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Workshop C: CNS and Immune System, Abstract C1 bis C20
Workshop C CNS and the Immune System
Max-Planck-Institut für physiologische und klinische Forschung, Bad Nauheim, Germany
C. 1 Expression of ELC (CCL19) and SLC (CCL 21) at the blood-brain barrier during experimental autoimmune encephalomyelitis suggests their involvement in lymphocyte recruitment into the central nervous system C. ALT and B. ENGELHARDT Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated autoimmune disease of the central nervous system (CNS), where CD4+ autoaggressive T cells gain access to the CNS and start the molecular events leading to inflammation, edema and demyelination. Based on our recent observation that T cell recruitment across the blood-brain barrier (BBB) does require Gprotein mediated signals in vivo we have started to search for the chemokines promoting leukocyte arrest on the BBB endothelium as chemokine expression at the BBB has not been reported. By performing in situ hybridizations for a large panel of chemokines ELC (CCL 19) and SLC (CCL 21) were the only chemokines expression of which could be localized to cerebral vessels surrounded by perivascular cuffs in brain and spinal cord sections derived from mice afflicted with EAE. The receptor for ELC and SLC, CCR7 was detected in inflammatory cells surrounding cerebral vessels. In addition expression of ELC (CCL 19) was detected in cerebral vessels of healthy control mice. Preliminary data confirm the presence of ELC and SLC protein in cerebral vessels. The spatial correlation of the expression of CCL19 and CCL21 at the BBB and CCR7 on inflammatory cells in inflammatory cuffs during EAE observed here implies a not yet appreciated involvement of these constitutive chemokines in regulating lymphocyte homing into nonlymphoid tissues. This is supported by our in vitro observations that encephalitogenic T lymphocytes showed specific chemotaxis towards towards CCL 19 and CCL 21 in a concentration dependent manner and comparable to naïve lymphocytes. Our data suggest that ELC and SLC besides regulating lymphocyte homing into lymphoid organs additionally regulate T cell homing into the immunoprivileged CNS.
32nd Annual Meeting of the German Society of Immunology · 33 Institutes of 1Anatomy and Cell Biology and 3Immunology, Philipps-University, Marburg, Germany and 2Center of Neurovirology, Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, USA
C. 2 Accumulation of macrophage migration inhibitory factor in astrocytes of borna disease virus-infected rat brain is accompanied by inhibition of macrophage infiltration M. BETTE1, E. WEIHE1, B. DIETZSCHOLD2, D. GEMSA3, and M. BACHER3 To test the hypothesis that macrophage migration inhibitory factor (MIF) plays a role in macrophage invasion during virus-induced encephalitis, we analyzed the expression and cellular localization of MIF in the Borna Disease Virus (BDV)-infected rat brain, monitored monocyte/macrophage infiltration and evaluated the influence of anti-inflammatory treatment with dexamethasone. MIF mRNA expression was restricted to neurons and remained unchanged after BDV-infection or after dexamethasone treatment of either BDV-infected or uninfected control rats. In contrast, MIF protein immunoreactivity (ir) was not only seen in neurons but also in glia. After BDV-induced encephalitis and treatment of uninfected rats with dexamethasone, MIF-ir was only slightly altered in neurons but moderately enhanced in tanycytes, ependyma and choroid plexus epithelium and markedly increased or induced in astrocyte end-feet at the blood brain barrier (BBB). The increase in MIF-ir in astrocytes after BDV-infection was blocked by dexamethasone. The induction or enhancement of MIF-ir at the BBB significantly correlated with reduced numbers of infiltrating ED1-positive monocytes/macrophages after BDV-infection. Increased macrophage invasion was observed in regions where no astrocytic MIF was detected. The BDV- or dexamethasone-induced accumulation of MIF protein in astrocytes in vivo in absence of detectable astrocytic MIF mRNA expression is most likely due to MIF translocation from neurons rather than to a constitutive or induced MIF mRNA expression in astrocytes. In conclusion, we provide evidence that translocation of MIF from neurons or other extracellular sources into astrocytes is likely to modulate the inflammatory process during the course of virusinduced encephalitis by limiting monocyte/macrophage migration through the BBB.
1
Institut für Klinische Immunologie und Transfusionsmedizin, Universität Leipzig and 2Vita34 Gesellschaft für Zelltransplantate mbH, Leipzig, Germany
C. 3 Human umbilical cord blood: A source of neural stem cells? J. BOLTZE1, U. SACK1, D. EGGER2, and F. EMMRICH1 Neural stem cells, which display self-renewal and multilineage differentiation properties, have been isolated from embryonic and adult central nervous system (CNS) tissues of murine and even human origin to be expanded in vitro in recent years. After expansion these cells are able to differentiate into neurons and glial cells in vitro and in vivo. Therefore, neural stem cells are important regarding clinical applications, i.e. cell therapy in patients with CNS disorders.
34 · 32nd Annual Meeting of the German Society of Immunology Umbilical cord blood is a rich source of neonatal stem cells which are shown to give rise to blood and bone marrow cells and are expected to have further abilities. Hence, they are an important basis for autologous transplantation strategies avoiding immunologic rejections. Therefore, neonatal stem cells are investigated for neurogenerative properties. After collecting cord blood samples the mononuclear population containing neonatal stem cells was isolated by using a modified Ficoll density gradient centrifugation protocol. Further, cells are grown in ITSFn, a DMEM/F12 based medium with insulin, transferrin, selenite and fibronectin supplements which has been shown to strongly select for neural stem cells and progenitor cells. After 6 to 8 days in ITSFn, most of the cells were destroyed. Surviving cells were analyzed for the expression of mRNA coding for the nestin protein, an early marker for neural stem cells using LightCycler PCR. Alternatively they can be transferred to an expansion medium containing bFGF, EGF and a variety of further supplements in order to form neurospheres (free floating colonies of neural stem cells in medium) before differentiation into neurons and glia. In cell culture several indications were obtained that differentiation towards neuronal cells took place. Neurosphere-like clusters as well as branched-dendrites were observed. To verify these findings direct transfer of cord blood cells to CNS of immunodeficient mice will be performed.
1
Abteilung Neuroimmunologie, Max-Planck-Institut für Neurobiologie, Martinsried, Germany and 2Abteilung Neuroimmunologie, Institut für Hirnforschung, Wien, Österreich
C. 4 Myelin degeneration and autoimmune disease: Altered distribution of inflammatory lesions in hemizygous transgenic rats overexpressing proteolipid protein M. BRADL1, M. KLEIN1, J. BAUER2, H. LASSMANN2, and H. WEKERLE1 Adult hemizygous transgenic Lewis rats overexpressing the myelin proteolipid protein (PLP) develop low grade, subclinical myelin degeneration. Contrary to expectation, pathological changes rarely occur in the myelin-rich CNS white matter, but are almost exclusively confined to CNS gray matter. In this compartment, numerous oligodendrocytes retain and accumulate PLP, myelin oligodendrocyte glycoprotein (MOG) and myelin associated glycoprotein (MAG) in the endoplasmic reticulum (ER), but correctly synthesize myelin basic protein (MBP) in the cellular processes. Some of these oligodendrocytes become apoptotic. Apparantly as a consequence of oligodendrocyte stress and death in CNS gray matter, local microglia cells become activated. They upregulate the complement receptor type III, phagocytose oligodendrocyte debris and express MHC class II molecules. The accumulation of PLP and MOG, but not MBP in the oligodendrocytes ER, the resulting oligodendrocyte degeneration and microglia activation seem to jointly influence the distribution of inflammatory lesions in the course of experimental autoimmune encephalomyelitis (EAE): In PLP-specific T cell-induced EAE, effector T cells and inflammatory lesions are present in the meninges and (rarely)in the CNS white matter of wildtype animals, but almost exclusively infiltrate CNS gray matter of the transgenic rats. In MOGspecific T cell-induced EAE, T cells and lesions are found in CNS white and gray matter areas of wildtype rats, and more often in CNS gray matter areas of their transgenic counterparts. In MBPspecific T cell-induced EAE, effector T cells and inflammatory lesions are seen in identical places in wildtype and transgenic rats. These data demonstrate a link between myelin/oligodendrocyte
32nd Annual Meeting of the German Society of Immunology · 35 degeneration and the localization of inflammatory lesions in the CNS. It remains to be seen whether similar mechanisms act in human leukodystrophies with spontaneous T cell infiltration of the CNS.
Institut für Medizinische Mikrobiologie und Virologie, Heinrich-Heine-Universität, Düsseldorf, Germany
C. 5 Anti-viral effector mechanisms in human astrocytoma cells W. DÄUBENER, C. OBERDÖRFER, K. BESKEN, C.R. MACKENZIE, and O. ADAMS Encephalitis and meningitis caused by parasites, bacteria and viruses are the most dangerous infectious diseases of the brain. We have analyzed the capacity of human astrocytoma cells to restrict the growth of Toxoplasma gondii, group B streptococci and Herpes simplex virus. Human astrocytoma cells are capable of restricting the growth of Toxoplasma gondii and of group B streptococci after stimulation with IFN-g. We found that the antimicrobial effect of IFN-g-stimulated astrocytoma cells is mediated via an induction of indoleamine 2,3 dioxygenase (IDO). This enzyme degrades L-tryptophan to kynurenine, and this results in complete depletion of L-tryptophan, an essential amino acid for both pathogens. IDO activity was found in astrocytoma cells by the detection of IDO mRNA in RT-PCR, detection of enzyme protein 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 sufficient to inhibit the growth of Toxoplasma gondii and of group B streptococci, and this antimicrobial effect is blocked in the presence of excess amounts of L-tryptophan, indicating that IDO activity is indeed the active effector mechanism against both pathogens. In addition we found that IFN-g stimulated human astrocytoma cells are capable of restricting herpes simplex virus replication. Furthermore we found that IFN-a and IFN-b which were unable to induce an anti-parasitic or anti-bacterial effect in astrocytoma cells induce a strong anti-viral effect. Using a quantitative PCR protocol we found that an excess amount of L-tryptophan nearly completely abrogates the antiviral effect of astrocytoma cells against HSV-1 and HSV-2. We therefore suggest that IDO activation is involved in the anti-viral defense mediated by astrocytoma cells.
