TNF receptors shedding in multiple sclerosis patients

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P14.08

W08.03

ORGAN SPECIFICI~IXYrHELIAL CELL ~ ( X i l / ~ ' q ~ ' Y MAY REGULATECNS TROI~k( OF DIFFn~ENT STRAINSOF MOUSE I~PA'ITrls VIRUS (M~,'). J.Jol~h, R.Kim, K.$inbect, F.D, Leblin, C.Offeabech and R,L. Kaoble¢, Diviaion of

EXPRESSION OF PREPRO-ENKEPHALIN (PPE) mRNA PERIPHERAL BLOOD MONONUCLEAR CELLB

Neuroimmtmolosy,Deplutlmmtof Neg~-olosy,Jeffea~nMedicalCollege,lPailed¢lphht,PA 19107,USA. IntrodactLou: E~uthelial cdin form an ~ bctweea circulating blood mul orSmm and thus could ~¢v©Ill • barrkwto inl~utinllby virgins, Badothofial ¢al15are also I~t¢log¢l~oalin

J.S. Kamohuis. H. Eschar, A. Kavelsere, W. Kui= and C.J. Heijnan Dept. of Immunology, University Hospital for Children and Youth 'Hat Wilhelmina Kinderziekenhuis', Utrecht, The Netherlands

d i f f ~ t o r p a l and may exhinit Ideutlvityin permitting viral elltry, thul inflaea¢ing their fiche tropiua. ~ : la olrder to itudy the role of eaduth~ial cells in rogullKiag viral tropisms prima~ c ~ of _t~f~t~- mduthallal ¢~ll, ~ ~ cerabral eaduthdicl cells de¢ived from MHV mmceptibin SALVe rake wet-e iafe¢~l with MHV-3 or MEW-4 at an MOI of O. 1. Supefnatout~from iaf~tod ¢~altoreswet~ ¢ol|ectod, for performin a phtqoe ss~ys,at 0, 24, 4S, 72 sad 96 hotws ~ iaf~tioa. Cytol~thk effectl w~¢ aim monitored st those time points. Rmmlts: H ~ t t k mdetholial =dis ~ fotmd to m~t~rt the roplicatinu of the hepatutropk MHV3 but not the aeurOUelpicMltV-4 (2.9 x 105 WU/ml for MHV-3 vs. 0 EFU/ml for MllV-4 at 24 hours p~t infeutina). MHV-4 mplkution w u not obl~rved in hepatic e~dutheJialc#Jlsat uy of tho time point~ examined, whil© th© MHV-3 tlte~lpe'~ked ut 48 hours/poet infection(3.2 x I0~ P]FUIml). Cynic effectswere aico oblefved in hepatic ~sdutheSal ceils u early as 24 hours altar infectina with MHV-3 but not blHV-4. In contrut to the fmdinp for hepatic eadothalial cells, hqmtocytm were able to suplportthereplication of both MItV-3 and MHV-4. Cytoputhic ©ffeutlw~reevideat at 48 hour* after infection with MHV-3 and MHV-4. Gurabral cedothclicl c4dls demom~-atcd • delayed spp~mm~ of cytopsthic effecus following infcutinn with MHV-3, whe~l compared to hop=tic¢=lotkaliclcells. Furthermore, cerabral endothelial cells showed cytopathic effects and replicated virus following infection with the neurotropic MHV-4, unlike the hepatic eatdothellel cells. ~on¢lusion: Thoae results suggest that the e~dothdial cedisbut not hepatocytu, demonstrato differ~mtinlresponses to MHV-4 aud MHV-3 infection. I~dotheJielcellhetorol~meltythuscould play an important role in regulutln s organ tropism of MHV-3 and MHV-4.

