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Workshop V: Neuro-Endocrino-Immunology
Workshop V Neuro-Endocrino-Immunology
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Department of Internal Medicine, Charité, Humboldt-University, Berlin, Germany, Department of Gynecology, University of Pecs, Pecs, Hungary, and 3Department of Endocrinology, University of Edinburgh, Edinburgh, UK 2
V. 1 Endocrine-immune interaction in murine stress-triggered abortion: The progesterone derivate dydrogesterone induces a Th2 biased local immune response P.C. ARCK1, R.A. JOACHIM1, J. SZEKERS-BARTHO2, A. DOUGLAS3, A.C. ZENCLUSSEN1, S. FEST1, and B.F. KLAPP1 Problem: Stress is known to induce abortions in mice and human via increased levels of abortogenic Th1 cytokines, decrease of progesterone, or the pregnancy protective progesterone induced blocking factor (PIBF). Adequate levels of progesterone exert an antiabortive effect by induction of a Th2 biased immune response an by controlling NK activity. The aim of this study was to investigate the protective effect of the progesterone derivative dydrogesterone in stress triggered murine abortion. Methods: DBA/2J-mated CBA/J female mice were exposed to ultrasonic sound stress on day 5 of pregnancy. The mice were randomized and some were treated with dydrogesterone in different dosages. Results: On day 13 of pregnancy uteri were removed and the abortion rate was calculated. An abortion rate of 44.1% was detected in stressed mice compared to 10.7% in non-stressed control mice, accompanied by decreased levels of progesterone and PIBF. Injection of dydrogesterone significantly decreased the abortion rate in stressed mice. Flow cytometry of decidual cells revealed that dydrogesterone dramatically increased IL-4 positive decidual immune cells in stressed mice. Conclusion: These data suggest that dydrogesterone abrogates stress triggered abortion by inducing a Th2 biased local immune response.
Institut für Medizinische Mikrobiologie und Virologie, Heinrich-Heine-Universität, Düsseldorf, Germany
V. 2 Different anti-viral effector mechanisms induced by type 1 and type 2 interferons W. DÄUBENER, C. OBERDÖRFER, K. BESKEN, C.R. MACKENZIE, and O. ADAMS Human astrocytoma cells are capable of restricting the growth of Toxoplasma gondii and of group B streptococci after stimulation with IFN-g. We have described that the anti-microbial effect of IFN-g-stimulated astrocytoma cells is mediated via an induction of indoleamine-2,3-dioxygenase. This enzyme degrades L-tryptophan to kynurenine, and this results in complete depletion of
312 · 33rd Annual Meeting of the German Society of Immunology L-tryptophan, an essential amino acid for both pathogens. IDO activity was found in IFN-g activated astrocytoma cells by the detection of IDO mRNA in RT-PCR, detection of the enzyme with monoclonal antibodies in western blot, as well as by direct measurement of enzyme activity in the activated cells. IFN-g mediated IDO activity in human astrocytoma cells is also responsible for the inhibition of virus growth since this anti-viral effect is blocked in the presence of excess amounts of L-tryptophan. In addition we found that a co-stimulation of astrocytoma cells, and also of endothelial cells with IFN-g and TNF-a results in an enhanced anti-viral effect. This synergistic effect of TNF-a and IFN-g is mediated by an enhanced activation of IDO. The anti-viral effect induced in cells co-stimulated with IFN-g and TNF-a could be blocked by a supplementation with L-tryptophan. Similar to the anti-viral effect of IFN-g both type 1 interferons, IFN-a and IFN-b are also able to restrict herpes simplex growth in astrocytoma cells. Using a quantitative PCR protocol we found that all three interferons are able to induce IDO gene transcription, however IDO mRNA induction in astrocytoma cells stimulated with IFN-a or IFN-b is 10 to 20 fold lower than in IFN-g stimulated cells. Furthermore, the addition of an excess amount of L-tryptophan is not sufficient to inhibit the IFN-a-IFN-b induced anti-viral effect. We therefore conclude that the anti-viral effects mediated by type 1 and type 2 interferons are different.
Abteilung für Klinische Chemie und Molekulare Diagnostik, Klinikum der PhilippsUniversität, Marburg, Germany
V. 3 Expression of neurotrophins and neurotrophin receptors in mouse B cells J. EMMEL, B. SCHUHMANN, H. RENZ, and W.A. NOCKHER The neurotrophins (NT), including nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin- 3 (NT-3) and neurotrophin-4/5 (NT-4/5) play an important role in development and maintenance of the nervous system. NT-signaling is mediated by binding to ligand specific high affinity receptors (trk A, trk B, trk C) and to the low affinity pan-neurotrophin receptor p75NTR. In addition neurotrophins are also important mediators in the immune system. Recent studies have shown that neurothrophins influence the immune response by acting on various inflammatory cells. Furthermore, it has been recognized that several immune competent cells were also able to produce these factors. The aim of this study was to characterize the expression pattern of NTs and NT-receptors on B220 positive B cells in the bone marrow and spleen. Murine B cells from 17-days old C57/B6 mice were purified by B-220 immunoselection using a MoFlo cell sorter with a purity up to 90%. Expression of NGF, BDNF, NT-3 as well as expression of the NT-receptors trk A, trk B, trk C and p75NTR were detected by using RT-PCR in all analysed samples. Monitoring the trk B-receptor, we used primer pairs detecting the intracellular signal transduction domain or the extracellular ligand binding domain. We found that B220 positive B cells of bone marrow and spleen constitutively express mRNA for all NTs and NT-receptors except the active signal transducing form of the trk B. This receptor was only found in the brain. However, mouse B cells expressed the truncated form of the trk B receptor without the intracellular kinase domain. Preliminary experiments by real-time PCR with LightCycler reveal quantitative differences between B cells in bone marrow and spleen. We conclude that regulation of neurotrophin expression and signaling may be important during mouse B cell development.
33rd Annual Meeting of the German Society of Immunology · 313 1
Department of Neurology, University of Rostock, Rostock, 2Max-Delbrück-Center for Molecular Medicine, and 3Deutsches Rheuma-Forschungszentrum, Berlin, Germany
V. 4 Functional upregulation of CTLA-4 expression in T helper cells by astrocytes: A possible mechanism counteracting CNS inflammation U. GIMSA1, A. OREN2, and M.C. BRUNNER-WEINZIERL3 Astrocytes are the most abundant glial cells of the CNS whose main task is the trophic support and the maintenance of physiologic homeostasis for neurons. When activated in neuroinflammation, they can act as antigen-presenting cells since they express MHC-II and B7 molecules. Since astrocytes have been shown to exert both pro- and anti-inflammatory actions, we tested their influence on T helper cell function. In our experiments, astrocytes that were activated by IFN-g inhibited proliferation of MBP-specific Th1 and Th2 cells from T cell receptor (TCR) transgenic mice and inhibited IFN-g and IL-10 but not IL-4 production. The inhibition of proliferation was at least partly mediated by induction of CTLA-4 expression in T cells by astrocytes since anti-CTLA-4 treatment reversed the inhibition of proliferation partly. We found an upregulation of CTLA-4 in T cells induced by astrocytes which was independent of antigen-presentation but not of prior T cell activation. This upregulation of CTLA-4 occurred even in the absence of cell-cell contact between T cells and astrocytes showing that a soluble factor is responsible. The inhibition of proliferation by induction of CTLA-4 expression in CNS-invading T cells might be one mechanism how astrocytes downregulate T cell-mediated inflammation in the CNS.