1
Institute of Medical Microbiology and Hygiene and 2Department of Molecular Neuroscience, Institute of Anatomy and Cell Biology, Philipps-University, Marburg, Germany
C. 6 Microglia cells are activated by immunostimulatory CpG-DNA A.H. DALPKE1, M. SCHÄFER2, M. FREI1, S. ZIMMERMANN1, E. WEIHE2, and K. HEEG1 Bacterial DNA containing motifs of unmethylated CpG-dinucleotides (CpG-DNA) can be recognized by the innate immune system through the pattern recognition receptor TLR 9. Thus bacterial DNA possesses potent immunostimulatory effects on macrophages, dendritic cells and B lymphocytes and can contribute to inflammation during the course of bacterial infections.
36 · 32nd Annual Meeting of the German Society of Immunology Only limited information is available concerning the role of CpG-DNA in immune reactions of the CNS. To address this question we examined the effects of CpG-DNA on microglia cells in vitro and in vivo. Here we show that primary microglia cells obtained from neonatal brain cultures as well as microglia cell lines expressed mRNA encoding TLR 9 transcripts. CpG-DNA in vitro activated microglia cells to produce TNF-a, IL-12p40 and nitric oxide. In addition microglia displayed upregulation of MHC class II, B7–1, B7–2 and CD40 molecules after CpGDNA administration. Furthermore CpG-DNA increased phagocytosis by microglia cells. In vitro CpG-DNA also induced TNF-a and IL-12p40 transcripts in microglia cells after intracerebroventricular injection as shown by in situ hybridization and quantitative RT-PCR. CpG-antagonistic oligonucleotides containing guanosine rich regions inhibited CpG-DNA induced activation of microglia. Taken together these results indicate that the microglia 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 influence the immunostimulatory properties of the microglia system.
Max-Planck-Institut für physiologische und klinische Forschung, Bad Nauheim, Germany
C. 7 Tetracycline-inducible expression of adhesion molecules at the bloodbrain barrier in transgenic mice B. ENGELHARDT, T.M. SCHLÄGER, B. DEHOUCK, S. TAUBER, S. HENNIG, V. SCHMIDT, W. RISAU, and U. DEUTSCH We have investigated the expression and function of adhesion molecules on blood-brain barrier (BBB) endothelium during experimental autoimmune encephalomyelitis (EAE) in the SJL/N mouse. We have found that during EAE expression of E- and P-selectin is not induced in BBB endothelium and that E- and P-selectin are not required for the recruitment of inflammatory cells across the BBB. In contrast, VCAM-1 but not MAdCAM-1 is induced on BBB endothelium during EAE and actively involved in the recruitment of a4b1-integrin-positive inflammatory cells during the disease. In order to find out whether ectopic overexpression of the adhesion molecules lacking at the BBB is sufficient to overcome the selective block of recruitment for subsets of leukocytes across the BBB, we have generated transgenic mice, in which expression of E- and P-selectin and MAdCAM-1 can be induced at the BBB-endothelium using the «TET-OFF» system. The «TET-OFF» system consists of a binary system of transgenic activator and responder mice that allows for tetracycline-regulated expression of transgenes in double transgenic mice. Different types of activator mouse lines expressing the tetracycline-inducible transactivator (tTA) under the control of regulatory elements of the mouse Tie-2 receptor tyrosine kinase gene that is required for expression in all embryonic and adult vascular beds, have been established. Additionally, various types of responder mouse lines with expression of E- and P-selectin or MAdCAM-1 under the control of multiple tet-operators have been raised. Removal of tetracycline leads to induced expression of E-selectin and MAdCAM-1 in several combinations of double transgenic animals. These double transgenic mice exhibit endothelial cell-specific induction of the adhesion molecules E-selectin and MAdCAM-1 in the vessels of the CNS. Currently these mice are used to investigate the recruitment of inflammatory cells across the «altered» BBB.
32nd Annual Meeting of the German Society of Immunology · 37 1
Department of Immunology and Gnotobiology, Institute of Microbiology, Czech Academy of Sciences, and 2Biochemical Research Unit, 1st Medical Faculty and 3Department of Pharmacology, 3rd Medical Faculty, Charles University, Prague, Czech Republic
C. 8 Catecholaminergic system regulates immunity to tumors A. FISEROVA1, H. KOVARU2, L.E. VANNUCCI1, O. MATEJKOVA1, M. KULDOVA1, M. STAREC3, and M. POSPISIL1 Introduction: Bidirectional interactions between the immune and neuroendocrine systems are involved in the maintenance of cellular homeostasis and regulation of tumor growth (Chuluyan et al., NeuroImmunoModulation 8, 107, 2000). Engagement of neurotransmitter receptors expressed on lymphoid cells enables regulation of their growth and function induced by neuroendocrine mediators. Aim: To assess the effects evoked by these mediators represented by D1 (agroclavine) and D2 (terguride) dopamine receptor agonists on antitumor immune responses. Methods: Phenotypic and functional analysis of dopamine agonists treated tumor and MLTC generated cytotoxic lymphocyte subpopulations were followed in vitro. The study was completed by the detection of associated signaling pathways. For in vivo experiments the transplanted B16F10 melanoma in C57/bl6 mice was used. Results: In vitro obtained data demonstrated a stimulatory effect of D1 agonists on cytotoxic activity of NK cells (paralleled by IL-1b, IL-2, IFN-g cytokine production, and triggering of Gq-protein, ERK1/2 signal transmission), however D2 agonist had the opposite effect. The D1 agonist treatment of tumor cells increased their sensitivity to the NK cell effector function and D2 agonist evoked higher expression of MHC-I antigen (with lowered synthesis of IL-2, IL-6, IFN-g, and blocked signaling pathways). These results were confirmed in melanoma bearing mice in vivo. The mean survival time of mice transplanted with D1 conditioned melanomas was prolonged, while shortened after D2 treatment, in comparison to the wild type B16F10 cells. The best therapeutic effect (46% of surviving animals) was achieved by use of liposome-encapsulated D1 agonist (agroclavine). Conclusions: Cell-mediated antitumor response appears to be under dopaminergic control involving activation through D1-type and inhibition by D2-type of dopamine receptors. Supported by grants: 312/98/K034, 304/01/0850, GAUK 71/2001/C/3.LF, MSMT 111100001, and by Institutional Research Concept AV0Z5020903.
38 · 32nd Annual Meeting of the German Society of Immunology 1
Department of Neuroimmunology, Max-Planck-Institute of Neurobiology, Martinsried, Institute of Medical Immunology, Charité, Humboldt-University, Berlin, and 3Institute of Molecular Immunology, GSF National Research Center for Environment and Health, Munich, Germany 2
C. 9 Reactivation within the target organ determines the pathogenic potential of encephalitogenic T cells A. FLÜGEL1, N. KAWAKAMI1, Z.X. LI1, T. RITTER2, J.W. ELLWART3, C. LININGTON1, and H. WEKERLE1 Autoimmune inflammation of the CNS, e.g. in Multiple sclerosis, results in a wide spectrum of clinical disease types ranging from mild, self-limited to severe progressive disease. This is reflected in the T cell-mediated animal model of MS, the experimental autoimmune encephalomyelitis (EAE). There, the outcome of clinical disease depends on the target antigen of encephalitogenic T cells and the genetic background of the recipient animals. Thus, T cells specific to myelin basic protein (MBP) induce a severe paralyzing EAE in Lewis rats, whereas T cells specific for myelin oligodendrocyte protein (MOG) or the astrocytic antigen S100b evoke intense CNS inflammation without marked clinical symptoms. Likewise, in DA rats, MOG specific T cells (MOG-DA T cells) induce a severe clinical disease resembling MBP T cell-mediated EAE of the Lewis rat, while EAE induced by MOG specific T cells in Lewis rats (MOG-Lewis) is mild. We used retrovirally engineered T cells expressing the marker gene of green fluorescent protein (Flügel et al., 1999, Nat. Med. 5: 843–47) to examine the mechanisms, which determine the clinical outcome of adoptive transfer EAE. We found that all brain specific T cells caused a massive influx of effector cells into the CNS three to four days following transfer. However, only the highly pathogenic MBP and MOG-DA T cells up-regulated activatory markers such as IL-2-receptor and OX40 antigen. Further the activated T cells increased mRNA encoding the proinflammatory cytokines IFN-g and TNF-a. In contrast, low-pathogenic S100B and MOG-Lewis T cells did not show these activatory changes, but showed a moderate increase of IL-2 and IL-4 mRNA. We conclude that the level of activation within the target organ might critically determine the character of autoimmune CNS disease. The work was supported by the SFB 455.
Department of Immunology, Bernhard-Nocht-Institute of Tropical Medicine, Hamburg, Germany
C. 10 Murine malaria following blood-stage infection by P. berghei is exacerbated by CTLA-4 blockade T. JACOBS, S.E.B. GRAEFE, S. NIKNAFS, I. GAWORSKI, and B. FLEISCHER In this study we examined the role of CTLA-4 expression (CD152) in experimental blood-stage malaria. Infection of C57BL/6 mice with by P. berghei led to a strong increase of CTLA-4 expression on spleen cells. On day 9 after infection up to 10% of CD4+ T cells expressed CTLA-4,
32nd Annual Meeting of the German Society of Immunology · 39 whereas only a marginal expression was found on other cells. Spleen cells taken on day 9 after infection were suppressed in their proliferative response, which could not be reversed by addition of either anti-CTLA-4 or anti-TGF-b antibodies. On day 5 after infection only about 1% of CD4+ T cells expressed CTLA-4, however stimulation in the presence of anti-CTLA-4 led to an increased T cell proliferation. Furthermore, in vivo administration of anti-CTLA-4 antibodies and subsequent challenge with P. berghei led to an exacerbation of disease in comparison to control mice, which was accompanied by an increased proliferation of spleen cells. Histologic examination of brain sections from anti-CTLA-4 treated mice revealed pathologic changes characteristic for cerebral malaria like sequestration of parasitized erythrocytes, perivascular edema and stasis which were absent in control mice. Our data suggest that CTLA-4 expression on CD4+ T cells suppress pathological immune responses most probably by other mechanisms than TGF-b secretion.
