25 June 1997- Posterpresentations Neuroimmunology 465 Materials and Methods: A&antibodies to brain tissue were tested by ELISA and immunoblot. Homogenized, washed and iysed cortex, subcortex and cerebellum were used as a source of antigens. Antigen-antibody interac- tion was detected using biotinyiated antibodies to rat ig, IgG, IgM and IgA and streptavidin iabelled by peroxidase. The reaction was visualized by enhanced chemiiuminiscence in immunobiot. Rwultr: Autoantibodies to brain tissue of ail three Ig classes are present in substantial levels in ail normal rat sera. The prevalence of IgM antibodies in ail cases indicates the natural, physiological character of antibodies. Brain antigens were separated to about 25 fractions by SDS-PAGE. Autonatibodies of normal rat sera reacted with some 16 of them. The majority of antibodies reacted with antigen fractions having mol. wt in the range of 35-70 kDa. The most prominent antibody specifii corresponded to the antigen fraction of 70 kDa. Further dominant antibodies reacted with antigens having approximal mol. wt. of 60, 50,45, 40 and 35 kDa. No differences between normal rat sera and sera of rats expcsed to short (h) anoxia were found. Conclusion: The antibody detection by ELISA can not reveal small quantita- tive changes because of the relative high levels of physiological autoantibodies, and does not infom about the possible changes in the spectrum of the an- tibody specificities. The detection of differences in antibody heterogeneity by immunoblot saems to be more suitable method for possible diagnosis of the damage of neural tissue. P.5.20.02 In vhto enhancement of cytotoxic activity of mouse peritoneal macrophages by Met-enkephalin U/ml of IFN-y significantly induced MHC class I expression, and this effect was potentiated by TNFa (50 @ml), which was by itself ineffective. To correlate MHC class I expression with the transcriptional activation of the gene, a qualita- tive analysis of MHC class I promoter binding factors was performed by EMSA. MHC class I molecule expression is transctiptionaiiy regulated through at least two critical enhancer sequences present in the promoter region: the MHCI regulatory element (MHC-CRE) and the MHC-IRF-E. Using the MHCI-IRF-E sequence, we observed that IFN-y was able to induce the IRF-1 complex; the combined treatment with IFN-y and TNFa was even more effective than IFN-y alone in inducing such a complex. Using the MHC-CRE regulatory sequence, we demonstrated that NFkB p65/p50 heterodimers were significantly induced after the incubation of OL with both IFN-y and TNFa, but not with TNFa alone. As IRF-1 acts as an intermediate transcription factor to activate the MHC class I gene, we also analyzed the expression of the IRF-1 gene, which is known to be regulated by the KB and GAS sites. We found that IFN-y induced the IRF-1 gene through the activation of GAS element by STAT-1 homodimer, whereas TNFa significantly induced the binding of NF-KB to the KB sites in the IRF-1 promoter only when the cells were pretreated with IFN-y. Conclusion: These results indite that in a puttfied population of OL IFN-y and TNF-a synergistically induce IRF-1 and MHC class I gene expression via activation of specific transcriptional factors. The fact that TNFa activates NFkB only following IFN-y treatment raises the possibility that OL express only low levels of TNFa receptors and that IFN-y upregulates TNFa recep- tor expression on these cells. This possibility is currently under investigation. The synergism demonstrated in the present study highlights the importance of cytokine interactions in magnifying biological effects consequent to brain injury and inflammation. Supported by the Research Project on Multiple Sclerosis of the Italian Min- istety of Health. J. Gabrilovac, D. Breljak, T. Balog, T. Marotti. Ruder BoSkoviC Institute, Department of Biology and Medicine, PO. Box 1016, 10001 Zagreb, Croatia Intmductlon: Met-enkephalin modulates several functions of macrophages in vitro by binding to opioid receptors expressed on them. However, there is a paucity of data on its ability to alter macrophage functions in viva in this study, we examined the effect of single systemic (i/p) injection of Met-enkephalin (MENK) into CBA mice on the cytotoxic activity and Ne secretion of peritoneal macrophages. lnvoivement of opioid receptors in the MENK action was tested by using opioid receptor antagonist, naloxone (Nx). Mate&Is and Methods: Peritoneal macrophams were collected 5 days after glycogen injection (0.5 ml of the 0.12% s&&n, i/p), and 12 hours a&r MENK (2.5 m&g) injection. Nx (2.5, 5 or IO m&g, i/p) was administered 20 - _. min bef&e MENK. C&toxic activity was determined by using L-929 ceils as targets. After a 24 hour incubation with various concentrations of macrophages, the proportion of viable L-929 ceils was determined by a calorimetric method, using WST-1. NO’ was determined by the method of Nasiund et al. (inf. Immun., 63: 1296,1995). Reeuk MENK significantly enhanced cytotoxic activity of peritoneal macrophages. This was associated with enhanced