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Laboratory of Molecular Immunology, Institute of Genetics, University of Cologne, Cologne, Germany, 2Center for Blood Research, Boston MA, USA, and 3Department of Neuroimmunology, Brain Research Institute, University of Vienna, Vienna, Austria
V. 5 Conditional deletion reveals a role for Fas–Fas ligand interaction in brain inflammation N. HÖVELMEYER1, Z.-Y. HAO1, T. BUCH1, K. RAJEWSKY2, H. LASSMANN3, and A. WAISMAN1 Experimental autoimmune encephalomyelitis (EAE) is a widely used animal model for multiple sclerosis. The disease is manifested by an influx of T cells to the brain. EAE can be induced in C57BL/6 mice by immunization with a peptide derived from myelin oligodendrocyte glycoprotein (MOG). It was previously shown that mice deficient for the fas gene (lpr mice) are partially resistant to MOG-induced EAE. As lpr mice have a deficiency in the development of the immune system, it is not clear whether their inability to develop EAE is a result of compromised T cell development or from resistance of brain cells to Fas-mediated apoptosis. We have decided to circumvent this problem by making a mouse with a tissue specific deletion of the fas gene in the brain. To this end, we have crossed a mouse in which the fas gene is flanked by two loxP sites with a mouse that express the cre-recombinase specifically in oligodendrocytes utilizing the MOG promoter (MOG-Cre) developed in our laboratory. EAE was induced in mice with the loxP flanked fas gene that express the MOG-Cre and as wild type controls, in mice without the
314 · 33rd Annual Meeting of the German Society of Immunology MOG-Cre. We found that the mice in which Fas was inactivated in oligodendrocytes had a milder form of EAE compared to the control mice, manifested by lower level of paralysis. These clinical findings could be verified by pathological examination of the CNS, showing that the mice expressing MOG-Cre had less inflammatory infiltrate, demyelination and nerve cell apoptosis compared to control mice. Together, these results demonstrate that killing of oligodendrocytes by Fas–Fas ligand interaction contribute to the development of inflammatory autoimmune disease in the brain.
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Department of Molecular Neuroimmunology, Institute of Anatomy and Cell Biology and Institute of Pharmacology and Toxicology, Phillips-University, Marburg, Germany, 3 Department of Microbiology and Immunology, University of Leicester, Leicester and 4 Rheumatology Section, Hammersmith Campus, Imperial College School of Medicine, London, UK 2
V. 6 Functionally active C1q limits brain injury in a mouse model of permanent focal cerebral ischemia M. HRISTOVA1, C. CULMSEE2, V. JUNKER2, C. STOVER3, M. BOTTO4, N. BIKOV1, J. KRIEGLSTEIN2, W. SCHWAEBLE3, and E. WEIHE1 Complement activation may participate in neurodegenerative processes. Previous studies demonstrate that the classical pathway recognition molecule C1q is specifically up-regulated in brain microglia during transient global cerebral ischemia. To investigate the role of C1q in ischemic brain damage we induced permanent focal cerebral ischemia by left middle cerebral artery (MCA) electrocoagulation in male C1q-deficient, Factor B/C2-deficient and wildtype mice (SVJ129 genotype). The animals were sacrificed 2 and 7 days after MCA occlusion. The infarct size was determined by image analysis system (SCION). C1q mRNA and protein-expression were analysed by in situ hybridisation and immunocytochemistry. The infarct area in the C1q deficient mice was significantly increased 2 days post ischemia (p.i.) as compared to the C1q sufficient littermate controls. The infarct size in all groups was reduced 7 days p.i. when compared to 2 days p.i. The late reduction of infarcted tissue was significantly less pronounced in C1q Achain deficient mice in comparison to the wildtype control. The deletion of Factor B/C2 genes had no impact on the overall size of the infarct area. Significant increases in mRNA expression of all three C1q chains were observed 2 and 7 days p.i. C1q A-chain mRNA was undetectable in C1q A–/– mice resulting in total deficiency of C1q functional activity assessed in hemolytic assay. The deletion of the C1q A-chain gene however, did not affect the expression of C1q B- and Cchain genes. The ischemia-induced increase in C1q B- and C-chain mRNA abundance in the wildtype was also observed in C1q A-chain deficient mice suggesting that the three genes encoding C1q are regulated independently. This suggests that C1q contributes to limitation of brain injury after stroke. C1q and the classical complement pathway probably fulfil a crucial scavenger function in the cellular debris disposal in the course of neurodegeneration and neuroinflammation. Supported by DFG.
33rd Annual Meeting of the German Society of Immunology · 315 Departments of 1Internal Medicine and 2Pediatrics, Charité, Humboldt-University, Berlin, Germany
V. 7 Neurokinin 1 receptor mediates stress-induced increment on bronchoalveolar cell count in ovalbumin sensitized mice R.A. JOACHIM1, D. QUARCOO2, A.C. ZENCLUSSEN1, P.C. ARCK1, and B.F. KLAPP1 Airway inflammation is a leading symptom of bronchial asthma. Recent data is linking stress to the initiation and perpetuation of asthma symptoms. Tachykinins like substance P have been shown to play an important role in generating asthma symptoms and furthermore to be released in tissues in response to stress. The aim of the study was to determine 1) the influence of stress on murine allergen induced airway inflammation and 2) the involvement of tachykinins in stress mediation of airway inflammation. CBA/J mice were sensitized by i.p. injection of ovalbumin (OVA). Coinciding with the beginning of the first OVA aerosol airway provocation, one half of the mice was stressed by exposure to an ultrasonic sound stressor for 24 h. Part of the animals were additionally treated with a tachykinin NK1-receptor antagonist (NK1-RA) before airway challenges. 24 h after the second airway provocation, animals were sacrificed. Broncho-alveolar lavage fluid (BAL) was obtained and total cell numbers were determined. Cytospins were prepared for each sample and after staining differential cell counts were performed. A significantly higher total cell count was present in the BAL in stressed animals compared to unstressed control mice. This stress-induced increase was mainly caused by an increase of eosinophils and could be blocked by application of the NK1-RA, whereas NK1-RA did not influence airway inflammation in unstressed controls. Our data suggest that stress increases allergen induced airway inflammation. Furthermore it points to tachykinins like substance P as putative mediators in stress induced airway inflammation.
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Institute of Clinical Immunology and Transfusion Medicine and 4Ear, Nose and Throat Department, University of Leipzig, Leipzig, 2Ear, Nose and Throat Department, Klinikum Chemnitz, Chemnitz, and 3Institute of Immunology, Medical Faculty Carl Gustav Carus, Technical University of Dresden, Dresden, Germany
V. 8 Autoimmune findings in sudden hearing loss patients M. KAMPRAD1, K. DONAUBAUER2, K. CONRAD3, H. MÜLLER4, U. FICKWEILER4, and U. SACK1 Early diagnosis of immunologically relevant acute diseases of the inner ear such as sudden hearing loss can improve patient care and prevent complications. Recent publications describe candidate antigens for autoantibodies related to inner ear diseases. In this retrospective study, we have investigated diagnostic reliability of relevant autoantibodies for differential diagnosis of patients in an ENT department.