1
Department of Clinical Immunology, Medical School, Hannover, 2Department of Physiological Psychology, University of Marburg, Marburg, and 3Institute of Medical Psychology, University of Essen, Essen, Germany
C. 11 Immune response upon adrenaline infusion in patients with rheumatoid arthritis J.M. KITTNER1, C.R. PAWLAK2, M. SCHEDLOWSKI3, R.E. SCHMIDT1, and R. JACOBS1 Introduction: Clinical observations suggest the course of rheumatoid arthritis (RA) to be influenced by psychological stress. Reduced density of b2-adrenoceptors on lymphocytes in RA patients has been shown. Aim of this study was to investigate whether the immunological reaction to adrenaline is altered in RA patients. Methods: 14 female RA patients and 14 female healthy controls were infused with 1 mg/kg adrenaline over 20 min. Blood was drawn before, immediately after and one hour after end of the infusion. Surface molecules and cytokine production were determined by FACS analysis. Results: Patients and controls exhibited mild cardiovascular changes with an increase of the heart rate (plus 10%), an increase of the systolic (plus 5%) and a decrease (minus 10%) of the diastolic blood pressure. NK cells increased in both groups from 200 to 1000 cells/ml. CD3+CD8+ and CD4+ cells increased significantly in all individuals. Cytotoxic activity was significantly (p=0.025) higher in RA patients at all three time points. IL-2, IL-4, IFN-g, and TNF-a producing lymphocytes increased after the infusion in both groups. IL-10 producing monocytes of RA patients presented a significant higher increase after the infusion (interaction p=0.026). IL-8+ monocyte numbers were higher on baseline in patients (p=0.014). Monocytes increased by a factor of 1.6 in both groups, the subset of CD14+CD16+ cells increased by a factor of 2.6. Adrenaline caused a significant (p=0.012) increase of CD11a++CD8+CD56– T cells in both groups. The percentage of perforin expressing CD8+CD56– T cells in patients and controls increased significantly from 10% to 30% (p<0.0001) after infusion. All values returned to baseline one hour after end of the infusion. Discussion: These data demonstrate an altered reaction to adrenaline in patients with RA with both pro- and anti-inflammatory dissimilarities. Adrenaline infusion as a model for psychological stress recruits activated T cells and monocytes which may influence disease activity.
40 · 32nd Annual Meeting of the German Society of Immunology 1
Immunopathology, Institute of Rheumatic Diseases, Piestany and 2Institute of Experimental Endocrinology, Bratislava, Slovakia
C. 12 Prolactin, autoantibodies, and HLA-antigens in primary Sjögren’s syndrome D. KOZAKOVA1, J. ROVENSKY1, V. BOSAK1, L. CEBECAUER1, D. MICEKOVA1, and M. VIGAS2 Introduction: The immunoregulatory hormone prolactin plays a role in the pathogenesis of autoimmune diseases, such as systemic lupus erythematosus and Sjögren’s syndrome (SjS). Primary SjS is frequently associated with specific autoantibodies of the anti-Ro/anti-La type (SS-A/SS-B), as well as with HLA system antigens, mainly HLA-DR3. The objective of the study was to check upon possible connections between prolactin levels, autoantibodies and HLA system antigens in primary SjS. Methods: The group of patients comprised 40 unrelated women aged 25–71 years, meeting the California Diagnostic Criteria for prim SjS. Prolactin levels were determined using commercial IRMA kit (Immunotech, Praha). Antibodies anti-Ro/La were determined using counterimmunoelectrophoresis and immunoblotting, HLA antigens were determined using fluorescencemodified microlymphocytotoxic test. Results: As compared with healthy controls, the group of female patients with prim SjS showed significantly (p<0.05) increased frequencies of elevated PRL levels (PRL >20mg/l). Elevated PRL levels were measured in 15.0% of the patients. Correlation of elevated PRL levels with the presence of anti-Ro/La antibodies failed to confirm any significant relationship. In a group of 23 women with prim SjS, immunogenetic analysis confirmed an association between HLA-DR3 antigen and prim SjS (RR=5.7). The relationship with HLA-DR3 was more pronounced in patients with anti-Ro/La antibodies (DR3 present in 75.9%, p<0.001, RR=13.3). Antigen DR3 carriers were more likely to have elevated PRL levels (15.4%) compared to DR3negative patients (0%). The difference in the frequencies however was not statistically significant (p>0.05). Conclusion: Since the group of women with elevated PRL levels comprised in the present study was too small, additional analysis including larger groups of patients is needed.
1
Department of Neuroimmunology, Max-Planck-Institute of Neurobiology, Martinsried, Institute of Medical Immunology, Charité, Humboldt-University, Berlin and 3Institute of Molecular Immunology, GSF National Research Center for Environment and Health, Munich, Germany 2
C. 13 Molecular mechanism of high-dose antigen therapy in experimental autoimmune encephalomyelitis Z.X. LI1, N. KAWAKAMI1, T. RITTER2, J.W. ELLWART3, H. WEKERLE1, and A. FLÜGEL1 Intravenous administration of soluble myelin basic protein (MBP) protects animals from adoptive experimental autoimmune encephalomyelitis (tEAE) transferred by MBP specific T cells. We explored the molecular mechanisms of soluble antigen therapy, using retrovirally engineered
32nd Annual Meeting of the German Society of Immunology · 41 MBP-specific T cells expressing green fluorescent protein (GFP) as marker (Flügel et al., 1999, Nat. Med. 5: 843–47). Soluble MBP was intravenously injected just before development of clinical symptoms (60–72 h after T cells transfer). Following such treatment, MBP specific T cells were found to rapidly lose their ability to enter the CNS, with no CNS infiltration of recruited T cells and macrophages. In the periphery, more than 70% of antigen specific T cells disappeared within one hour. The remaining effector cells up-regulated activation markers such as IL-2-R and OX-40. Also mRNA levels of IL-2 and proinflammatory cytokines TNF-a, IFN-g, massively increased. The majority of ex vivo sorted soluble antigen treated GFP+ T cells became positive for annexin V and active caspase-3 when transferred to culture. We suggest that soluble antigen treatment leads to rapid clearance of effector T cells in the periphery most likely mediated by activation induced cell death. Alternatively, capture of reactivated effector cells in peripheral tissues can not be excluded. Further, recruitment of host-derived T cells and macrophages into the CNS in clinical EAE is critically dependent on encephalitogenic effector T cells. The work was supported by SFB 455.
1
Institute of Cell Biology and Immunology, University of Stuttgart, Stuttgart, Germany, Division of Neuroimmunology, Brain Research Institute, Vienna, Austria, and 3Laboratoire de Neurosciences, University Caen, Caen, France 2
a signaling in neurodegenerative diseases C. 14 TNF-a L. MARCHETTI1, M. KLEIN1, J. BAUER2, E. PETIT3, K. PFIZENMAIER1, and U. EISEL1 Glutamate is a major excitatory neurotransmitter in the Central Nervous System. Pharmacological studies in rodents and recent clinical studies in humans have shown, that the extracellular concentration of glutamate is raised to toxic levels for neurons in ischemia, and additionally glutamate is believed to contribute to neuronal damage in degenerative disorders such as stroke, traumatic brain injury, Multiple Sclerosis, Chorea Huntington, Alzheimer and Parkinson Disease. TNF-a is also thought to play an important role in neurodegeneration. We have generated a transgenic mouse model with a forebrain specific mTNF-a-expression under the control of the NR2B-promoter. In vitro these transgenic animals express elevated levels of IFN-g, LTb and bfl-1 (an anti-apoptotic bcl-2-family-member) and seem to be protected in focal cerebral ischemia experiments. In in vitro studies with primary cortical cultures the transgenic, mTNF-a expressing neurons were less vulnerable to glutamate induced excitotoxic insults in comparison to wild type cells. This neuroprotective effect could be partially reproduced by pre-stimulating wildtype cells with mTNF-a. In a model of retinal ischemia using wildtype, TNF –/–, TNFR1–/– or TNFR2–/– animals we were able to deduce an TNFR2-depending neuroprotective pathway as the cell loss was increased in TNFR2–/– animals compared to wildtype or TNF –/– controls. TNFR1–/– animals on the other hand exhibited strong protection from neuronal cell loss, which could be correlated to strong Akt/PKB-phosphorylation 6h after reperfusion indicating an antagonistic function of TNF receptor signaling in ischemia.
42 · 32nd Annual Meeting of the German Society of Immunology Departments of 1Internal Medicine I and 2Orthopedic Surgery, University Hospital Regensburg, Regensburg, Germany
C. 15 Norepinephrine from synovial tyrosine hydroxylase and CD163 positive macrophages are strong indicators of synovial inflammation in rheumatoid arthritis L.E. MILLER1, J. GRIFKA2, J. SCHÖLMERICH1, and R.H. STRAUB1 Recently, we have demonstrated that the density of sympathetic nerve fibers in synovial tissue was significantly lower in patients with rheumatoid arthritis (RA) as compared to osteoarthritis (OA). This was accompanied by marked norepinephrine (NE) release from synovial tyrosine hydroxylase positive macrophages (TH+ MAC). Synovial tissue of 34 patients with RA and 36 patients with OA was characterized using immunohistochemistry and a synovial tissue superfusion technique, respectively. In separate culture experiments with mixed synoviocytes, the effect of NE on secretion of interleukin (IL)-6, IL-8, tumor necrosis factor (TNF), and matrix metalloproteinase-3 (MMP-3) was investigated. Tissue density of TH+ MAC was higher in RA as compared to OA (63.9 vs 34.2 cells/mm2, p=0.017) which was also found for synovial cellularity, T cells, CD163+ macrophages, and cells of the synovial lining layer. Basal NE release from synovial tissue highly significantly correlated with density of TH+ MAC in RA (RRank=0.573, p=0.001) but not in OA. Basal NE release correlated with the degree of inflammation in RA (RRank=0.420, p=0.021) but not in OA, and with spontaneous IL-8 secretion in RA (RRank=0.581, p=0.001) but not in OA. Only in RA, density of TH+ MAC positively correlated with spontaneous secretion of IL-6, IL-8, and MMP-3. We confirmed the extensive loss of sympathetic nerve fibers in RA as compared to OA (0.32 vs 3.1 nerve fiber/mm2, p<0.001). TH+ MAC and release of NE are strongly linked to a higher degree of synovial inflammation. Culture experiments indicate that NE has anti-inflammatory properties at higher concentrations (10–6M). NE secretion of TH+ MAC will probably be an anti-inflammatory mechanism to counteract local inflammation. Thus, TH+ MAC-derived NE is an important local factor of immunomodulation in synovial inflammation.
