulation of LLO-dependent listerial escape
from macrophages.
O17
ENZYMATICALLY DEGRADED LDL PREFERENTIALLY BINDS TO
CD14high CD16+ MONOCYTES AND INDUCES FOAM CELL FORMATION
MEDIATED ONLY IN PART BY THE CLASS B SCAVENGER RECEPTOR CD36
Michael Torzewski, Michael Kapinsky, Christa Büchler, Chinh Quoc Duong, Gregor Rothe,
Gerd Schmitz
Institute of Clinical Chemistry and Laboratory Medicine, University of Regensburg, Germany
(E-mail: [email protected])
Objective: Heterogeneity of peripheral blood monocytes is characterized by specific patterns in membrane
expression of Fc-receptor FcRIII/CD16 and LPS (Lipopolysaccharide)- receptor CD14. The aim was to
analyze the correlation of these phenotypic differences to the early interaction of freshly isolated monocytes with
modified lipoproteins using either E-LDL (enzymatically degraded LDL), ac-LDL (acetylated LDL), ox-LDL
(oxidized LDL) or native LDL.
Methods: Flow cytometry, lipid analysis, gel electrophoresis, immunoblotting, RNA-expression analysis.
Results: Highest E-LDL binding was observed on CD14highCD16+ monocytes suggesting a selective
interaction of E-LDL with distinct subpopulations of monocytes. E-LDL induced rapid foam cell formation both
in predifferentiated monocyte-derived macrophages and, in contrast to ac-LDL or ox-LDL, also in freshly
isolated monocytes. This was accompanied by upregulation of the class B scavenger receptors CLA-1/SR-BI and
CD36. Cellular binding and uptake of E-LDL was neither competed by ac-LDL nor the class A scavenger
receptor inhibitor polyinosinic acid but was partially inhibited by an excess of ox-LDL. and an anti CD36
antibody.
Conclusions: Foam cell formation induced by enzymatically degraded LDL is mediated only in part through
charge and motif recognition by the class B scavenger receptor CD36.
O18
MIGRATION OF MONOCYTES IN RESPONSE TO CC-CHEMOKINE
PRODUCTION OF PRIMARY HUMAN MACROPHAGES AND ASTROCYTES.
H.A. Smits, K. Rutten, J. Verhoef and H.S.L.M. Nottet.
Eijkman-Winkler Institute, University Hospital Utrecht, Heidelberglaan 100, NL-3584 CX, Utrecht, The
Netherlands. (E-mail: [email protected])
Neuritic plaques in Alzheimer‘s disease are surrounded by microglia and astrocytes. Amyloid-beta together
with the pro-inflammatory products released by these microglia and astrocytes are held responsible for neuronal
dead. Moreover, microglia and astrocytes are held responsible for the continuation of the inflammatory process.
There are far more glial cells surrounding the plaque than located elsewhere in the brain. Chemotaxis might be
responsible for attracting these cells towards the neuritic plaque. Previously we have shown that amyloid-beta is
able to induce MIP-1a, MIP-1b and MCP-1 production in macrophages and astrocytes. Here we studied the
capabilities of amyloid-b to induce chemotaxis of monocytes towards macrophages, astrocytes and cocultures of
macrophages and astrocytes. Results show that there is a concentration-dependant increase in chemotaxis of
monocytes towards amyloid-beta-stimulated macrophages. We found, however, no increased chemotaxis of
monocytes towards amyloid-beta-stimulated astrocytes. Cocultures showed an increased chemotaxis of
monocytes when compared to cells alone. By using neutralizing antibodies we want to identify the
CC-chemokines involved in the migration of the monocytes. Results show that MCP-1 is involved in the
migration of the monocytes. Future plans include the identification of other CC-chemokines
O19 MRP8 AND MRP14, TWO S100-PROTEINS EXPRESSED IN INFILTRATING
MACROPHAGES, IN JUVENILE RHEUMATOID ARTHRITIS
J. Roth, M. Frosch, A. Strey, S. Seeliger, C. Sunderkötter, T. Vogl, and C. Sorg
Institute of Experimental Dermatology and Department of Paediatrics, University of Münster, Germany
(E-mail: [email protected])
Objective: MRP8 and MRP14 are two calcium-binding proteins of the S100-family which are expressed in
infiltrating macrophages and neutrophils in various inflammatory diseases and under some conditions in
activated keratinocytes. In the present study we investigated functional aspects of macrophages in a systemic
autoimmune disease.