316 · 33rd Annual Meeting of the German Society of Immunology 58 patients with inner ear diseases and 62 controls with other ENT diagnosis have been investigated for the presence of the following autoantibodies: ANA screen, ENA screen including RNP, Sm, SS-A, SS-B, Jo-1, Scl-70 and CenpB, autoantibodies to endothelial cells, to cardiolipin, to 68 kD heat shock protein, to type II collagen, and rheumatoid factors. In sera of sudden hearing loss patients, only sometimes ANA and never ENA could be detected. Autoantibodies to 68 kD heat shock protein and type II collagen were less frequently detectable in comparison to the controls. Interestingly, antibodies to endothelial cells correlated with sudden hearing loss when detected on HUVEC cells but not by indirect immunofluorescence at lung tissue. Anti-cardiolipin antibodies and rheumatoid factors, particularly IgM, could be common found in patients with inner ear diseases. Although none of the parameters correlated significantly with sudden hearing loss, frequency of any autoantibody positivity is enhanced (p=0.012). By calculating a computerized classification based on positive and negative correlations between single parameters and sudden hearing loss, groups could be separated with p<0.001. We conclude that parameters such as ANA, ENA, antibodies to 68 kD heat shock protein and to type II collagen are rather absent in sudden inner ear diseases. By including a whole panel of parameters, classification of sudden hearing loss patients can be performed in a substantial significant manner. Nevertheless, a single significant parameter to identify such diseases has yet to be found.
Institute of Anatomy and Cell Biology, Philipps-University, Marburg, Germany
a expression in mouse brain V. 9 Systemic LPS but not SEB induces TNF-a spreading from meninges and circumventricular organs into the cerebral parenchyma O. KAUT, M. BETTE, and E. WEIHE The role of TNF-a within the immune system-to-brain communication under conditions of bacterial lipopolysaccharide (LPS)- or staphylococcal enterotoxin B (SEB)-evoked toxemia is still not fully understood. The aim of our study was to monitor the constitutive expression of TNF-a and to differentiate the effects of a macrophage activation induced by LPS from a T cell specific activation induced by SEB on TNF-a expression in the brain. Under basal conditions in situ hybridization revealed no expression of TNF-a mRNA in the brain. At 1h post injection (p.i.) LPS caused an increase of TNF-a serum levels, of c-fos expression in the hypothalamic paraventricular nucleus (PVN), and of TNF-a expression in the circumventricular organs (CVOs), meninges and choroid plexus. At 4h and later stages p.i., TNF-a mRNA disappeared from the CVOs, meninges and choroid plexus and was translocated in regions adjacent to the CVOs and to brain parenchyma. SEB challenge caused an elevation of TNF-a in the serum, an increase of c-fos expression in the PVN, but in contrast to LPS no induction of cerebral TNF-a mRNA. Thus we demonstrate and conclude that: i.) elevated TNF-a serum levels are not automatically paralleled by an induction of TNF-a expression in the brain ii.) peripheral T cell activation is not causing cerebral induction of TNF-a iii.) the expression of cerebral TNF-a within the immune system-to-brain communication is dependent on the specific immune stimulus. Supported by SFB 297
33rd Annual Meeting of the German Society of Immunology · 317 Department of Internal Medicine I, University Hospital, Regensburg, Germany
V. 10 Presence of the sympathetic nerve repellent Semaphorin 3C and lack of the sensory nerve repellent Semaphorin 3A in RA synovium may reduce sympathetic innervation and may perpetuate inflammation L.E. MILLER, J. GRIFKA, W. FALK, J. SCHÖLMERICH, and R.H. STRAUB In terms of the chronic inflammation that occurs during rheumatoid arthritis (RA), the sensory nervous system tends to produce proinflammatory stimuli, while the sympathetic nervous system tends to induce anti-inflammatory effects. We have recently shown a distinct reduction of the sympathetic nerve fibers in the synovial tissue of RA patients (FASEB J 2000 14:2097–2107). The presence of selective nerve repellents, i.e. mediators of the semaphorin family, could be responsible for the observed reduction of the sympathetic innervation. In order to investigate the presence of mRNA of different semaphorins in synovial tissue, Diglabeled RNA probes were constructed. These Dig-labeled RNA probes were used for in situ hybridizations within frozen tissue sections from patients with RA and OA. Control hybridizations with probes directed against fibronectin and vimentin were carried out in parallel to the semaphorin hybridizations. In situ hybridizations showed that semaphorin 3C mRNA, a nerve repellent of sympathetic nerve fibers, was present in the synovial tissue of all investigated patients with RA (N=5) and two investigated patients with OA. In contrast, semaphorin 3A mRNA, directed against sensory nerve fibers, could not be detected in the synovial tissue of patients with RA (N=4) and OA (N=4). The parallel control hybridizations with probes against vimentin (RA, N=6; OA, N=2) and fibronectin (RA, N=2; OA, N=1) were always positive and therefore confirmed technically successful hybridization conditions. Control hybridizations with the sense probes were always negative. These findings suggest that semaphorin 3C, which is selectively directed against sympathetic nerve fibers, may be responsible for the reduced sympathetic innervation in the synovial tissue of patients with RA. The inability of the sympathetic nervous system to re-innervate the synovial tissue of patients with RA could be contributing to the chronic inflammatory nature of RA.
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Department of Neurology, Eberhard-Karls-University, Tübingen, 2Department of Anthropology and Human Genetics, 3Genzentrum and Friedrich-Baur Institut, and 4Institute of Clinical Neuroimmunology, Ludwig Maximilians University, Munich, Germany
V. 11 The nonclassical MHC molecule HLA-G protects human muscle cells from immune-mediated lysis: Implications for myoblast transplantation and gene therapy M. MITSDÖRFFER1, V. HOFMEISTER2, S. KRAUSE3, E.H. WEISS2, H. LOCHMÜLLER3, R. HOHLFELD4, A. MELMS1, M. WELLER1, and H. WIENDL1 HLA-G is a nonclassical MHC class I molecule with highly limited tissue distribution which has been attributed chiefly immune regulatory functions. We have previously reported that HLA-G
318 · 33rd Annual Meeting of the German Society of Immunology is expressed in inflamed muscle in vivo and by cultured myoblasts in vitro. Here, we used the in vitro models of human myoblasts or TE671 muscle rhabdomyosarcoma cells to characterize the functional role of HLA-G for muscle immune cell interactions. Gene transfer of the two major isoforms of HLA-G (transmembranous HLA-G1, soluble HLA-G5) into TE671 rendered these cells resistant against direct alloreactive lysis and killing by antigen-specific cytotoxic T cells. HLA-G not only inhibited NK cells but also prevented alloreactive lysis by CD4 and CD8 T cells. Further, HLA-G obviated effective priming of antigen-specific cytotoxic T cells and reduced antigen specific lysis. HLA-G pre-induced on cultured myoblasts inhibited lysis by alloreactive peripheral blood mononuclear cells. This protection was reversed by a neutralizing HLA-G antibody. Interestingly, few HLA-G-positive cells within a population of HLA-G negative muscle target cells conveyed significant inhibitory effects on alloreactive lysis. Our results reveal further insights into the immunobiology of muscle and suggest that ectopic expression of HLA-G may promote the survival of transplanted myoblasts in the future treatment of hereditary muscle diseases. Further, HLA-G could represent a novel self-derived anti-inflammatory principle applicable in strategies against inflammatory aggression.