Department of Cell Biology and Neurobiology, Charité, Berlin, Germany
C. 16 Nerve growth factor is produced by astrocytes following interaction with Th1 and Th2 lymphocytes A. OREN, R. NITSCH, and U. GIMSA Introduction: Neuroinflammatory diseases like multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) are associated with T cell infiltration of the central nervous system. Nerve growth factor (NGF) has been shown to be neuroprotective in EAE. Astrocytic production of NGF is increased during inflammation in the central nervous system. We have studied how the interaction between astrocytes and Th1 and Th2 lymphocytes influences the production of astrocytic NGF.
32nd Annual Meeting of the German Society of Immunology · 43 Methods: Cultures of primary cortical astrocytes were made from new-born B10.PL mice and the Th1/Th2 cell lines from adult MBP-T cell receptor-transgenic mice of a B10.PL background. Lymphocytes were co-cultured with or without contact to either resting or pre-activated (100 U/ml IFN-g for 24h) astrocytes. Culture supernatants were collected and spun down to remove cells and stored frozen. NGF was subsequently detected by ELISA. Results: NGF was up-regulated in co-cultures of astrocytes with either Th1 or Th2 cells provided antigen-presentation by astrocytes was allowed. It was not produced in co-cultures of T cells with irradiated splenic antigen-presenting cells. Conclusions: Th1 and Th2 cells induce NGF production by astrocytes following cognate interaction. This might be a mechanism of neuroprotection in the brain following invasion of T cells. Astrocytes may thus counteract destructive effects of inflammatory processes in the brain. This study was supported by the Gemeinnützige Hertie-Stiftung (1.319.110–01–04).
1 Department of Internal Medicine I, University Hospital, Regensburg, 2Department of Clinical Immunology, Medical School, Hannover, and 4Institute of Medical Psychology, University of Essen, Essen, Germany and 3Department of Pediatric Immunology, Wilhelmina Children’s Hospital, University Medical Center, Utrecht, The Netherlands
C. 17 Infusion of adrenaline decreases serum levels of cortisol and 17hydroxy-progesterone in patients with rheumatoid arthritis but not in healthy subjects R.H. STRAUB1, J. KITTNER2, C. HEIJNEN3, M. SCHEDLOWSKI4, R.E. SCHMIDT2, and R. JACOBS2 This study was initiated in order to investigate the pituitary and adrenal hormone response after an intravenous adrenaline challenge in patients with rheumatoid arthritis (RA) and healthy subjects (HS). Fifteen untreated female RA patients (age: 51.5±3.2 yr) and 7 female HS (48.0±4.3 yr) were infused with adrenaline (0.05 mg/kg/min) for approx. 20 min. Plasma levels of adrenocorticotropic hormone (ACTH), and serum levels of cortisol, 17-hydroxyprogesterone (17OHP), and dehydroepiandrosterone sulfate (DHEAS) were analyzed at baseline and shortly after cessation of adrenaline infusion (20 min). At baseline and after adrenaline infusion, serum levels of cortisol (p=0.045) and 17OHP (p=0.021) were higher in healthy subjects as compared to RA patients. In contrast, at baseline and after adrenaline infusion, plasma levels of ACTH and serum levels of DHEAS were similar in HS as compared to RA patients. After adrenaline infusion, only patients with RA as compared to HS demonstrated a significant decrease of serum cortisol (p=0.026) and serum 17OHP (p=0.026). Plasma levels of ACTH (p=0.073) and serum levels of DHEAS (p=0.055) tended to decrease. This study demonstrates that serum cortisol and 17OHP (cortisol precursor) were lower in patients with RA as compared to HS despite similar ACTH levels. Furthermore, simulation of an adrenomedullary stress response by adrenaline infusion decreased serum cortisol and 17OHP only in RA patients but not in HS. Such a response may play an unfavorable role during a typical stress reaction in RA patients which may lead to a more proinflammatory situation.
44 · 32nd Annual Meeting of the German Society of Immunology 1
Department of Internal Medicine I, University Hospital, Regensburg, Germany and 2Orton Hospital, Invalid Foundation and 3Division of Rheumatology, Department of Medicine, Helsinki University Central Hospital, Helsinki, Finland
C. 18 Inadequately low serum levels of steroid hormones in relation to IL-6 and TNF in untreated patients with early rheumatoid arthritis and reactive arthritis R.H. STRAUB1, L. PAIMELA2, R. PELTOMAA3, J. SCHÖLMERICH1, and M. LEIRISALO-REPO3 In order to compare levels of steroid hormones in relation to cytokines and to study levels of cortisol or dehydroepiandrosterone (DHEA) in relation to other adrenal hormones, untreated patients with early rheumatoid arthritis (RA) and reactive arthritis (ReA) were compared to healthy controls. In a retrospective study with 34 patients with RA, 46 with ReA, and 112 healthy subjects (HS), we measured serum levels of interleukin (IL)-6, tumor necrosis factor (TNF), cortisol, 17OH-progesterone, androstenedione, DHEA, and DHEA sulfate (DHEAS). Patients with RA had higher serum levels of IL-6, TNF, cortisol, and DHEA as compared to ReA and HS but no difference was noticed with respect to DHEAS. However, in RA and ReA as compared to HS, serum levels of cortisol, androstenedione, DHEAS, and 17OH-progesterone were markedly lower in relation to IL-6 and TNF. Furthermore, the number of swollen joints inversely correlated with the ratio of serum cortisol/serum IL-6 in RA (RRank=-0.582, p=0.001) and, to a lesser extent, in ReA (RRank=-0.417, p=0.011). Furthermore, in these untreated patients with RA and ReA, there was a significant shift of hormone secretion from 17OH-progesterone, androstenedione, and DHEAS to DHEA and cortisol. This study indicates that cortisol is relatively low in relation to IL-6 and TNF in untreated patients with early RA and ReA as compared to HS. It further demonstrates that there is a hormone shift to DHEA and cortisol but not to DHEAS. This study emphasizes that adrenal steroid secretion is inadequately low in relation to inflammation. This is partly caused by an adrenal hormone shift from cortisol to DHEA. Although changes of hormone levels are similar in RA and ReA, alteration of the steroidogenesis is more pronounced in patients with RA as compared to ReA.
1
Department of Neurology and 2Section of Transplantation Immunology and Immunohematology, University of Tübingen, and 3EMC microcollections GmbH, Tübingen, Germany
C. 19 T cell reactivity towards myelin oligodendrocyte glycoprotein in multiple sclerosis R. WEISSERT1, J. KUHLE1, W. WIENHOLD1, C.A. MÜLLER2, K.-H. WIESMÜLLER3, and A. MELMS1 Multiple sclerosis is an inflammatory, demyelinating and neurodegenerative disease of the central nervous system. There is strong evidence for an autoimmune pathogenesis driven by myelin-specific T cells. Myelin-oligodendrocyte-glycoprotein induces in animals experimental autoimmune encephalomyelitis, a very multiple sclerosis-like disease with demyelinating central nervous sys-
32nd Annual Meeting of the German Society of Immunology · 45 tem lesions and axonal loss underscoring its potential role in multiple sclerosis pathogenesis. We performed a T cell reactivity pattern analysis of multiple sclerosis patients and controls that were stratified for the genetic risk factor HLA-DRB1*1501. We show for the first time that there is a promiscuous dominant T cell epitope within the intracellular part of myelin oligodendrocyte glycoprotein comprising amino acids 146–154. Surprisingly controls had broader T cell reactivity patterns towards myelin oligodendrocyte glycoprotein compared to multiple sclerosis patients and the trans-membrane and intracellular parts of myelin oligodendrocyte glycoprotein were much more immunogenic compared to the extracellular part. These data underscore that myelin oligodendrocyte glycoprotein is a target molecule in multiple sclerosis immunopathogenesis.
Department of Neuroimmunology, Max-Planck-Institute of Neurobiology, Martinsried, Germany
C. 20 Enhancement of experimental autoimmune encephalomyelitis by B cell transfer T. ZIEMSSEN, H. WEKERLE, and A. IGLESIAS Introduction: B cells seem to have a crucial role in the pathogenesis of myelin autoimmune disease. Methods: We transferred B cells from transgenic TH mice expressing a targeted heavy chain of the anti myelin oligodendrocyte glycoprotein (MOG) 8.18c5 antibody into mice with subclinical or mild experimental autoimmune encephalomyelitis (EAE). Using green fluorescent protein (GFP)- and TH double transgenic mice, we were able to follow simultaneously clinical disease, anti-MOG serum titer and trafficking of the transferred cells. Results: The transfer of LPS-activated, but not of resting TH or activated wild type B cells or the injection of large amounts of MOG-specific 8.18c5 antibodies lead to an exacerbated and accelerated form of EAE in mice treated to develop mild or subclinical EAE. In untreated mice activated TH B cells or MOG-specific antibodies have no effects. TH/GFP B cells peak at day 4 after transfer, when they appear predominantly in spleen and peripheral blood, but also in lymph node and bone marrow. 16 days post transfer, only few TH/GFP B cells are still detectable bearing mostly a plasmacytoid phenotype. MOG-specific IgM is detectable 1 day and peaks 4 days after transfer, while MOG-specific IgG appears at day 4 and its concentration raises continuously through days 16 and 29 after transfer. Thus transgenic TH B cells undergo isotype switch in recipient animals. However, there is no increase in the number of B cells in CNS infiltrates of TH transgenic and B cell transferred mice with exacerbated EAE. Instead, the large inflammatory infiltrates observed in these animals include increased numbers of cells of the myeloid lineage. Conclusions: MOG-specific B cells and antibodies are not pathogenic alone, but accelerate and exacerbate an ongoing mild EAE. We are currently investigating the possibility that MOGspecific antibodies mediate enhanced recruitment of monocytes and neutrophils into the inflamed site of the CNS.