Methods: Using immunohistochemistry, in situ hybridisation, and ELISA we analyzed endothelial activation,
leukocyte recruitment and the expression of MRP8 and MRP14 in systemic onset juvenile rheumatoid arthritis
(SOJRA). In vitro experiments were performed in an endothelium-monocyte co-culture system.
Results: Analyzing skin biopsies of patients with SOJRA we found that beside activation of vascular
endothelium infiltration of monocytes/macrophages expressing MRP8 and MRP14 is an initial phenomenon of
this disease. Keratinocytes of SOJRA-patients also express these two proteins. MRP8 and MRP14 are
specifically secreted by monocytes during interaction with inflammatory activated endothelial cells. Serum
concentrations of both proteins reflect disease activity of SOJRA significantly better than conventional markers
as C-reactive protein or erythrocyte sedimentation rate. Complexes of MRP8/MRP14 directly alter permeability
of endothelial monolayers in vitro.
Conclusion: Since MRP8/MRP14 are highly expressed in SOJRA and promote leukocyte recruitment at
concentrations found in these patients our data point to a novel, macrophage-dependent pathomechanism in a
systemic autoimmune disease.
O20
EXPRESSION OF CHEMOKINES AND THEIR RECEPTORS DURING
MACROPHAGE INVASION AFTER SCIATIC NERVE AXOTOMY
H. Siebert; U. Michel*; A. Keller; D. Woellner; S. Bunkowski*; W.A. Kuziel; W. Brück
Institute of Neuropathology, Charité Berlin, Augustenburger Platz 1, 13353 Berlin; *Dept. Neurology,
University Hospital Göttingen, Robert-Koch-Str. 40, 37075 Göttingen; Dept. Microbiology, University of Texas
in Austin, USA (E-mail: [email protected])
+
Objective: The transection of axons in the central or peripheral nervous system results in Wallerian degeneration
of the distal part of the nerve. Peripheral blood cells invade the nerve 2 or 3 days after axotomy. Axons and
myelin become massively degraded. Then the cell debris and myelin ovoids are removed by macrophages. The
recruitment of macrophages, leukocytes and granulocytes is due to different mediators such as adhesion
molecules (e.g. ICAM), cytokines and chemokines. Members of the CC-chemokines seem to play an important
role for the attraction of monocytes to the injury site. We and others could show that chemokines (MCP-1) and
their receptors (CCR2) are also involved in inflamma-tory processes during peripheral nerve degeneration.
Methods: We investigated the expression and regulation of five different chemokines (MCP-1, MCP-3,
RANTES; MIP-1, MIP-1) and their receptors CCR2 and CCR5 after transection of the sciatic nerve in mice at
different timepoints. Chemokines and receptors were analyzed in mRNA of dissected nerves 12hours, 24h, 48h,
4days, and 6d after axotomy by RT-PCR. Immunohistochemistry for these chemokines and receptors were done
on longitudinal nerve sections at the same timepoints.
Results: We found that m RNAs for MCP-1, MIP-1 and MIP-1 are highly expressed at early timepoints from
12h until 48h, followed by a moderate expression at later timepoints. MCP-3 and RANTES were expressed all
the time as well as both receptors CCR2 and CCR5. Immunohistochemistry localized MCP-1 in Schwann cells
and its receptor CCR2 was expressed by macrophages. CCR5 is also expressed by macrophages. After 6 days
Schwann cells also were positive for CCR2 and for MIP1. All chemokines were detected in cells accumulating
around the transection site mainly consisting of macrophages and granulocytes.
Conclusions: These data suggest that chemokines and their receptors play an important role for the recruitment
of peripheral macrophages to injury sites of the peripheral nervous system. It can be concluded that macrophages
and Schwann cells as the main actors during Wallerian degeneration are able to change the expression pattern of
chemokines.
This work is supported by DFG grant 1274/5-1
O21
IMMUNOSTIUMLATION OF MURINE MICROGLIA CELLS BY CPG-DNA
A. Dalpke*, M. Schäfer#, M. Frei*, S. Zimmermann*, E. Weihe# and K. Heeg*
*Institute of Medical Microbiology, Philipps University Marburg, Pilgrimstein 2, 35037 Marburg #Dept. of
Mol. Neuroscience, Institute of Anatomy and Cell Biology, Philipps University Marburg, Robert-Koch-Str. 6,
35033 Marburg (E-mail: [email protected])
Bacterial DNA containing motifs of unmethylated CpG-dinucleotides (CpG-DNA) triggers innate immune cells
through the pattern recognition receptor TLR 9 and thus possesses potent immunostimulatory effects on
macrophages, dendritic cells and B-lymphocytes. Therefore bacterial CpG-DNA contributes to inflammation
during the course of bacterial infections. In contrast to other TLR ligands CpG-DNA is a strong inducer of IL-12
and thus acts as a Th1-polarizing agent that can be utilized as vaccine adjuvant. To assess the role of CpG-DNA
in immune reactions in the CNS we examined the effects of CpG-DNA on microglia cells in vitro and in vivo.