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Department of Experimental and Physiological Psychology and 2Institute of Theoretical Surgery, University of Marburg, Marburg, Germany, and 3Department of Psychology, Chung Shan Medical University, Taiwan, ROC
V. 12 Relationships between anxiety and basal IL-2 mRNA levels in the striatum of the rat C.R. PAWLAK1, A. BAUHOFER2, Y.J. HO3, and R.K.W. SCHWARTING1 Pharmacological interventions in the periphery or brain with LPS, cyclosporin A, or interleukins (IL), have been shown to affect behavior, e.g. anxiety or exploration. In contrast, it is largely unknown whether endogenous levels of cytokines in specific brain compartments may be related to such behaviors. Our previous experiments have shown that male Wistar rats, although identical in strain, sex, and age can differ systematically in their anxiety, or exploration behavior. Furthermore, we found anxiety to be related to the neurotransmitter serotonin in the ventral striatum, whereas exploration was related to the neurotransmitter dopamine and acetylcholine. Therefore, we asked whether behavioral individuality may also be related to immunological function in the brain. First, we tested a sample of male adult outbred Wistar rats (n=34) in our routine testing procedure, that is a novel open field (exploration), followed by the elevated plus-maze (anxiety). Based on the behavioural analyses in a novel open field, the rats were divided into two subgroups according to their exploration behavior expressed as low rearing (LRA), or high rearing activity (HRA). In parallel, the same animals were divided into low (LA), or high anxiety (HA) rats based on time spent on the closed arm in an elevated plus-maze. Then, IL-1b, IL-2, IL-6, and TNF-a cDNA levels were measured post mortem in striatum tissues using semi-quantitative, competitive, reverse transcription polymerase chain reaction (RT-PCR). We observed no significant differences between LRA and HRA rats (novel open field behavior) and the analysed cytokines. However, HA compared to LA rats showed significantly higher IL-2 mRNA levels (p<.05), but no altered IL-1b, IL-6, and TNF-a mRNA expression. Additionally, pronounced individual differences in all cytokine levels in the striatum were observed. These results suggest basal levels of IL-2 mRNA expression in the striatum to be associated with anxiety behavior in adult male Wistar rats.
33rd Annual Meeting of the German Society of Immunology · 319 Institutes of 1Applied Psychology and 2Clinical Immunology and Transfusion Medicine, University of Leipzig, Leipzig, Germany
V. 13 Psycho-immunological process evaluation of a stress preventive intervention program for teachers M. STÜCK1, K. MEYER1, K. BAUER2, and U. SACK2 Immunological and psychological parameters reflecting stress management capabilities are closely connected and have been shown to interact in a highly reproducible manner. With our study we wanted to examine this correlation between self-regulative therapeutic stimuli and immunoglobulin A in saliva. As part of the integrative stress management training-concept for schools, a ten-week stress management training was carried out with teachers. It was the aim of this intervention to enable teachers to better cope with their daily pressures. The effects of the intervention methods were to be determined in a accompanying process-evaluation study. For this purpose, health-psychological (stress-relevant psychological variables; work related behavioural and experience patterns) as well as psychological (blood pressure, skin resistance) and immunological variables (immunoglobulin A) were taken. For the immunological variables we took immunoglobulin A from the saliva of 12 teachers before and after the sessions of both intervention studies. Parallel to this different emotional parameters were ascertained. For both methods overwhelming effects in improving stress resistance could be verified. In saliva, increase of immunoglobulin A could be found in persons with a positive response to our training. Furthermore, a significant positive correlation between the subjective sensation of relaxation or other health parameters and IgA could be measured. Our ten-week stress management training could be shown to be effective in improving stress management capabilities. Psychological and subjective parameters could be found to correlate well with immunoglobulin A concentration in saliva. Therefore, measurement of this simple immunological parameter could be proved to be a reliable indicator for stress resistance as well as training effects.
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Department of Medicine II, University of Leipzig, Leipzig and 2Department of Rheumatology and Clinical Immunology, Charité, Humboldt-University, Berlin, Germany
V. 14 Altered functional response to stimulation of b2-adrenergic receptors in CD4 and CD8 positive human T cells following preincubation with IL-2 M. WAHLE1, I. NAUMANN1, A. KRAUSE2, H. HÄNTZSCHEL1, and C.G.O. BAERWALD1 Human CD4 and CD8 positive T lymphocytes express b2-adrenergic receptors (b2-R). However, the expression of b2-R is about fourfold higher on CD8 positive T cells than on CD4 positive T cells. In addition, previous investigations revealed that IL-2 increased b2-R expression significantly in CD8 compared to CD4 positive T cells. To further evaluate the effect of IL-2 on b2-R function, CD4 and CD8 positive T cells were isolated and preincubated with IL-2 before stimulation and activation of b2-R. CD4 and CD8 positive lymphocytes from healthy donors were isolated by magnetic cell separation (MACS) and activated with anti-CD3 and anti-CD28 antibody together with IL-2. Aliquots of CD4 or CD8 positive T cells were incubated with 0.5 or 5 ng/ml
320 · 33rd Annual Meeting of the German Society of Immunology IL-2 for 16 or 72 h prior to stimulation. Assessment of b2-R function was determined by the suppression of IFN-g (IFN-g) production following stimulation with the b2-R agonist epinephrine (10 mM). The concentration of IFN-g was determined in culture supernatant after 24 and 48 h using a sandwich ELISA. Stimulation of T cells induced a time dependent increase in IFN-g production of CD4 and CD8 positive T cells. Epinephrine inhibited the synthesis of IFN-g by 40% (CD4) and 50% (CD8), respectively (p<0.05). Preincubation with IL-2 for 16 h, but not 72 h, abrogated the functional response to epinephrine. In contrast, the IFN-g concentration in the culture supernatant of stimulated cells even increased upon co-incubation with epinephrine (5 ng/ml IL-2, not significant). In conclusion, the function of b2-R on CD4 and CD8 positive T cells is altered by IL-2 time and dose dependently. It may be speculated that IL-2 additionally induces the functional expression of alpha-adrenergic receptors on human T cells.
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Department of Neurology, Eberhard-Karls-University, Tübingen, 3Department of Neurology, Heinrich-Heine-Universität, Düsseldorf, Germany, and 2Department of Neurology, KarlFranzens-Universität, Graz, Austria,
V. 15 Functional characterization of the CD28-related molecule ICOS in multiple sclerosis: A possible target for selective immune intervention? H. WIENDL1, O. NEUHAUS2, M. MEHLING1, S. WINTTERLE1, B. SCHREINER1, M. MITSDÖRFFER1, R. WEISSERT1, M. WELLER1, H.P. HARTUNG3, E. TOLOSA1, and A. MELMS1 ICOS has recently been described as an inducible CD28 related costimulatory molecule with relevance for T cell differentiation and effector function and a possible role in autoimmune diseases like multiple sclerosis. This study was aimed at characterizing expression and functional role of ICOS costimulation in multiple sclerosis. After nonspecific or antigen-specific stimulation, ICOS was shown to be preferentially expressed on CD4+ Th2 T cells. Experiments with antigen-specific T cell lines as well as purified SAg-stimulated CD4 T cells demonstrated that ICOS-costimulation affected T cell proliferation and production of both Th1 and Th2 cytokines, whereas expression of T cell activation markers or chemokine receptors were unaffected. Interestingly, the effects on cytokine secretion were observable in the presence and the absence of B7 costimulation, therefore suggesting that ICOS costimulation can modulate cytokine secretion also independently of CD28. Levels of constitutive and inducible ICOS expression on human T cell subsets from peripheral blood were quantified in healthy donors (n=10) and patients with multiple sclerosis (acute relapse or chronic disease; n=10). Constitutive expression of ICOS on T cells varied between 0.1 to 42.3%. No significant differences were noted between both groups concerning the baseline expression or inducibility on T cells. Cells constitutively expressing ICOS did not represent the CD4+CD25+ regulatory T cell subset. ICOS expression on CSF T lymphocytes in patients with acute MS relapses or acute neuroborreliosis showed that expression of ICOS was not elevated compared to peripheral blood. Neither IFN-b or glatiramer acetate did alter ICOS-expression, whereas the anti-inflammatory cytokine IL-10 downregulated ICOS. In summary, ICOS costimulation affects both Th1 and Th2 cytokine production in the presence and absence of B7-costimulation. Since ICOS is rapidly induced on T cells after antigenspecific stimulation, this molecule qualifies as a suitable target aimed at modulating T cell cytokine responses in CNS inflammation.