Max-Planck-Institut für physiologische und klinische Forschung, Bad Nauheim, Germany
C. 1 Expression of ELC (CCL19) and SLC (CCL 21) at the blood-brain barrier during experimental autoimmune encephalomyelitis suggests their involvement in lymphocyte recruitment into the central nervous system C. ALT and B. ENGELHARDT Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated autoimmune disease of the central nervous system (CNS), where CD4+ autoaggressive T cells gain access to the CNS and start the molecular events leading to inflammation, edema and demyelination. Based on our recent observation that T cell recruitment across the blood-brain barrier (BBB) does require Gprotein mediated signals in vivo we have started to search for the chemokines promoting leukocyte arrest on the BBB endothelium as chemokine expression at the BBB has not been reported. By performing in situ hybridizations for a large panel of chemokines ELC (CCL 19) and SLC (CCL 21) were the only chemokines expression of which could be localized to cerebral vessels surrounded by perivascular cuffs in brain and spinal cord sections derived from mice afflicted with EAE. The receptor for ELC and SLC, CCR7 was detected in inflammatory cells surrounding cerebral vessels. In addition expression of ELC (CCL 19) was detected in cerebral vessels of healthy control mice. Preliminary data confirm the presence of ELC and SLC protein in cerebral vessels. The spatial correlation of the expression of CCL19 and CCL21 at the BBB and CCR7 on inflammatory cells in inflammatory cuffs during EAE observed here implies a not yet appreciated involvement of these constitutive chemokines in regulating lymphocyte homing into nonlymphoid tissues. This is supported by our in vitro observations that encephalitogenic T lymphocytes showed specific chemotaxis towards towards CCL 19 and CCL 21 in a concentration dependent manner and comparable to naïve lymphocytes. Our data suggest that ELC and SLC besides regulating lymphocyte homing into lymphoid organs additionally regulate T cell homing into the immunoprivileged CNS.
32nd Annual Meeting of the German Society of Immunology · 33 Institutes of 1Anatomy and Cell Biology and 3Immunology, Philipps-University, Marburg, Germany and 2Center of Neurovirology, Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, USA
C. 2 Accumulation of macrophage migration inhibitory factor in astrocytes of borna disease virus-infected rat brain is accompanied by inhibition of macrophage infiltration M. BETTE1, E. WEIHE1, B. DIETZSCHOLD2, D. GEMSA3, and M. BACHER3 To test the hypothesis that macrophage migration inhibitory factor (MIF) plays a role in macrophage invasion during virus-induced encephalitis, we analyzed the expression and cellular localization of MIF in the Borna Disease Virus (BDV)-infected rat brain, monitored monocyte/macrophage infiltration and evaluated the influence of anti-inflammatory treatment with dexamethasone. MIF mRNA expression was restricted to neurons and remained unchanged after BDV-infection or after dexamethasone treatment of either BDV-infected or uninfected control rats. In contrast, MIF protein immunoreactivity (ir) was not only seen in neurons but also in glia. After BDV-induced encephalitis and treatment of uninfected rats with dexamethasone, MIF-ir was only slightly altered in neurons but moderately enhanced in tanycytes, ependyma and choroid plexus epithelium and markedly increased or induced in astrocyte end-feet at the blood brain barrier (BBB). The increase in MIF-ir in astrocytes after BDV-infection was blocked by dexamethasone. The induction or enhancement of MIF-ir at the BBB significantly correlated with reduced numbers of infiltrating ED1-positive monocytes/macrophages after BDV-infection. Increased macrophage invasion was observed in regions where no astrocytic MIF was detected. The BDV- or dexamethasone-induced accumulation of MIF protein in astrocytes in vivo in absence of detectable astrocytic MIF mRNA expression is most likely due to MIF translocation from neurons rather than to a constitutive or induced MIF mRNA expression in astrocytes. In conclusion, we provide evidence that translocation of MIF from neurons or other extracellular sources into astrocytes is likely to modulate the inflammatory process during the course of virusinduced encephalitis by limiting monocyte/macrophage migration through the BBB.
1
Institut für Klinische Immunologie und Transfusionsmedizin, Universität Leipzig and 2Vita34 Gesellschaft für Zelltransplantate mbH, Leipzig, Germany
C. 3 Human umbilical cord blood: A source of neural stem cells? J. BOLTZE1, U. SACK1, D. EGGER2, and F. EMMRICH1 Neural stem cells, which display self-renewal and multilineage differentiation properties, have been isolated from embryonic and adult central nervous system (CNS) tissues of murine and even human origin to be expanded in vitro in recent years. After expansion these cells are able to differentiate into neurons and glial cells in vitro and in vivo. Therefore, neural stem cells are important regarding clinical applications, i.e. cell therapy in patients with CNS disorders.
34 · 32nd Annual Meeting of the German Society of Immunology Umbilical cord blood is a rich source of neonatal stem cells which are shown to give rise to blood and bone marrow cells and are expected to have further abilities. Hence, they are an important basis for autologous transplantation strategies avoiding immunologic rejections. Therefore, neonatal stem cells are investigated for neurogenerative properties. After collecting cord blood samples the mononuclear population containing neonatal stem cells was isolated by using a modified Ficoll density gradient centrifugation protocol. Further, cells are grown in ITSFn, a DMEM/F12 based medium with insulin, transferrin, selenite and fibronectin supplements which has been shown to strongly select for neural stem cells and progenitor cells. After 6 to 8 days in ITSFn, most of the cells were destroyed. Surviving cells were analyzed for the expression of mRNA coding for the nestin protein, an early marker for neural stem cells using LightCycler PCR. Alternatively they can be transferred to an expansion medium containing bFGF, EGF and a variety of further supplements in order to form neurospheres (free floating colonies of neural stem cells in medium) before differentiation into neurons and glia. In cell culture several indications were obtained that differentiation towards neuronal cells took place. Neurosphere-like clusters as well as branched-dendrites were observed. To verify these findings direct transfer of cord blood cells to CNS of immunodeficient mice will be performed.
1
Abteilung Neuroimmunologie, Max-Planck-Institut für Neurobiologie, Martinsried, Germany and 2Abteilung Neuroimmunologie, Institut für Hirnforschung, Wien, Österreich
C. 4 Myelin degeneration and autoimmune disease: Altered distribution of inflammatory lesions in hemizygous transgenic rats overexpressing proteolipid protein M. BRADL1, M. KLEIN1, J. BAUER2, H. LASSMANN2, and H. WEKERLE1 Adult hemizygous transgenic Lewis rats overexpressing the myelin proteolipid protein (PLP) develop low grade, subclinical myelin degeneration. Contrary to expectation, pathological changes rarely occur in the myelin-rich CNS white matter, but are almost exclusively confined to CNS gray matter. In this compartment, numerous oligodendrocytes retain and accumulate PLP, myelin oligodendrocyte glycoprotein (MOG) and myelin associated glycoprotein (MAG) in the endoplasmic reticulum (ER), but correctly synthesize myelin basic protein (MBP) in the cellular processes. Some of these oligodendrocytes become apoptotic. Apparantly as a consequence of oligodendrocyte stress and death in CNS gray matter, local microglia cells become activated. They upregulate the complement receptor type III, phagocytose oligodendrocyte debris and express MHC class II molecules. The accumulation of PLP and MOG, but not MBP in the oligodendrocytes ER, the resulting oligodendrocyte degeneration and microglia activation seem to jointly influence the distribution of inflammatory lesions in the course of experimental autoimmune encephalomyelitis (EAE): In PLP-specific T cell-induced EAE, effector T cells and inflammatory lesions are present in the meninges and (rarely)in the CNS white matter of wildtype animals, but almost exclusively infiltrate CNS gray matter of the transgenic rats. In MOGspecific T cell-induced EAE, T cells and lesions are found in CNS white and gray matter areas of wildtype rats, and more often in CNS gray matter areas of their transgenic counterparts. In MBPspecific T cell-induced EAE, effector T cells and inflammatory lesions are seen in identical places in wildtype and transgenic rats. These data demonstrate a link between myelin/oligodendrocyte
32nd Annual Meeting of the German Society of Immunology · 35 degeneration and the localization of inflammatory lesions in the CNS. It remains to be seen whether similar mechanisms act in human leukodystrophies with spontaneous T cell infiltration of the CNS.
Institut für Medizinische Mikrobiologie und Virologie, Heinrich-Heine-Universität, Düsseldorf, Germany
C. 5 Anti-viral effector mechanisms in human astrocytoma cells W. DÄUBENER, C. OBERDÖRFER, K. BESKEN, C.R. MACKENZIE, and O. ADAMS Encephalitis and meningitis caused by parasites, bacteria and viruses are the most dangerous infectious diseases of the brain. We have analyzed the capacity of human astrocytoma cells to restrict the growth of Toxoplasma gondii, group B streptococci and Herpes simplex virus. Human astrocytoma cells are capable of restricting the growth of Toxoplasma gondii and of group B streptococci after stimulation with IFN-g. We found that the antimicrobial effect of IFN-g-stimulated astrocytoma cells is mediated via an induction of indoleamine 2,3 dioxygenase (IDO). This enzyme degrades L-tryptophan to kynurenine, and this results in complete depletion of L-tryptophan, an essential amino acid for both pathogens. IDO activity was found in astrocytoma cells by the detection of IDO mRNA in RT-PCR, detection of enzyme protein 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 sufficient to inhibit the growth of Toxoplasma gondii and of group B streptococci, and this antimicrobial effect is blocked in the presence of excess amounts of L-tryptophan, indicating that IDO activity is indeed the active effector mechanism against both pathogens. In addition we found that IFN-g stimulated human astrocytoma cells are capable of restricting herpes simplex virus replication. Furthermore we found that IFN-a and IFN-b which were unable to induce an anti-parasitic or anti-bacterial effect in astrocytoma cells induce a strong anti-viral effect. Using a quantitative PCR protocol we found that an excess amount of L-tryptophan nearly completely abrogates the antiviral effect of astrocytoma cells against HSV-1 and HSV-2. We therefore suggest that IDO activation is involved in the anti-viral defense mediated by astrocytoma cells.