W17.04

R E G U L A T I O N O F T H E M E T A B O L I S M OF I N F L A M M A T O R Y M E D I A T O R S IN BRAIN-DERIVED ENDOTHELIAL CELLS L. Juillesat-Jearmeret Division of Neuropathology and Division of Pneumology, University of Lausanne, Lausanne, Switzerland Introduction. Activation of endothelial cells of most organs is regulated by inflammatory mediators and is involved in the adhesion and migration of hloodborne cells into tissues. As brain-derived endothelial cells (bEC) forming the blond-brain barrier express particular properties and enzyme systems in comparison with endothelial cells of other origin, we have analyzed in bEC the mechanisms involved in the regulation of secretion of nitric oxide and interleuldn-6, and of the expression of peptidases involved in the metabolism of vasuactive hormones regulating vascular permeability and tone. Methods. bEC were grown in tissue culture and exposed to cellular effectors. N O and interleuldn-6 were deterrmned in cell culture supernatants. Enzymatic activities were measured in cell extracts. Results. Cytokines, cAMP and LPS increased N O release and N O synthase activity in bEC, which were down-regulated by dexamethasune. Bioactive interleukin-6 was secreted exclusively as a 25 kD peptide and its secretion in bEC was increased by cAMP and a serine-prntease inhibitor, but not LPS. and decreased by dexamethasone. Dexaarethasone, but not cAMP, spocificaUy regulated the expression of aminopoptidases, aminodipoptidase and eathoxydipeptidase in bEC. Conclusion. These results demonstrate that the release and metabolism of inflammatcry mediators in bEC are under the control of cAMP and glucocorticoid hormones and involve the regulation o f specific enzymes expressed by these cells.

WO4.O4 "BIF RECEPTORSSHEOOING IN NULTIPI~ SCLEROSIS PATIENTS A.3urewicz, A.Walczak, Z.Nowak, K.Selmaj L a b o r a t o r y o f Neurotrmunoiogy, Department o f Neurology, H e d i c a l Academy o f Lodz, Poland. I n t r o d u c t i o n = I n t h i s study we e v a l u a t e d the g e n e r a t i o n o f TNF i n h i b i t o r s i n HS p a t i e n t s . H a t e r t a l and methods=The INF-Rs shedding from u n s t t m u l a ted and PHA-,LPS-,HBP-stlmulated p e r i p h e r a l blood moann u c l e a r cells(PBMC) was assessed by measuring l e v e l s o f sTNF-Rs w i t h monoclonal a n t i b o d i e s a g a i n s t sTNF-Rp55 and sTNF-Rp75 and ELISA technique. Results:PBHC o f c h r o n i c p r o g r e s s i v e HS p a t i e n t s and H5 p a t i e n t s i n r e l a p s e generated spontaneously h i g h e r l e v e I s o f sTNF-Rp75(Z,4 and 1,Sng/ml r e s p e c t i v e l y ) than PBHC o f HS p a t i e n t s In r m a l s s l o n ( 1 , 0 n g / m l ) and a l l o f them have h i g h e r I e v e l s In comparisOn w i t h c o n t r o l PBNC(0,4ng/ml). S i m i l a r l y PHA-sttmulated c o n t r o l PBHC generated lower amounts o f sTNF-Rp75(2,4ng/ml) than PBNC o f MS p a t i e n t s i n reladse(4,4ng/ml).PHA-tnduced PBHC o f c h r o n i c p r o g r e s s i v e H5 o a t t e n t s and HS p a t i e n t s I n remission generated sTNF-Rp75 at comparable l e v e l to c o n t r o l PSHC.However, the r a t i o PHA-induced/spontaneous sTNF-Rp75 g e n e r a t i o n was s i g n i f i c a n t l y h i g h e r in c o n t r o l PBHC(Sx) than i n k15 g r o u p s ( 1 , 5 - 2 , 9 ) . D i f f e r e n c e s i n shedding o f sTNF-Rp55 were i n s i g n i f i c a n t i n a l l I n v e s t i g a t e d groups. Conclusion:Disturbances i n shedding o f TNF-Rp75 occur in H5 p a t i e n t s .