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Department of Internal Medicine, Charité, Humboldt-University, Berlin, Germany, Department of Gynecology, University of Pecs, Pecs, Hungary, and 3Department of Endocrinology, University of Edinburgh, Edinburgh, UK 2
V. 1 Endocrine-immune interaction in murine stress-triggered abortion: The progesterone derivate dydrogesterone induces a Th2 biased local immune response P.C. ARCK1, R.A. JOACHIM1, J. SZEKERS-BARTHO2, A. DOUGLAS3, A.C. ZENCLUSSEN1, S. FEST1, and B.F. KLAPP1 Problem: Stress is known to induce abortions in mice and human via increased levels of abortogenic Th1 cytokines, decrease of progesterone, or the pregnancy protective progesterone induced blocking factor (PIBF). Adequate levels of progesterone exert an antiabortive effect by induction of a Th2 biased immune response an by controlling NK activity. The aim of this study was to investigate the protective effect of the progesterone derivative dydrogesterone in stress triggered murine abortion. Methods: DBA/2J-mated CBA/J female mice were exposed to ultrasonic sound stress on day 5 of pregnancy. The mice were randomized and some were treated with dydrogesterone in different dosages. Results: On day 13 of pregnancy uteri were removed and the abortion rate was calculated. An abortion rate of 44.1% was detected in stressed mice compared to 10.7% in non-stressed control mice, accompanied by decreased levels of progesterone and PIBF. Injection of dydrogesterone significantly decreased the abortion rate in stressed mice. Flow cytometry of decidual cells revealed that dydrogesterone dramatically increased IL-4 positive decidual immune cells in stressed mice. Conclusion: These data suggest that dydrogesterone abrogates stress triggered abortion by inducing a Th2 biased local immune response.
Institut für Medizinische Mikrobiologie und Virologie, Heinrich-Heine-Universität, Düsseldorf, Germany
V. 2 Different anti-viral effector mechanisms induced by type 1 and type 2 interferons W. DÄUBENER, C. OBERDÖRFER, K. BESKEN, C.R. MACKENZIE, and O. ADAMS Human astrocytoma cells are capable of restricting the growth of Toxoplasma gondii and of group B streptococci after stimulation with IFN-g. We have described that the anti-microbial effect of IFN-g-stimulated astrocytoma cells is mediated via an induction of indoleamine-2,3-dioxygenase. This enzyme degrades L-tryptophan to kynurenine, and this results in complete depletion of
312 · 33rd Annual Meeting of the German Society of Immunology L-tryptophan, an essential amino acid for both pathogens. IDO activity was found in IFN-g activated astrocytoma cells by the detection of IDO mRNA in RT-PCR, detection of the enzyme with monoclonal antibodies in western blot, as well as by direct measurement of enzyme activity in the activated cells. IFN-g mediated IDO activity in human astrocytoma cells is also responsible for the inhibition of virus growth since this anti-viral effect is blocked in the presence of excess amounts of L-tryptophan. In addition we found that a co-stimulation of astrocytoma cells, and also of endothelial cells with IFN-g and TNF-a results in an enhanced anti-viral effect. This synergistic effect of TNF-a and IFN-g is mediated by an enhanced activation of IDO. The anti-viral effect induced in cells co-stimulated with IFN-g and TNF-a could be blocked by a supplementation with L-tryptophan. Similar to the anti-viral effect of IFN-g both type 1 interferons, IFN-a and IFN-b are also able to restrict herpes simplex growth in astrocytoma cells. Using a quantitative PCR protocol we found that all three interferons are able to induce IDO gene transcription, however IDO mRNA induction in astrocytoma cells stimulated with IFN-a or IFN-b is 10 to 20 fold lower than in IFN-g stimulated cells. Furthermore, the addition of an excess amount of L-tryptophan is not sufficient to inhibit the IFN-a-IFN-b induced anti-viral effect. We therefore conclude that the anti-viral effects mediated by type 1 and type 2 interferons are different.
Abteilung für Klinische Chemie und Molekulare Diagnostik, Klinikum der PhilippsUniversität, Marburg, Germany
V. 3 Expression of neurotrophins and neurotrophin receptors in mouse B cells J. EMMEL, B. SCHUHMANN, H. RENZ, and W.A. NOCKHER The neurotrophins (NT), including nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin- 3 (NT-3) and neurotrophin-4/5 (NT-4/5) play an important role in development and maintenance of the nervous system. NT-signaling is mediated by binding to ligand specific high affinity receptors (trk A, trk B, trk C) and to the low affinity pan-neurotrophin receptor p75NTR. In addition neurotrophins are also important mediators in the immune system. Recent studies have shown that neurothrophins influence the immune response by acting on various inflammatory cells. Furthermore, it has been recognized that several immune competent cells were also able to produce these factors. The aim of this study was to characterize the expression pattern of NTs and NT-receptors on B220 positive B cells in the bone marrow and spleen. Murine B cells from 17-days old C57/B6 mice were purified by B-220 immunoselection using a MoFlo cell sorter with a purity up to 90%. Expression of NGF, BDNF, NT-3 as well as expression of the NT-receptors trk A, trk B, trk C and p75NTR were detected by using RT-PCR in all analysed samples. Monitoring the trk B-receptor, we used primer pairs detecting the intracellular signal transduction domain or the extracellular ligand binding domain. We found that B220 positive B cells of bone marrow and spleen constitutively express mRNA for all NTs and NT-receptors except the active signal transducing form of the trk B. This receptor was only found in the brain. However, mouse B cells expressed the truncated form of the trk B receptor without the intracellular kinase domain. Preliminary experiments by real-time PCR with LightCycler reveal quantitative differences between B cells in bone marrow and spleen. We conclude that regulation of neurotrophin expression and signaling may be important during mouse B cell development.
33rd Annual Meeting of the German Society of Immunology · 313 1
Department of Neurology, University of Rostock, Rostock, 2Max-Delbrück-Center for Molecular Medicine, and 3Deutsches Rheuma-Forschungszentrum, Berlin, Germany
V. 4 Functional upregulation of CTLA-4 expression in T helper cells by astrocytes: A possible mechanism counteracting CNS inflammation U. GIMSA1, A. OREN2, and M.C. BRUNNER-WEINZIERL3 Astrocytes are the most abundant glial cells of the CNS whose main task is the trophic support and the maintenance of physiologic homeostasis for neurons. When activated in neuroinflammation, they can act as antigen-presenting cells since they express MHC-II and B7 molecules. Since astrocytes have been shown to exert both pro- and anti-inflammatory actions, we tested their influence on T helper cell function. In our experiments, astrocytes that were activated by IFN-g inhibited proliferation of MBP-specific Th1 and Th2 cells from T cell receptor (TCR) transgenic mice and inhibited IFN-g and IL-10 but not IL-4 production. The inhibition of proliferation was at least partly mediated by induction of CTLA-4 expression in T cells by astrocytes since anti-CTLA-4 treatment reversed the inhibition of proliferation partly. We found an upregulation of CTLA-4 in T cells induced by astrocytes which was independent of antigen-presentation but not of prior T cell activation. This upregulation of CTLA-4 occurred even in the absence of cell-cell contact between T cells and astrocytes showing that a soluble factor is responsible. The inhibition of proliferation by induction of CTLA-4 expression in CNS-invading T cells might be one mechanism how astrocytes downregulate T cell-mediated inflammation in the CNS.