1
Institute of Medical Microbiology and Hygiene and 2Department of Molecular Neuroscience, Institute of Anatomy and Cell Biology, Philipps-University, Marburg, Germany
C. 6 Microglia cells are activated by immunostimulatory CpG-DNA A.H. DALPKE1, M. SCHÄFER2, M. FREI1, S. ZIMMERMANN1, E. WEIHE2, and K. HEEG1 Bacterial DNA containing motifs of unmethylated CpG-dinucleotides (CpG-DNA) can be recognized by the innate immune system through the pattern recognition receptor TLR 9. Thus bacterial DNA possesses potent immunostimulatory effects on macrophages, dendritic cells and B lymphocytes and can contribute to inflammation during the course of bacterial infections.
36 · 32nd Annual Meeting of the German Society of Immunology Only limited information is available concerning the role of CpG-DNA in immune reactions of the CNS. To address this question we examined the effects of CpG-DNA on microglia cells in vitro and in vivo. Here we show that primary microglia cells obtained from neonatal brain cultures as well as microglia cell lines expressed mRNA encoding TLR 9 transcripts. CpG-DNA in vitro activated microglia cells to produce TNF-a, IL-12p40 and nitric oxide. In addition microglia displayed upregulation of MHC class II, B7–1, B7–2 and CD40 molecules after CpGDNA administration. Furthermore CpG-DNA increased phagocytosis by microglia cells. In vitro CpG-DNA also induced TNF-a and IL-12p40 transcripts in microglia cells after intracerebroventricular injection as shown by in situ hybridization and quantitative RT-PCR. CpG-antagonistic oligonucleotides containing guanosine rich regions inhibited CpG-DNA induced activation of microglia. Taken together these results indicate that the microglia 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 influence the immunostimulatory properties of the microglia system.
Max-Planck-Institut für physiologische und klinische Forschung, Bad Nauheim, Germany
C. 7 Tetracycline-inducible expression of adhesion molecules at the bloodbrain barrier in transgenic mice B. ENGELHARDT, T.M. SCHLÄGER, B. DEHOUCK, S. TAUBER, S. HENNIG, V. SCHMIDT, W. RISAU, and U. DEUTSCH We have investigated the expression and function of adhesion molecules on blood-brain barrier (BBB) endothelium during experimental autoimmune encephalomyelitis (EAE) in the SJL/N mouse. We have found that during EAE expression of E- and P-selectin is not induced in BBB endothelium and that E- and P-selectin are not required for the recruitment of inflammatory cells across the BBB. In contrast, VCAM-1 but not MAdCAM-1 is induced on BBB endothelium during EAE and actively involved in the recruitment of a4b1-integrin-positive inflammatory cells during the disease. In order to find out whether ectopic overexpression of the adhesion molecules lacking at the BBB is sufficient to overcome the selective block of recruitment for subsets of leukocytes across the BBB, we have generated transgenic mice, in which expression of E- and P-selectin and MAdCAM-1 can be induced at the BBB-endothelium using the «TET-OFF» system. The «TET-OFF» system consists of a binary system of transgenic activator and responder mice that allows for tetracycline-regulated expression of transgenes in double transgenic mice. Different types of activator mouse lines expressing the tetracycline-inducible transactivator (tTA) under the control of regulatory elements of the mouse Tie-2 receptor tyrosine kinase gene that is required for expression in all embryonic and adult vascular beds, have been established. Additionally, various types of responder mouse lines with expression of E- and P-selectin or MAdCAM-1 under the control of multiple tet-operators have been raised. Removal of tetracycline leads to induced expression of E-selectin and MAdCAM-1 in several combinations of double transgenic animals. These double transgenic mice exhibit endothelial cell-specific induction of the adhesion molecules E-selectin and MAdCAM-1 in the vessels of the CNS. Currently these mice are used to investigate the recruitment of inflammatory cells across the «altered» BBB.
32nd Annual Meeting of the German Society of Immunology · 37 1
Department of Immunology and Gnotobiology, Institute of Microbiology, Czech Academy of Sciences, and 2Biochemical Research Unit, 1st Medical Faculty and 3Department of Pharmacology, 3rd Medical Faculty, Charles University, Prague, Czech Republic
C. 8 Catecholaminergic system regulates immunity to tumors A. FISEROVA1, H. KOVARU2, L.E. VANNUCCI1, O. MATEJKOVA1, M. KULDOVA1, M. STAREC3, and M. POSPISIL1 Introduction: Bidirectional interactions between the immune and neuroendocrine systems are involved in the maintenance of cellular homeostasis and regulation of tumor growth (Chuluyan et al., NeuroImmunoModulation 8, 107, 2000). Engagement of neurotransmitter receptors expressed on lymphoid cells enables regulation of their growth and function induced by neuroendocrine mediators. Aim: To assess the effects evoked by these mediators represented by D1 (agroclavine) and D2 (terguride) dopamine receptor agonists on antitumor immune responses. Methods: Phenotypic and functional analysis of dopamine agonists treated tumor and MLTC generated cytotoxic lymphocyte subpopulations were followed in vitro. The study was completed by the detection of associated signaling pathways. For in vivo experiments the transplanted B16F10 melanoma in C57/bl6 mice was used. Results: In vitro obtained data demonstrated a stimulatory effect of D1 agonists on cytotoxic activity of NK cells (paralleled by IL-1b, IL-2, IFN-g cytokine production, and triggering of Gq-protein, ERK1/2 signal transmission), however D2 agonist had the opposite effect. The D1 agonist treatment of tumor cells increased their sensitivity to the NK cell effector function and D2 agonist evoked higher expression of MHC-I antigen (with lowered synthesis of IL-2, IL-6, IFN-g, and blocked signaling pathways). These results were confirmed in melanoma bearing mice in vivo. The mean survival time of mice transplanted with D1 conditioned melanomas was prolonged, while shortened after D2 treatment, in comparison to the wild type B16F10 cells. The best therapeutic effect (46% of surviving animals) was achieved by use of liposome-encapsulated D1 agonist (agroclavine). Conclusions: Cell-mediated antitumor response appears to be under dopaminergic control involving activation through D1-type and inhibition by D2-type of dopamine receptors. Supported by grants: 312/98/K034, 304/01/0850, GAUK 71/2001/C/3.LF, MSMT 111100001, and by Institutional Research Concept AV0Z5020903.
38 · 32nd Annual Meeting of the German Society of Immunology 1
Department of Neuroimmunology, Max-Planck-Institute of Neurobiology, Martinsried, Institute of Medical Immunology, Charité, Humboldt-University, Berlin, and 3Institute of Molecular Immunology, GSF National Research Center for Environment and Health, Munich, Germany 2
C. 9 Reactivation within the target organ determines the pathogenic potential of encephalitogenic T cells A. FLÜGEL1, N. KAWAKAMI1, Z.X. LI1, T. RITTER2, J.W. ELLWART3, C. LININGTON1, and H. WEKERLE1 Autoimmune inflammation of the CNS, e.g. in Multiple sclerosis, results in a wide spectrum of clinical disease types ranging from mild, self-limited to severe progressive disease. This is reflected in the T cell-mediated animal model of MS, the experimental autoimmune encephalomyelitis (EAE). There, the outcome of clinical disease depends on the target antigen of encephalitogenic T cells and the genetic background of the recipient animals. Thus, T cells specific to myelin basic protein (MBP) induce a severe paralyzing EAE in Lewis rats, whereas T cells specific for myelin oligodendrocyte protein (MOG) or the astrocytic antigen S100b evoke intense CNS inflammation without marked clinical symptoms. Likewise, in DA rats, MOG specific T cells (MOG-DA T cells) induce a severe clinical disease resembling MBP T cell-mediated EAE of the Lewis rat, while EAE induced by MOG specific T cells in Lewis rats (MOG-Lewis) is mild. We used retrovirally engineered T cells expressing the marker gene of green fluorescent protein (Flügel et al., 1999, Nat. Med. 5: 843–47) to examine the mechanisms, which determine the clinical outcome of adoptive transfer EAE. We found that all brain specific T cells caused a massive influx of effector cells into the CNS three to four days following transfer. However, only the highly pathogenic MBP and MOG-DA T cells up-regulated activatory markers such as IL-2-receptor and OX40 antigen. Further the activated T cells increased mRNA encoding the proinflammatory cytokines IFN-g and TNF-a. In contrast, low-pathogenic S100B and MOG-Lewis T cells did not show these activatory changes, but showed a moderate increase of IL-2 and IL-4 mRNA. We conclude that the level of activation within the target organ might critically determine the character of autoimmune CNS disease. The work was supported by the SFB 455.
Department of Immunology, Bernhard-Nocht-Institute of Tropical Medicine, Hamburg, Germany
C. 10 Murine malaria following blood-stage infection by P. berghei is exacerbated by CTLA-4 blockade T. JACOBS, S.E.B. GRAEFE, S. NIKNAFS, I. GAWORSKI, and B. FLEISCHER In this study we examined the role of CTLA-4 expression (CD152) in experimental blood-stage malaria. Infection of C57BL/6 mice with by P. berghei led to a strong increase of CTLA-4 expression on spleen cells. On day 9 after infection up to 10% of CD4+ T cells expressed CTLA-4,
32nd Annual Meeting of the German Society of Immunology · 39 whereas only a marginal expression was found on other cells. Spleen cells taken on day 9 after infection were suppressed in their proliferative response, which could not be reversed by addition of either anti-CTLA-4 or anti-TGF-b antibodies. On day 5 after infection only about 1% of CD4+ T cells expressed CTLA-4, however stimulation in the presence of anti-CTLA-4 led to an increased T cell proliferation. Furthermore, in vivo administration of anti-CTLA-4 antibodies and subsequent challenge with P. berghei led to an exacerbation of disease in comparison to control mice, which was accompanied by an increased proliferation of spleen cells. Histologic examination of brain sections from anti-CTLA-4 treated mice revealed pathologic changes characteristic for cerebral malaria like sequestration of parasitized erythrocytes, perivascular edema and stasis which were absent in control mice. Our data suggest that CTLA-4 expression on CD4+ T cells suppress pathological immune responses most probably by other mechanisms than TGF-b secretion.