IN

HUMAN

In recent years, it has been shown that apart from the neuro-andocrine system, the immune system is also capable of producing neuropagtides. It is known that opioids are produced at the site of inflammation in rats with arthritis. Peripheral nerve endings in the joint express opioid receptors. Moreover, opioid receptors are slao exprelaed on cells of the immune system. This leads to the assumption that production of these enkephalins at the site of the inflammatory reaction can modulate pain perception as well as immunologic responses. We have recently shown that human peripheral blood mononuclear cells (PBMC) can secrete the precursor protein (proenkephalin) as well as the processed pentapaptida met-anksphalin. In the present study we investigated the regulation of mRNA of PPE in human PBMC by PCR and Southern Blot analysis. Our data show that stimuli such as the nitric oxide donor sodium nitro prusaide can induce mRNA of PPE expression. Moreover we could show that various cytokines, like the inhibiting cytokine IL-4, are capable of inducing PPE mRNA expression. The latter finding suggests a possible anti-inflammatory function of enkephalins produced at the site of inflammation, since they are induced by inhibiting mediators of the immune system.

Wll .01 PREVENTION OF TMEV-INDUCED DEMYELINATING DISEASE BY IN VIVO TOLERANCE INDUCTION. William J. Kamus. Jeffrev D. Paterson. Jonathan G. Pone. and Stanhen D. Miller. Northwastam University Medical School, Chicago, IL USA 60611. Theiler's murine encephalomyelitis virus (TMEV) induces an immune mediated, chronic demyelinating disease of the central nervous system (CNS) charactenzed by mononuclear ceil infiltration that appears to correlate with the development of virus-specific delayed type hypersensitivity (DTH) in the absence of neuroantigen-specific autoimmune responses. The result is bystander demyelination and development of clinical paralysis. TMEV-specific tolerance induction effectively induces a inhibition of TMEV-specific DTH, T cell proliferation, IL-2 and IFN-7 secretion, and Thl (IL-2, IFN-¥, and LT/'I'NF-I~) but not Th2 cytokine gane expression (IL-4 and 11--6). Moreover, tolerance induction results in the decrease of virus-specific IgG2a, but an increase in virus-specific IgG1. Treatment of SJL/J mice with a single administration of TMEV-couplad spleen cells prior to infection significantly delays the onset of symptoms, decreases disease incidence and clinical severity. Moreover, tolerance induction significantly reduces the number of CD4+IL-2R+ T cells and MOMA + macrophages that infiltrate into the CNS. These data suggest that in vivo tolerance induction prevents clinical paralysis by anergizing virusspecific Thl responses and preventing the infiltration of activated CD4 + T cells into the CNS, while not affecting virus-specific Th2 responses.

W15.06 PREVENTION AND TREATMENT OF PLP PEPTIDE-INDUCED EAE BY ANTI-MIP-lot ADMINISTRATION William J. Karmm. Iqichobm W. Lukec& Bradford L. McRaa. Flobert M. ~rkller. Slewn L. Kunkai. and Stanhen D, Miller. Northwestern University Medical School, Chicago, IL 60611 and University of Michigan, Ann Arbor, MI 48109. Experimental autoimmune encephalomyelitis (EAE) is a T celtmediated, inflammatory demyelinating disease of the central nervous system (CNS) that servas all a model for the human deelyaiinatlng disease, mulliple sclerce~. We and ofhere have demonairated that a relapsing-remitting form of EAE can be induced in SJL/J n'dce by the transfer of PLP139.151-specific T cells. In the present report we investigated the role of the chemokine MIP-la in development of EAE. Groups of SJL mice were given PLPf39.151-speciflc T cells and the recipients were treated with either anti-MIP-l~t or normal rabbit serum concomitant with T cell tmsefer. Mice that received antkMIP-lot failed to develop EAE whereas control-treated mice developed eevere EAE. In vitro treatment of PLP139.151 T cells with antI-MIP-lot did not abrogate the ability to transfer EAE. Moreover,/n vffro neutraltzaRlionof MIP-la did not have an effect on the expre~don of IL-2, IL-4, IFN% or TNF-ot production. Administration of anti-MIP-la after the onset of clinical EAE decreased the severity of paralyt~ relative to control treatment. These results auggset that MIP-lot plays an important role in the recruitment of inflammatory cells into the CNS.