1
Laboratory of Molecular Immunology, Institute of Genetics, University of Cologne, Cologne, Germany, 2Center for Blood Research, Boston MA, USA, and 3Department of Neuroimmunology, Brain Research Institute, University of Vienna, Vienna, Austria
V. 5 Conditional deletion reveals a role for Fas–Fas ligand interaction in brain inflammation N. HÖVELMEYER1, Z.-Y. HAO1, T. BUCH1, K. RAJEWSKY2, H. LASSMANN3, and A. WAISMAN1 Experimental autoimmune encephalomyelitis (EAE) is a widely used animal model for multiple sclerosis. The disease is manifested by an influx of T cells to the brain. EAE can be induced in C57BL/6 mice by immunization with a peptide derived from myelin oligodendrocyte glycoprotein (MOG). It was previously shown that mice deficient for the fas gene (lpr mice) are partially resistant to MOG-induced EAE. As lpr mice have a deficiency in the development of the immune system, it is not clear whether their inability to develop EAE is a result of compromised T cell development or from resistance of brain cells to Fas-mediated apoptosis. We have decided to circumvent this problem by making a mouse with a tissue specific deletion of the fas gene in the brain. To this end, we have crossed a mouse in which the fas gene is flanked by two loxP sites with a mouse that express the cre-recombinase specifically in oligodendrocytes utilizing the MOG promoter (MOG-Cre) developed in our laboratory. EAE was induced in mice with the loxP flanked fas gene that express the MOG-Cre and as wild type controls, in mice without the
314 · 33rd Annual Meeting of the German Society of Immunology MOG-Cre. We found that the mice in which Fas was inactivated in oligodendrocytes had a milder form of EAE compared to the control mice, manifested by lower level of paralysis. These clinical findings could be verified by pathological examination of the CNS, showing that the mice expressing MOG-Cre had less inflammatory infiltrate, demyelination and nerve cell apoptosis compared to control mice. Together, these results demonstrate that killing of oligodendrocytes by Fas–Fas ligand interaction contribute to the development of inflammatory autoimmune disease in the brain.
1
Department of Molecular Neuroimmunology, Institute of Anatomy and Cell Biology and Institute of Pharmacology and Toxicology, Phillips-University, Marburg, Germany, 3 Department of Microbiology and Immunology, University of Leicester, Leicester and 4 Rheumatology Section, Hammersmith Campus, Imperial College School of Medicine, London, UK 2
V. 6 Functionally active C1q limits brain injury in a mouse model of permanent focal cerebral ischemia M. HRISTOVA1, C. CULMSEE2, V. JUNKER2, C. STOVER3, M. BOTTO4, N. BIKOV1, J. KRIEGLSTEIN2, W. SCHWAEBLE3, and E. WEIHE1 Complement activation may participate in neurodegenerative processes. Previous studies demonstrate that the classical pathway recognition molecule C1q is specifically up-regulated in brain microglia during transient global cerebral ischemia. To investigate the role of C1q in ischemic brain damage we induced permanent focal cerebral ischemia by left middle cerebral artery (MCA) electrocoagulation in male C1q-deficient, Factor B/C2-deficient and wildtype mice (SVJ129 genotype). The animals were sacrificed 2 and 7 days after MCA occlusion. The infarct size was determined by image analysis system (SCION). C1q mRNA and protein-expression were analysed by in situ hybridisation and immunocytochemistry. The infarct area in the C1q deficient mice was significantly increased 2 days post ischemia (p.i.) as compared to the C1q sufficient littermate controls. The infarct size in all groups was reduced 7 days p.i. when compared to 2 days p.i. The late reduction of infarcted tissue was significantly less pronounced in C1q Achain deficient mice in comparison to the wildtype control. The deletion of Factor B/C2 genes had no impact on the overall size of the infarct area. Significant increases in mRNA expression of all three C1q chains were observed 2 and 7 days p.i. C1q A-chain mRNA was undetectable in C1q A–/– mice resulting in total deficiency of C1q functional activity assessed in hemolytic assay. The deletion of the C1q A-chain gene however, did not affect the expression of C1q B- and Cchain genes. The ischemia-induced increase in C1q B- and C-chain mRNA abundance in the wildtype was also observed in C1q A-chain deficient mice suggesting that the three genes encoding C1q are regulated independently. This suggests that C1q contributes to limitation of brain injury after stroke. C1q and the classical complement pathway probably fulfil a crucial scavenger function in the cellular debris disposal in the course of neurodegeneration and neuroinflammation. Supported by DFG.
33rd Annual Meeting of the German Society of Immunology · 315 Departments of 1Internal Medicine and 2Pediatrics, Charité, Humboldt-University, Berlin, Germany
V. 7 Neurokinin 1 receptor mediates stress-induced increment on bronchoalveolar cell count in ovalbumin sensitized mice R.A. JOACHIM1, D. QUARCOO2, A.C. ZENCLUSSEN1, P.C. ARCK1, and B.F. KLAPP1 Airway inflammation is a leading symptom of bronchial asthma. Recent data is linking stress to the initiation and perpetuation of asthma symptoms. Tachykinins like substance P have been shown to play an important role in generating asthma symptoms and furthermore to be released in tissues in response to stress. The aim of the study was to determine 1) the influence of stress on murine allergen induced airway inflammation and 2) the involvement of tachykinins in stress mediation of airway inflammation. CBA/J mice were sensitized by i.p. injection of ovalbumin (OVA). Coinciding with the beginning of the first OVA aerosol airway provocation, one half of the mice was stressed by exposure to an ultrasonic sound stressor for 24 h. Part of the animals were additionally treated with a tachykinin NK1-receptor antagonist (NK1-RA) before airway challenges. 24 h after the second airway provocation, animals were sacrificed. Broncho-alveolar lavage fluid (BAL) was obtained and total cell numbers were determined. Cytospins were prepared for each sample and after staining differential cell counts were performed. A significantly higher total cell count was present in the BAL in stressed animals compared to unstressed control mice. This stress-induced increase was mainly caused by an increase of eosinophils and could be blocked by application of the NK1-RA, whereas NK1-RA did not influence airway inflammation in unstressed controls. Our data suggest that stress increases allergen induced airway inflammation. Furthermore it points to tachykinins like substance P as putative mediators in stress induced airway inflammation.
1
Institute of Clinical Immunology and Transfusion Medicine and 4Ear, Nose and Throat Department, University of Leipzig, Leipzig, 2Ear, Nose and Throat Department, Klinikum Chemnitz, Chemnitz, and 3Institute of Immunology, Medical Faculty Carl Gustav Carus, Technical University of Dresden, Dresden, Germany
V. 8 Autoimmune findings in sudden hearing loss patients M. KAMPRAD1, K. DONAUBAUER2, K. CONRAD3, H. MÜLLER4, U. FICKWEILER4, and U. SACK1 Early diagnosis of immunologically relevant acute diseases of the inner ear such as sudden hearing loss can improve patient care and prevent complications. Recent publications describe candidate antigens for autoantibodies related to inner ear diseases. In this retrospective study, we have investigated diagnostic reliability of relevant autoantibodies for differential diagnosis of patients in an ENT department.