Here we show that primary microglia cells as well as microglia cell lines expressed mRNA encoding TLR9
transcripts. In vitro CpG-DNA was capable to activate microglia cells and induced TNFa and IL-12p40 as well
as upregulation of MHC-class II , B7-1, B7-2 and CD40 molecules. After intracerebroventricular injection of
CpG-DNA microglia cells were activated to produce TNF- and IL-12p40 transcripts as shown by in situ
hybridization.. Furthermore CpG antagonistic oligonucleotides containing guanosine rich regions inhibited
CpG-DNA induced immunostimulation. These results indicate that the microglial compartment is sensitive to
bacterial DNA containing CpG-motifs. Thus CpG-DNA could play an important role in infections of the central
nervous system and could contribute to the immunstimulatory properties of the microglial system.
O22 AUTOLOGOUS ACTIVATED MACROPHAGES: A NOVEL THERAPY FOR
ACUTE COMPLETE SPINAL CORD INJURY
V. Fulga, R. Bakimer, L. Reich
Proneuron Biotechnologies, Ness-Ziona, Israel
(E-mail: [email protected])
Objective: The development of an effective therapy for spinal cord injury is a significant challenge. Based on
pre-clinical studies in which administration of activated macrophage into the injured spinal cord of rats induced
partial motor function recovery, we have developed a Phase I clinical protocol to assess the safety of the therapy
for complete spinal cord injury in human subjects.
Methods: Cells – Monocytes were isolated from peripheral blood of patients with spinal cord injury and
activated by co-incubation with autologous skin. The culture was analyzed for cellular make-up and
biochemical characteristics, using flow cytometry and enzyme linked immunosorbent assay (ELISA). The
morphology of the activated monocyte/macrophage was assessed microscopically. The activated cells were
administered into the injured spinal cord. Patients – Patients diagnosed with Acute Complete Spinal Cord Injury
(no sensory and/or motor function below the injury level) are treated within 14 days from the injury by
administration of the autologous activated macrophages directly into the spinal cord parenchyma. The follow-up
continues for 12 months during which the safety and efficacy of the treatment are evaluated by clinical
examination of sensory and motor function below the injury site. Electrophysiological and MRI analyses are also
performed.
Results: A total of three patients with complete spinal cord injury have been enrolled in this phase I clinical
study. All three patients were diagnosed before treatment as complete injuries and classified as ASIA A based on
the American Spinal Cord Injury Association Impairment Scale. After 6-10 months follow-up, all three patients
show significant improvement in neurological functions (sensory recovery in 3 patients and motor recovery in
two patients), no longer being diagnosed with complete neurological loss. Thus, they can be re-classified as
ASIA C (2 patients) and ASIA B (1 patient).
Conclusion: Based on the interim clinical results, autologous activated macrophages may be developed as a
treatment for spinal cord injury in human subjects. Larger clinical studies will be conducted to assess the
potential safety and effect of this therapy.