1
Department of Clinical Immunology, Medical School, Hannover, 2Department of Physiological Psychology, University of Marburg, Marburg, and 3Institute of Medical Psychology, University of Essen, Essen, Germany
C. 11 Immune response upon adrenaline infusion in patients with rheumatoid arthritis J.M. KITTNER1, C.R. PAWLAK2, M. SCHEDLOWSKI3, R.E. SCHMIDT1, and R. JACOBS1 Introduction: Clinical observations suggest the course of rheumatoid arthritis (RA) to be influenced by psychological stress. Reduced density of b2-adrenoceptors on lymphocytes in RA patients has been shown. Aim of this study was to investigate whether the immunological reaction to adrenaline is altered in RA patients. Methods: 14 female RA patients and 14 female healthy controls were infused with 1 mg/kg adrenaline over 20 min. Blood was drawn before, immediately after and one hour after end of the infusion. Surface molecules and cytokine production were determined by FACS analysis. Results: Patients and controls exhibited mild cardiovascular changes with an increase of the heart rate (plus 10%), an increase of the systolic (plus 5%) and a decrease (minus 10%) of the diastolic blood pressure. NK cells increased in both groups from 200 to 1000 cells/ml. CD3+CD8+ and CD4+ cells increased significantly in all individuals. Cytotoxic activity was significantly (p=0.025) higher in RA patients at all three time points. IL-2, IL-4, IFN-g, and TNF-a producing lymphocytes increased after the infusion in both groups. IL-10 producing monocytes of RA patients presented a significant higher increase after the infusion (interaction p=0.026). IL-8+ monocyte numbers were higher on baseline in patients (p=0.014). Monocytes increased by a factor of 1.6 in both groups, the subset of CD14+CD16+ cells increased by a factor of 2.6. Adrenaline caused a significant (p=0.012) increase of CD11a++CD8+CD56– T cells in both groups. The percentage of perforin expressing CD8+CD56– T cells in patients and controls increased significantly from 10% to 30% (p<0.0001) after infusion. All values returned to baseline one hour after end of the infusion. Discussion: These data demonstrate an altered reaction to adrenaline in patients with RA with both pro- and anti-inflammatory dissimilarities. Adrenaline infusion as a model for psychological stress recruits activated T cells and monocytes which may influence disease activity.
40 · 32nd Annual Meeting of the German Society of Immunology 1
Immunopathology, Institute of Rheumatic Diseases, Piestany and 2Institute of Experimental Endocrinology, Bratislava, Slovakia
C. 12 Prolactin, autoantibodies, and HLA-antigens in primary Sjögren’s syndrome D. KOZAKOVA1, J. ROVENSKY1, V. BOSAK1, L. CEBECAUER1, D. MICEKOVA1, and M. VIGAS2 Introduction: The immunoregulatory hormone prolactin plays a role in the pathogenesis of autoimmune diseases, such as systemic lupus erythematosus and Sjögren’s syndrome (SjS). Primary SjS is frequently associated with specific autoantibodies of the anti-Ro/anti-La type (SS-A/SS-B), as well as with HLA system antigens, mainly HLA-DR3. The objective of the study was to check upon possible connections between prolactin levels, autoantibodies and HLA system antigens in primary SjS. Methods: The group of patients comprised 40 unrelated women aged 25–71 years, meeting the California Diagnostic Criteria for prim SjS. Prolactin levels were determined using commercial IRMA kit (Immunotech, Praha). Antibodies anti-Ro/La were determined using counterimmunoelectrophoresis and immunoblotting, HLA antigens were determined using fluorescencemodified microlymphocytotoxic test. Results: As compared with healthy controls, the group of female patients with prim SjS showed significantly (p<0.05) increased frequencies of elevated PRL levels (PRL >20mg/l). Elevated PRL levels were measured in 15.0% of the patients. Correlation of elevated PRL levels with the presence of anti-Ro/La antibodies failed to confirm any significant relationship. In a group of 23 women with prim SjS, immunogenetic analysis confirmed an association between HLA-DR3 antigen and prim SjS (RR=5.7). The relationship with HLA-DR3 was more pronounced in patients with anti-Ro/La antibodies (DR3 present in 75.9%, p<0.001, RR=13.3). Antigen DR3 carriers were more likely to have elevated PRL levels (15.4%) compared to DR3negative patients (0%). The difference in the frequencies however was not statistically significant (p>0.05). Conclusion: Since the group of women with elevated PRL levels comprised in the present study was too small, additional analysis including larger groups of patients is needed.
1
Department of Neuroimmunology, Max-Planck-Institute of Neurobiology, Martinsried, Institute of Medical Immunology, Charité, Humboldt-University, Berlin and 3Institute of Molecular Immunology, GSF National Research Center for Environment and Health, Munich, Germany 2
C. 13 Molecular mechanism of high-dose antigen therapy in experimental autoimmune encephalomyelitis Z.X. LI1, N. KAWAKAMI1, T. RITTER2, J.W. ELLWART3, H. WEKERLE1, and A. FLÜGEL1 Intravenous administration of soluble myelin basic protein (MBP) protects animals from adoptive experimental autoimmune encephalomyelitis (tEAE) transferred by MBP specific T cells. We explored the molecular mechanisms of soluble antigen therapy, using retrovirally engineered
32nd Annual Meeting of the German Society of Immunology · 41 MBP-specific T cells expressing green fluorescent protein (GFP) as marker (Flügel et al., 1999, Nat. Med. 5: 843–47). Soluble MBP was intravenously injected just before development of clinical symptoms (60–72 h after T cells transfer). Following such treatment, MBP specific T cells were found to rapidly lose their ability to enter the CNS, with no CNS infiltration of recruited T cells and macrophages. In the periphery, more than 70% of antigen specific T cells disappeared within one hour. The remaining effector cells up-regulated activation markers such as IL-2-R and OX-40. Also mRNA levels of IL-2 and proinflammatory cytokines TNF-a, IFN-g, massively increased. The majority of ex vivo sorted soluble antigen treated GFP+ T cells became positive for annexin V and active caspase-3 when transferred to culture. We suggest that soluble antigen treatment leads to rapid clearance of effector T cells in the periphery most likely mediated by activation induced cell death. Alternatively, capture of reactivated effector cells in peripheral tissues can not be excluded. Further, recruitment of host-derived T cells and macrophages into the CNS in clinical EAE is critically dependent on encephalitogenic effector T cells. The work was supported by SFB 455.
1
Institute of Cell Biology and Immunology, University of Stuttgart, Stuttgart, Germany, Division of Neuroimmunology, Brain Research Institute, Vienna, Austria, and 3Laboratoire de Neurosciences, University Caen, Caen, France 2
a signaling in neurodegenerative diseases C. 14 TNF-a L. MARCHETTI1, M. KLEIN1, J. BAUER2, E. PETIT3, K. PFIZENMAIER1, and U. EISEL1 Glutamate is a major excitatory neurotransmitter in the Central Nervous System. Pharmacological studies in rodents and recent clinical studies in humans have shown, that the extracellular concentration of glutamate is raised to toxic levels for neurons in ischemia, and additionally glutamate is believed to contribute to neuronal damage in degenerative disorders such as stroke, traumatic brain injury, Multiple Sclerosis, Chorea Huntington, Alzheimer and Parkinson Disease. TNF-a is also thought to play an important role in neurodegeneration. We have generated a transgenic mouse model with a forebrain specific mTNF-a-expression under the control of the NR2B-promoter. In vitro these transgenic animals express elevated levels of IFN-g, LTb and bfl-1 (an anti-apoptotic bcl-2-family-member) and seem to be protected in focal cerebral ischemia experiments. In in vitro studies with primary cortical cultures the transgenic, mTNF-a expressing neurons were less vulnerable to glutamate induced excitotoxic insults in comparison to wild type cells. This neuroprotective effect could be partially reproduced by pre-stimulating wildtype cells with mTNF-a. In a model of retinal ischemia using wildtype, TNF –/–, TNFR1–/– or TNFR2–/– animals we were able to deduce an TNFR2-depending neuroprotective pathway as the cell loss was increased in TNFR2–/– animals compared to wildtype or TNF –/– controls. TNFR1–/– animals on the other hand exhibited strong protection from neuronal cell loss, which could be correlated to strong Akt/PKB-phosphorylation 6h after reperfusion indicating an antagonistic function of TNF receptor signaling in ischemia.
42 · 32nd Annual Meeting of the German Society of Immunology Departments of 1Internal Medicine I and 2Orthopedic Surgery, University Hospital Regensburg, Regensburg, Germany
C. 15 Norepinephrine from synovial tyrosine hydroxylase and CD163 positive macrophages are strong indicators of synovial inflammation in rheumatoid arthritis L.E. MILLER1, J. GRIFKA2, J. SCHÖLMERICH1, and R.H. STRAUB1 Recently, we have demonstrated that the density of sympathetic nerve fibers in synovial tissue was significantly lower in patients with rheumatoid arthritis (RA) as compared to osteoarthritis (OA). This was accompanied by marked norepinephrine (NE) release from synovial tyrosine hydroxylase positive macrophages (TH+ MAC). Synovial tissue of 34 patients with RA and 36 patients with OA was characterized using immunohistochemistry and a synovial tissue superfusion technique, respectively. In separate culture experiments with mixed synoviocytes, the effect of NE on secretion of interleukin (IL)-6, IL-8, tumor necrosis factor (TNF), and matrix metalloproteinase-3 (MMP-3) was investigated. Tissue density of TH+ MAC was higher in RA as compared to OA (63.9 vs 34.2 cells/mm2, p=0.017) which was also found for synovial cellularity, T cells, CD163+ macrophages, and cells of the synovial lining layer. Basal NE release from synovial tissue highly significantly correlated with density of TH+ MAC in RA (RRank=0.573, p=0.001) but not in OA. Basal NE release correlated with the degree of inflammation in RA (RRank=0.420, p=0.021) but not in OA, and with spontaneous IL-8 secretion in RA (RRank=0.581, p=0.001) but not in OA. Only in RA, density of TH+ MAC positively correlated with spontaneous secretion of IL-6, IL-8, and MMP-3. We confirmed the extensive loss of sympathetic nerve fibers in RA as compared to OA (0.32 vs 3.1 nerve fiber/mm2, p<0.001). TH+ MAC and release of NE are strongly linked to a higher degree of synovial inflammation. Culture experiments indicate that NE has anti-inflammatory properties at higher concentrations (10–6M). NE secretion of TH+ MAC will probably be an anti-inflammatory mechanism to counteract local inflammation. Thus, TH+ MAC-derived NE is an important local factor of immunomodulation in synovial inflammation.