316 · 33rd Annual Meeting of the German Society of Immunology 58 patients with inner ear diseases and 62 controls with other ENT diagnosis have been investigated for the presence of the following autoantibodies: ANA screen, ENA screen including RNP, Sm, SS-A, SS-B, Jo-1, Scl-70 and CenpB, autoantibodies to endothelial cells, to cardiolipin, to 68 kD heat shock protein, to type II collagen, and rheumatoid factors. In sera of sudden hearing loss patients, only sometimes ANA and never ENA could be detected. Autoantibodies to 68 kD heat shock protein and type II collagen were less frequently detectable in comparison to the controls. Interestingly, antibodies to endothelial cells correlated with sudden hearing loss when detected on HUVEC cells but not by indirect immunofluorescence at lung tissue. Anti-cardiolipin antibodies and rheumatoid factors, particularly IgM, could be common found in patients with inner ear diseases. Although none of the parameters correlated significantly with sudden hearing loss, frequency of any autoantibody positivity is enhanced (p=0.012). By calculating a computerized classification based on positive and negative correlations between single parameters and sudden hearing loss, groups could be separated with p<0.001. We conclude that parameters such as ANA, ENA, antibodies to 68 kD heat shock protein and to type II collagen are rather absent in sudden inner ear diseases. By including a whole panel of parameters, classification of sudden hearing loss patients can be performed in a substantial significant manner. Nevertheless, a single significant parameter to identify such diseases has yet to be found.
Institute of Anatomy and Cell Biology, Philipps-University, Marburg, Germany
a expression in mouse brain V. 9 Systemic LPS but not SEB induces TNF-a spreading from meninges and circumventricular organs into the cerebral parenchyma O. KAUT, M. BETTE, and E. WEIHE The role of TNF-a within the immune system-to-brain communication under conditions of bacterial lipopolysaccharide (LPS)- or staphylococcal enterotoxin B (SEB)-evoked toxemia is still not fully understood. The aim of our study was to monitor the constitutive expression of TNF-a and to differentiate the effects of a macrophage activation induced by LPS from a T cell specific activation induced by SEB on TNF-a expression in the brain. Under basal conditions in situ hybridization revealed no expression of TNF-a mRNA in the brain. At 1h post injection (p.i.) LPS caused an increase of TNF-a serum levels, of c-fos expression in the hypothalamic paraventricular nucleus (PVN), and of TNF-a expression in the circumventricular organs (CVOs), meninges and choroid plexus. At 4h and later stages p.i., TNF-a mRNA disappeared from the CVOs, meninges and choroid plexus and was translocated in regions adjacent to the CVOs and to brain parenchyma. SEB challenge caused an elevation of TNF-a in the serum, an increase of c-fos expression in the PVN, but in contrast to LPS no induction of cerebral TNF-a mRNA. Thus we demonstrate and conclude that: i.) elevated TNF-a serum levels are not automatically paralleled by an induction of TNF-a expression in the brain ii.) peripheral T cell activation is not causing cerebral induction of TNF-a iii.) the expression of cerebral TNF-a within the immune system-to-brain communication is dependent on the specific immune stimulus. Supported by SFB 297
33rd Annual Meeting of the German Society of Immunology · 317 Department of Internal Medicine I, University Hospital, Regensburg, Germany
V. 10 Presence of the sympathetic nerve repellent Semaphorin 3C and lack of the sensory nerve repellent Semaphorin 3A in RA synovium may reduce sympathetic innervation and may perpetuate inflammation L.E. MILLER, J. GRIFKA, W. FALK, J. SCHÖLMERICH, and R.H. STRAUB In terms of the chronic inflammation that occurs during rheumatoid arthritis (RA), the sensory nervous system tends to produce proinflammatory stimuli, while the sympathetic nervous system tends to induce anti-inflammatory effects. We have recently shown a distinct reduction of the sympathetic nerve fibers in the synovial tissue of RA patients (FASEB J 2000 14:2097–2107). The presence of selective nerve repellents, i.e. mediators of the semaphorin family, could be responsible for the observed reduction of the sympathetic innervation. In order to investigate the presence of mRNA of different semaphorins in synovial tissue, Diglabeled RNA probes were constructed. These Dig-labeled RNA probes were used for in situ hybridizations within frozen tissue sections from patients with RA and OA. Control hybridizations with probes directed against fibronectin and vimentin were carried out in parallel to the semaphorin hybridizations. In situ hybridizations showed that semaphorin 3C mRNA, a nerve repellent of sympathetic nerve fibers, was present in the synovial tissue of all investigated patients with RA (N=5) and two investigated patients with OA. In contrast, semaphorin 3A mRNA, directed against sensory nerve fibers, could not be detected in the synovial tissue of patients with RA (N=4) and OA (N=4). The parallel control hybridizations with probes against vimentin (RA, N=6; OA, N=2) and fibronectin (RA, N=2; OA, N=1) were always positive and therefore confirmed technically successful hybridization conditions. Control hybridizations with the sense probes were always negative. These findings suggest that semaphorin 3C, which is selectively directed against sympathetic nerve fibers, may be responsible for the reduced sympathetic innervation in the synovial tissue of patients with RA. The inability of the sympathetic nervous system to re-innervate the synovial tissue of patients with RA could be contributing to the chronic inflammatory nature of RA.
1
Department of Neurology, Eberhard-Karls-University, Tübingen, 2Department of Anthropology and Human Genetics, 3Genzentrum and Friedrich-Baur Institut, and 4Institute of Clinical Neuroimmunology, Ludwig Maximilians University, Munich, Germany
V. 11 The nonclassical MHC molecule HLA-G protects human muscle cells from immune-mediated lysis: Implications for myoblast transplantation and gene therapy M. MITSDÖRFFER1, V. HOFMEISTER2, S. KRAUSE3, E.H. WEISS2, H. LOCHMÜLLER3, R. HOHLFELD4, A. MELMS1, M. WELLER1, and H. WIENDL1 HLA-G is a nonclassical MHC class I molecule with highly limited tissue distribution which has been attributed chiefly immune regulatory functions. We have previously reported that HLA-G
318 · 33rd Annual Meeting of the German Society of Immunology is expressed in inflamed muscle in vivo and by cultured myoblasts in vitro. Here, we used the in vitro models of human myoblasts or TE671 muscle rhabdomyosarcoma cells to characterize the functional role of HLA-G for muscle immune cell interactions. Gene transfer of the two major isoforms of HLA-G (transmembranous HLA-G1, soluble HLA-G5) into TE671 rendered these cells resistant against direct alloreactive lysis and killing by antigen-specific cytotoxic T cells. HLA-G not only inhibited NK cells but also prevented alloreactive lysis by CD4 and CD8 T cells. Further, HLA-G obviated effective priming of antigen-specific cytotoxic T cells and reduced antigen specific lysis. HLA-G pre-induced on cultured myoblasts inhibited lysis by alloreactive peripheral blood mononuclear cells. This protection was reversed by a neutralizing HLA-G antibody. Interestingly, few HLA-G-positive cells within a population of HLA-G negative muscle target cells conveyed significant inhibitory effects on alloreactive lysis. Our results reveal further insights into the immunobiology of muscle and suggest that ectopic expression of HLA-G may promote the survival of transplanted myoblasts in the future treatment of hereditary muscle diseases. Further, HLA-G could represent a novel self-derived anti-inflammatory principle applicable in strategies against inflammatory aggression.