NO* production. MENK-induced effects were completely (NO’ release) or partly (cytotoxicity) reversed by naloxone (10 m#g). However, naioxone by itself acted as an ag- onist, enhancing macrophage cytotoxicity. Lower Nx doses (2.5 and 5 m@g) had lower agonistic activity, but were inactive with regard to the antagonistic ability. The necessity for a relatively high naioxone concentration for the rever- sion of the MENK-induced enhancement of macrophage cytotoxicity suggests involvement of 6 opioid receptors. Conclusions: In viw administration of MENK stimulates the cytotoxicity and release by peritoneal macrophages. Both effects could be at least partly reversed by naioxone in high concentration, suggesting involvement of S opioid receptors. Agonistic activity of Nx suggests tonic inhibitory role of endogenous opioid peptides on macrophage functions in viw. P.5.20.03 TNF-a and lFN+nediated signal transduction pathways: Effects on IRF-1 and MHC class I gene expression in oligodendrocytes C. Agresti, A. Bemardo, N. Del Russo, G. Marziali, A. Battistini, F. Aioisi, G. Levi, EM Coccia. Mtuto Sup&ore di Sanitd, Rome, ltaty Introduction: The role of cytokines in causing damage of myelin and oiigoden- drocytes during CNS inflammatory diseases, such as multiple sclerosis, is still poorly defined. We have recently shown that two pro-inflammatory cytokines (IFN-y and TNFa) synergize in inhibiting the proliferation and differentiation of oiigodendrocyte (OL) progenitor cells. To characterize the intracellular mecha- nisms that undertine the action of these two cytokines in OL, in the present study we analyzed the signal transduction pathways induced by IFN-y and TNFa by focusing on the regulation of MHC dass I expression in OL. Mate&Is and Methods: Purified oiigodendrocyte cultures were obtained from neonatal rat brains. The activation of transcriptional factors by IFN-y (STAT-l and IRF-1) and by TNFa (NF-KB) was analyzed in nuclear extracts utiiizino DNA eiectroohoretic mobiiitv shift assav (EMSA). I.. . 1 P 5 20 04 Effect of interferon beta treatment on the TNF-a and IFNy expression In multlple sclerosis patients A. Gayo, L. Mozo, A. Sulrez, A. TuA6n, C. Lahoz, C. GutiBrrez. Department of Immunolqy and Department of Neurolog)! Hospital Central de Asturias, Universidad de Oviedo, Spain Multiple sclerosis (MS) is a neurological disease of the central netvous system associated with a dysregulation of cytokine expression. Treatment with IFN-p lessens the overall frequency of MS attacks in patients with relapsing-remitting multiple sderosis (RRMS). The aim of our study was to analyze the effect of IFN-@ treatment on the expression of the proinflammatory cytokines TNF-(r e IFN-y-in RRMS patients. Samples were obtained from sixteen patients before and after three and six months of a subcutaneus administration of 6 MU of IFN-fl. We anaiized the TNF-crand IFN-y mRNA levels in PBMC by RT-PCR. PCR products were mea- sured by Southern blot hybridization with an internal 32P-labeiedoligonucieotide and the signal quantified by densitometric scanning. Serum levels and protein concentration induced by PHA + PMA were determined by ELISA. Treatment with IFN-B resulted in a decreased mRNA expression of TNFa and IFN-y, although in this last case the results were not statistically significant. Protein levels of these cytokines in serum before and after IFN-fi treatment were below the detection limit of the ELISA assay. Approximately half of these patients responded to PHA + PMA stimulation in vitm by producing high amounts of TNFa and IFN-)I which did not change after IFN-,¶treatment. However, the rest of the patients, who showed a slight response to mitogen stimulation before treatment, secreted large amounts of TNF-cr and IFN-y after three and six months of treatment with IFN-p. Taken together these data indicate that IFN-p could exert its benefidai effect in viw by modulating the spontaneous expression of proinflammatory cytokines. The mitogen induced increased production of these molecules in a group of patients may be due to a synergistic effect between IFN+J and other factors induced by PHA + PMA. 1 P.5.20.05 ) Differential susceptibility of young and aged rat muscle to antibcdy-mediated AChR degradation in experimental autolmmune myasthenia gravis (EAMG) A. Hoedemaekers, J. Bessereau ‘, Y. Graus’, T. Guyon 3, J. Changeux ‘, S. Berrih-Aknin 3, P. van Breda Vdesman, M. De Baets. Dept. of Immunok)g~ Maastricht Univenitv. PO. Box 616, 6200 MD Maastricht. The Netherland& ’ lnstitut Pasteur. L&i& de Neurobi&gie Moleculaire, 25 Rue du DI. Roux, 75724 Paris Cedex 15, France, ’ The Netherlands Cancer Institute, Dept. of lmmunolw, Plesmanlaan 121, 1066 CXAmstetdam, The Nelherlands, 3 H8pital Marie Lannelongue, 133 Avenue de la RBsistance, 92350 Le Plessis Robinson, France R&ults: in bashi culture conditions OL -db not express MHC class I molecules, as shown by immunocytcchemistry. incubation of OL with l-100 Introduction: Passive transfer EAMG is an excellent model to study the re- sponse of the acetyichoiine receptor (AChR) to the antibody-mediated immune