O23
STRUCTURE AND REGULATION OF THE HUMAN TOLL-LIKE
RECEPTOR 2 GENE IN MYELOID CELLS
Viola Hähnel, Lucia Schwarzfischer, Matthew J. Fenton*, Michael Rehli
Department of Hematology and Oncology, University Hospital, Regensburg, Germany and *Pulmonary Center,
Boston University School of Medicine, Boston, MA, USA (E-mail: [email protected])
The protein product of the Toll-like receptor (TLR) 2 gene has been implicated in the signal transduction events
induced by various microbial products, e.g. peptidoglycans, lipopeptides and lipoarabinomannan. Expression of
TLR2 mRNA in mice and humans is highly regulated and seems restricted to a small number of cell types,
primarily phagocytes and granulocytes. To understand the molecular basis for basal and regulated TLR2
expression in myeloid cells, we started to characterize structure and regulation of the human TLR2 gene. First
we determined the full 5`-sequence of TLR2 mRNA in human monocytes by RLM-PCR and identified the
proximal promoter and the transcriptional start side. The hTLR2 gene consists of three exons with only one start
codon in exon III. Alternative splicing of exon II was detected primarily in fresh isolated monocytes. The
putative 5`-promoter contains several binding sites for transcription factors, including Sp-1, ets- and NF-kB
family members, but no TATA-box. Reporter gene analysis in myeloid (THP 1) and non-myeloid (HeLa) cell
lines identified a minimal promoter of -220 bp which was activated by Sp1, Sp3 and PU.1 in Drosophila
Schneider cells. Interestingly, the murine and the human TLR2 show no significant homology in their 5’-gene
sequences, which results in different expression and activation patterns. In contrast to murine TLR2, both the
promoter and mRNA levels of its human counterpart are not induced by LPS or mycoplasmal lipopeptide
MALP-2 activated NF-kB in human monocytes or macrophages. Northern blot analysis of fresh isolated human
blood monocytes showed an upregulation of TLR2 mRNA after 3h adherence and no further induction by
microbial stimuli. In conclusion our data indicate profound differences in gene expression and regulation of
TLR2 in man and mouse.
O24
LPS-ANTAGONISTS BLOCK PG-INDUCED ACTIVATION IN HUMAN
MONOCYTES, BUT NOT IN TLR2/CD14 TRANSFECTED HEK AND CHO CELLS
Holger Heine, Andra Schromm ,Ulrich Zähringer, and Artur J. Ulmer
Research Center Borstel, 23845 Borstel, Germany (E-mail: [email protected])
Peptidoglycan (PG) is thought to be a major mediator of Gram-positive septic responses. Activation of human
monocytes by PG is mediated by CD14 and requires the expression of TLR2, whereas stimulation by LPS is
mediated by CD14 and TLR4/MD-2. Genetic studies strongly implied that lipid A- as well as LPS antagonists
such as lipid IVa or the synthetic compound B1233 directly block the response of the cells at the level of TLR4.
Interestingly, these LPS antagonists can also inhibit activation of human monocytes by PG. We tried to analyze
the molecular basis for this inhibition using transfected HEK or CHO cells. Under all investigated conditions,
using transfection with CD14, TLR2, TLR4, and/or MD-2, LPS antagonists failed to block PG activation.
Moreover, the expression of CD14 was not required for the signaling induced by PG. Therefore, we hypothesize
that the composition of the receptor complex responsible for transmitting the PG-induced signal in human
monocytes differs from the receptor complex in transfected HEK and CHO cells, thus resulting in a different
sensitivity to LPS antagonists.
This work has been supported by the DFG (SFB367, project C5)
O25
ENDOGENOUS AND EXOGENOUS HOST DEFENSE USE IDENTICAL
MECHANISMS FOR LIGAND SPECIFIC CLUSTERING OF THE INNATE
IMMUNITY RECEPTOR IN RAFT DOMAINS
A. Götz, M. Kapinsky, E. Orsó, G. Schütz*, H.G. Schindler*, W. Drobnik, G. Rothe, G. Schmitz
Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, 93042 Regensburg,
Germany. * Institute of Biophysics, University of Linz, Austria
(E-mail: [email protected])
The goal of the present study was to further investigate the CD14 dependent defense mechanisms. By using
fluorescence resonance energy transfer (FRET) technology. We could show that a multimeric receptor complex
is formed upon LPS activation in human monocytes, including CD14, complement receptor 3 (CR3), Fc-RIII,
CD36, TAPA, decay accelerating factor (DAF) and Toll-like receptor 4 (TLR4). In contrast, the stimulation of
cells with lipoteichoic acid (LTA) preferentially clustered TLR-2 instead of TLR-4. Moreover, we provide
evidence that long chain ceramides which may act as endogenous inflammatory mediators are also ligands of
CD14 and promote the formation of receptor clustering. However, the ceramide-induced receptor cluster differs
from that of LPS, as it involves CD14, CR3, CD36, CD47 integrin associated protein (IAP) and CD55 (DAF).
The LPS and ceramide induced co-assembly of CD14 with CD11b was totally blocked by cholesterol loading of
the plasma membrane, while cholesterol depletion reduced the co-association of CD11b and CD14. In addition,
the LPS antagonist lipid IVa significantly reduced both LPS and ceramide induced receptor clustering. These
data indicate the exogenous as well as endogenous host defense systems engage similar mechanisms, that are
related to an intact raft structure and the formation of ligand specific multimeric receptor complexes, followed by
a ligand specific integrated signaling response