1
Department of Experimental and Physiological Psychology and 2Institute of Theoretical Surgery, University of Marburg, Marburg, Germany, and 3Department of Psychology, Chung Shan Medical University, Taiwan, ROC
V. 12 Relationships between anxiety and basal IL-2 mRNA levels in the striatum of the rat C.R. PAWLAK1, A. BAUHOFER2, Y.J. HO3, and R.K.W. SCHWARTING1 Pharmacological interventions in the periphery or brain with LPS, cyclosporin A, or interleukins (IL), have been shown to affect behavior, e.g. anxiety or exploration. In contrast, it is largely unknown whether endogenous levels of cytokines in specific brain compartments may be related to such behaviors. Our previous experiments have shown that male Wistar rats, although identical in strain, sex, and age can differ systematically in their anxiety, or exploration behavior. Furthermore, we found anxiety to be related to the neurotransmitter serotonin in the ventral striatum, whereas exploration was related to the neurotransmitter dopamine and acetylcholine. Therefore, we asked whether behavioral individuality may also be related to immunological function in the brain. First, we tested a sample of male adult outbred Wistar rats (n=34) in our routine testing procedure, that is a novel open field (exploration), followed by the elevated plus-maze (anxiety). Based on the behavioural analyses in a novel open field, the rats were divided into two subgroups according to their exploration behavior expressed as low rearing (LRA), or high rearing activity (HRA). In parallel, the same animals were divided into low (LA), or high anxiety (HA) rats based on time spent on the closed arm in an elevated plus-maze. Then, IL-1b, IL-2, IL-6, and TNF-a cDNA levels were measured post mortem in striatum tissues using semi-quantitative, competitive, reverse transcription polymerase chain reaction (RT-PCR). We observed no significant differences between LRA and HRA rats (novel open field behavior) and the analysed cytokines. However, HA compared to LA rats showed significantly higher IL-2 mRNA levels (p<.05), but no altered IL-1b, IL-6, and TNF-a mRNA expression. Additionally, pronounced individual differences in all cytokine levels in the striatum were observed. These results suggest basal levels of IL-2 mRNA expression in the striatum to be associated with anxiety behavior in adult male Wistar rats.
33rd Annual Meeting of the German Society of Immunology · 319 Institutes of 1Applied Psychology and 2Clinical Immunology and Transfusion Medicine, University of Leipzig, Leipzig, Germany
V. 13 Psycho-immunological process evaluation of a stress preventive intervention program for teachers M. STÜCK1, K. MEYER1, K. BAUER2, and U. SACK2 Immunological and psychological parameters reflecting stress management capabilities are closely connected and have been shown to interact in a highly reproducible manner. With our study we wanted to examine this correlation between self-regulative therapeutic stimuli and immunoglobulin A in saliva. As part of the integrative stress management training-concept for schools, a ten-week stress management training was carried out with teachers. It was the aim of this intervention to enable teachers to better cope with their daily pressures. The effects of the intervention methods were to be determined in a accompanying process-evaluation study. For this purpose, health-psychological (stress-relevant psychological variables; work related behavioural and experience patterns) as well as psychological (blood pressure, skin resistance) and immunological variables (immunoglobulin A) were taken. For the immunological variables we took immunoglobulin A from the saliva of 12 teachers before and after the sessions of both intervention studies. Parallel to this different emotional parameters were ascertained. For both methods overwhelming effects in improving stress resistance could be verified. In saliva, increase of immunoglobulin A could be found in persons with a positive response to our training. Furthermore, a significant positive correlation between the subjective sensation of relaxation or other health parameters and IgA could be measured. Our ten-week stress management training could be shown to be effective in improving stress management capabilities. Psychological and subjective parameters could be found to correlate well with immunoglobulin A concentration in saliva. Therefore, measurement of this simple immunological parameter could be proved to be a reliable indicator for stress resistance as well as training effects.
1
Department of Medicine II, University of Leipzig, Leipzig and 2Department of Rheumatology and Clinical Immunology, Charité, Humboldt-University, Berlin, Germany
V. 14 Altered functional response to stimulation of b2-adrenergic receptors in CD4 and CD8 positive human T cells following preincubation with IL-2 M. WAHLE1, I. NAUMANN1, A. KRAUSE2, H. HÄNTZSCHEL1, and C.G.O. BAERWALD1 Human CD4 and CD8 positive T lymphocytes express b2-adrenergic receptors (b2-R). However, the expression of b2-R is about fourfold higher on CD8 positive T cells than on CD4 positive T cells. In addition, previous investigations revealed that IL-2 increased b2-R expression significantly in CD8 compared to CD4 positive T cells. To further evaluate the effect of IL-2 on b2-R function, CD4 and CD8 positive T cells were isolated and preincubated with IL-2 before stimulation and activation of b2-R. CD4 and CD8 positive lymphocytes from healthy donors were isolated by magnetic cell separation (MACS) and activated with anti-CD3 and anti-CD28 antibody together with IL-2. Aliquots of CD4 or CD8 positive T cells were incubated with 0.5 or 5 ng/ml
320 · 33rd Annual Meeting of the German Society of Immunology IL-2 for 16 or 72 h prior to stimulation. Assessment of b2-R function was determined by the suppression of IFN-g (IFN-g) production following stimulation with the b2-R agonist epinephrine (10 mM). The concentration of IFN-g was determined in culture supernatant after 24 and 48 h using a sandwich ELISA. Stimulation of T cells induced a time dependent increase in IFN-g production of CD4 and CD8 positive T cells. Epinephrine inhibited the synthesis of IFN-g by 40% (CD4) and 50% (CD8), respectively (p<0.05). Preincubation with IL-2 for 16 h, but not 72 h, abrogated the functional response to epinephrine. In contrast, the IFN-g concentration in the culture supernatant of stimulated cells even increased upon co-incubation with epinephrine (5 ng/ml IL-2, not significant). In conclusion, the function of b2-R on CD4 and CD8 positive T cells is altered by IL-2 time and dose dependently. It may be speculated that IL-2 additionally induces the functional expression of alpha-adrenergic receptors on human T cells.
1
Department of Neurology, Eberhard-Karls-University, Tübingen, 3Department of Neurology, Heinrich-Heine-Universität, Düsseldorf, Germany, and 2Department of Neurology, KarlFranzens-Universität, Graz, Austria,
V. 15 Functional characterization of the CD28-related molecule ICOS in multiple sclerosis: A possible target for selective immune intervention? H. WIENDL1, O. NEUHAUS2, M. MEHLING1, S. WINTTERLE1, B. SCHREINER1, M. MITSDÖRFFER1, R. WEISSERT1, M. WELLER1, H.P. HARTUNG3, E. TOLOSA1, and A. MELMS1 ICOS has recently been described as an inducible CD28 related costimulatory molecule with relevance for T cell differentiation and effector function and a possible role in autoimmune diseases like multiple sclerosis. This study was aimed at characterizing expression and functional role of ICOS costimulation in multiple sclerosis. After nonspecific or antigen-specific stimulation, ICOS was shown to be preferentially expressed on CD4+ Th2 T cells. Experiments with antigen-specific T cell lines as well as purified SAg-stimulated CD4 T cells demonstrated that ICOS-costimulation affected T cell proliferation and production of both Th1 and Th2 cytokines, whereas expression of T cell activation markers or chemokine receptors were unaffected. Interestingly, the effects on cytokine secretion were observable in the presence and the absence of B7 costimulation, therefore suggesting that ICOS costimulation can modulate cytokine secretion also independently of CD28. Levels of constitutive and inducible ICOS expression on human T cell subsets from peripheral blood were quantified in healthy donors (n=10) and patients with multiple sclerosis (acute relapse or chronic disease; n=10). Constitutive expression of ICOS on T cells varied between 0.1 to 42.3%. No significant differences were noted between both groups concerning the baseline expression or inducibility on T cells. Cells constitutively expressing ICOS did not represent the CD4+CD25+ regulatory T cell subset. ICOS expression on CSF T lymphocytes in patients with acute MS relapses or acute neuroborreliosis showed that expression of ICOS was not elevated compared to peripheral blood. Neither IFN-b or glatiramer acetate did alter ICOS-expression, whereas the anti-inflammatory cytokine IL-10 downregulated ICOS. In summary, ICOS costimulation affects both Th1 and Th2 cytokine production in the presence and absence of B7-costimulation. Since ICOS is rapidly induced on T cells after antigenspecific stimulation, this molecule qualifies as a suitable target aimed at modulating T cell cytokine responses in CNS inflammation.