Department of Cell Biology and Neurobiology, Charité, Berlin, Germany
C. 16 Nerve growth factor is produced by astrocytes following interaction with Th1 and Th2 lymphocytes A. OREN, R. NITSCH, and U. GIMSA Introduction: Neuroinflammatory diseases like multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) are associated with T cell infiltration of the central nervous system. Nerve growth factor (NGF) has been shown to be neuroprotective in EAE. Astrocytic production of NGF is increased during inflammation in the central nervous system. We have studied how the interaction between astrocytes and Th1 and Th2 lymphocytes influences the production of astrocytic NGF.
32nd Annual Meeting of the German Society of Immunology · 43 Methods: Cultures of primary cortical astrocytes were made from new-born B10.PL mice and the Th1/Th2 cell lines from adult MBP-T cell receptor-transgenic mice of a B10.PL background. Lymphocytes were co-cultured with or without contact to either resting or pre-activated (100 U/ml IFN-g for 24h) astrocytes. Culture supernatants were collected and spun down to remove cells and stored frozen. NGF was subsequently detected by ELISA. Results: NGF was up-regulated in co-cultures of astrocytes with either Th1 or Th2 cells provided antigen-presentation by astrocytes was allowed. It was not produced in co-cultures of T cells with irradiated splenic antigen-presenting cells. Conclusions: Th1 and Th2 cells induce NGF production by astrocytes following cognate interaction. This might be a mechanism of neuroprotection in the brain following invasion of T cells. Astrocytes may thus counteract destructive effects of inflammatory processes in the brain. This study was supported by the Gemeinnützige Hertie-Stiftung (1.319.110–01–04).
1 Department of Internal Medicine I, University Hospital, Regensburg, 2Department of Clinical Immunology, Medical School, Hannover, and 4Institute of Medical Psychology, University of Essen, Essen, Germany and 3Department of Pediatric Immunology, Wilhelmina Children’s Hospital, University Medical Center, Utrecht, The Netherlands
C. 17 Infusion of adrenaline decreases serum levels of cortisol and 17hydroxy-progesterone in patients with rheumatoid arthritis but not in healthy subjects R.H. STRAUB1, J. KITTNER2, C. HEIJNEN3, M. SCHEDLOWSKI4, R.E. SCHMIDT2, and R. JACOBS2 This study was initiated in order to investigate the pituitary and adrenal hormone response after an intravenous adrenaline challenge in patients with rheumatoid arthritis (RA) and healthy subjects (HS). Fifteen untreated female RA patients (age: 51.5±3.2 yr) and 7 female HS (48.0±4.3 yr) were infused with adrenaline (0.05 mg/kg/min) for approx. 20 min. Plasma levels of adrenocorticotropic hormone (ACTH), and serum levels of cortisol, 17-hydroxyprogesterone (17OHP), and dehydroepiandrosterone sulfate (DHEAS) were analyzed at baseline and shortly after cessation of adrenaline infusion (20 min). At baseline and after adrenaline infusion, serum levels of cortisol (p=0.045) and 17OHP (p=0.021) were higher in healthy subjects as compared to RA patients. In contrast, at baseline and after adrenaline infusion, plasma levels of ACTH and serum levels of DHEAS were similar in HS as compared to RA patients. After adrenaline infusion, only patients with RA as compared to HS demonstrated a significant decrease of serum cortisol (p=0.026) and serum 17OHP (p=0.026). Plasma levels of ACTH (p=0.073) and serum levels of DHEAS (p=0.055) tended to decrease. This study demonstrates that serum cortisol and 17OHP (cortisol precursor) were lower in patients with RA as compared to HS despite similar ACTH levels. Furthermore, simulation of an adrenomedullary stress response by adrenaline infusion decreased serum cortisol and 17OHP only in RA patients but not in HS. Such a response may play an unfavorable role during a typical stress reaction in RA patients which may lead to a more proinflammatory situation.
44 · 32nd Annual Meeting of the German Society of Immunology 1
Department of Internal Medicine I, University Hospital, Regensburg, Germany and 2Orton Hospital, Invalid Foundation and 3Division of Rheumatology, Department of Medicine, Helsinki University Central Hospital, Helsinki, Finland
C. 18 Inadequately low serum levels of steroid hormones in relation to IL-6 and TNF in untreated patients with early rheumatoid arthritis and reactive arthritis R.H. STRAUB1, L. PAIMELA2, R. PELTOMAA3, J. SCHÖLMERICH1, and M. LEIRISALO-REPO3 In order to compare levels of steroid hormones in relation to cytokines and to study levels of cortisol or dehydroepiandrosterone (DHEA) in relation to other adrenal hormones, untreated patients with early rheumatoid arthritis (RA) and reactive arthritis (ReA) were compared to healthy controls. In a retrospective study with 34 patients with RA, 46 with ReA, and 112 healthy subjects (HS), we measured serum levels of interleukin (IL)-6, tumor necrosis factor (TNF), cortisol, 17OH-progesterone, androstenedione, DHEA, and DHEA sulfate (DHEAS). Patients with RA had higher serum levels of IL-6, TNF, cortisol, and DHEA as compared to ReA and HS but no difference was noticed with respect to DHEAS. However, in RA and ReA as compared to HS, serum levels of cortisol, androstenedione, DHEAS, and 17OH-progesterone were markedly lower in relation to IL-6 and TNF. Furthermore, the number of swollen joints inversely correlated with the ratio of serum cortisol/serum IL-6 in RA (RRank=-0.582, p=0.001) and, to a lesser extent, in ReA (RRank=-0.417, p=0.011). Furthermore, in these untreated patients with RA and ReA, there was a significant shift of hormone secretion from 17OH-progesterone, androstenedione, and DHEAS to DHEA and cortisol. This study indicates that cortisol is relatively low in relation to IL-6 and TNF in untreated patients with early RA and ReA as compared to HS. It further demonstrates that there is a hormone shift to DHEA and cortisol but not to DHEAS. This study emphasizes that adrenal steroid secretion is inadequately low in relation to inflammation. This is partly caused by an adrenal hormone shift from cortisol to DHEA. Although changes of hormone levels are similar in RA and ReA, alteration of the steroidogenesis is more pronounced in patients with RA as compared to ReA.
1
Department of Neurology and 2Section of Transplantation Immunology and Immunohematology, University of Tübingen, and 3EMC microcollections GmbH, Tübingen, Germany
C. 19 T cell reactivity towards myelin oligodendrocyte glycoprotein in multiple sclerosis R. WEISSERT1, J. KUHLE1, W. WIENHOLD1, C.A. MÜLLER2, K.-H. WIESMÜLLER3, and A. MELMS1 Multiple sclerosis is an inflammatory, demyelinating and neurodegenerative disease of the central nervous system. There is strong evidence for an autoimmune pathogenesis driven by myelin-specific T cells. Myelin-oligodendrocyte-glycoprotein induces in animals experimental autoimmune encephalomyelitis, a very multiple sclerosis-like disease with demyelinating central nervous sys-
32nd Annual Meeting of the German Society of Immunology · 45 tem lesions and axonal loss underscoring its potential role in multiple sclerosis pathogenesis. We performed a T cell reactivity pattern analysis of multiple sclerosis patients and controls that were stratified for the genetic risk factor HLA-DRB1*1501. We show for the first time that there is a promiscuous dominant T cell epitope within the intracellular part of myelin oligodendrocyte glycoprotein comprising amino acids 146–154. Surprisingly controls had broader T cell reactivity patterns towards myelin oligodendrocyte glycoprotein compared to multiple sclerosis patients and the trans-membrane and intracellular parts of myelin oligodendrocyte glycoprotein were much more immunogenic compared to the extracellular part. These data underscore that myelin oligodendrocyte glycoprotein is a target molecule in multiple sclerosis immunopathogenesis.
Department of Neuroimmunology, Max-Planck-Institute of Neurobiology, Martinsried, Germany
C. 20 Enhancement of experimental autoimmune encephalomyelitis by B cell transfer T. ZIEMSSEN, H. WEKERLE, and A. IGLESIAS Introduction: B cells seem to have a crucial role in the pathogenesis of myelin autoimmune disease. Methods: We transferred B cells from transgenic TH mice expressing a targeted heavy chain of the anti myelin oligodendrocyte glycoprotein (MOG) 8.18c5 antibody into mice with subclinical or mild experimental autoimmune encephalomyelitis (EAE). Using green fluorescent protein (GFP)- and TH double transgenic mice, we were able to follow simultaneously clinical disease, anti-MOG serum titer and trafficking of the transferred cells. Results: The transfer of LPS-activated, but not of resting TH or activated wild type B cells or the injection of large amounts of MOG-specific 8.18c5 antibodies lead to an exacerbated and accelerated form of EAE in mice treated to develop mild or subclinical EAE. In untreated mice activated TH B cells or MOG-specific antibodies have no effects. TH/GFP B cells peak at day 4 after transfer, when they appear predominantly in spleen and peripheral blood, but also in lymph node and bone marrow. 16 days post transfer, only few TH/GFP B cells are still detectable bearing mostly a plasmacytoid phenotype. MOG-specific IgM is detectable 1 day and peaks 4 days after transfer, while MOG-specific IgG appears at day 4 and its concentration raises continuously through days 16 and 29 after transfer. Thus transgenic TH B cells undergo isotype switch in recipient animals. However, there is no increase in the number of B cells in CNS infiltrates of TH transgenic and B cell transferred mice with exacerbated EAE. Instead, the large inflammatory infiltrates observed in these animals include increased numbers of cells of the myeloid lineage. Conclusions: MOG-specific B cells and antibodies are not pathogenic alone, but accelerate and exacerbate an ongoing mild EAE. We are currently investigating the possibility that MOGspecific antibodies mediate enhanced recruitment of monocytes and neutrophils into the inflamed site of the CNS.