Expression of MHC class II and costimulatory molecules by endothelial cells of the blood brain barrier: Effect on antigen presentation

Poster Abstracts / Journal of Neuroimmunology 90 (1998) 13-105

95 C~4[~1 but not cq[~ is Critically Involved in the Development of EAE in the SJL/J Mouse: Evidence for a Role in T Cell Migration and Activation B. EngelhardtM. Sehulz,Max-Pland¢-InsmutfurKlinischeu.PhysiologischeForschung, Germany, U . Sam ulowitz, J. Vestwebeg, ZMBE, University ofMunsk, Germany, G. Hoeh, Max-Planck-lnstitutfur Klini,¢he u. Physiologische Forschung, Germany

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98 The Anaphylatoxin Receptor for Complement C3a is Expressed by Rat Mieroglia and Rat Oligodendrocytes. Role in Cell Chemotaxis and Cell Activation. P. Gagque, S.K. Singhrao, J.W. Neal, UWCM,U.K., J.D. Sedgwick, Centenary Institute of CancerMedicine & CellBiolo~, Australia

Based on the observation that the application of monoclonal antibodies (mAbs) directed against ct4-integrin and VCAM-I inhibit the development of experimental autoimmune encephalomyelitis (EAE) in vivo it has been concluded that the successful therapeutic effect is due to interference of the mAbs with aal]I/VCAM-I mediated interaction of autoaggressive T ceils with the blood-brain barrier (BBB). However. a possible role of ~x4[~7-integrin. which also binds to VCAM-I, or interference with other T cell mediated events during the pathogenesis of EAE has not been considered. We have compared the effects of mAb therapy on the development of EAE in the SJL/J mouse, using a large panel of mAbs directed against the ct4-subunit, the l~l-subunit, the !~7-subunit, the a4ta7-integrin and against VCAM-I, Although encephalitogenic T cells express both ct4-integrins, mAbs directed against ct,q~7or the flT-suhunit did not intertere with the development of EAE. In contrast, mAbs directed against a4 and VCAM-I clearly inhibited or diminished clinical and histopathological signs of EAE. Therefore, ct41~lbut not ct4137is critically involved in the development of EAE. Furthermore, our in vitro studies suggest that the therapeutic effect of anti-a4-treatment of EAE might not merely be due to inhibition of asI~I/VCAM-I mediated T cell interaction with the BBB. The possible influence of anti-ct4-treatment on T cell function in EAE will be discussed.

C3a is an i m p o r t a n t c o m p o n e n t of the innate immune s y s t e m r e l e a s e d in the v i c i n i t y of the i n f l a m m a t o r y site after a c t i v a t i o n of t h e c o m p l e m e n t system. C 3 a m e d i a t e s its a c t i v i t y b y a t t r a c t i n g [i.e., chemotaxis) and a c t i v a t i n g phagocytes to p r o d u c e c y t o k i n e s and chemokines. Rat a s t r o c y t e s express the C 3 a R (H.Akatsu and P. Gasque) but l i t t l e is k n o w n a b o u t the e x p r e s s i o n and the role of C 3 a R on o t h e r glial cella a n d neurones. We h e r e report that rat microglia and oligodendrocytes express c o n s t i t u t i v e l y C3aR.FACS and double immunofluorescence revealed that m o r e t h a n 95% of C D l l b / c + cells and o n l y 40-70% of GC+ ceils w e r e e x p r e s s i n g C3aR. E x p r e s s i o n of C 3 a R w a s c o n f i r m e d b y w e s t e r n b l o t a n a l y s i s of cell lysate. M l c r o g l i a w e r e s o r t e d on the basis of their cell s c a t t e r and t h e i r relative e x p r e s s i o n of C D 4 5 and C D l l b / c a n d total R N A w e r e p r e p a r e d for RTPCR. C 3 a R m R N A was e x p r e s s e d c o n s t i t u t i v e l y b y m i c r o g l i a and the i d e n t i t y of the c D N A w a s c o n f i r m e d b y s e q u e n c i n g and southern blot. The effect of C3a on mlcroglia and o l ± g o d e n d r o c y t e has b e e n assessed b y c a l c i u m f l u x and c h e m o t a x i s experiments using recombinant C3a and agonist C3a peptide. In corltrol rat brains, C3aK was charact~rised on microglia, astrecytes, o l i g o d e n d r o c y t e s and few neurones. C3a plays c e r t a i n l y an impertant role in brain inflammation by recruiting and a c t i v a t i n g glial cells.

96 The Role of Antigen Specificity in Induction of T CellAccumulation in the CNS Z. Fabry.Z. Qing,M.N. Hart,M. Sandor,University of Wisconsin, USA

99 Expression of MHC Class II and Costimulatory Molecules by Endothelial Cells of the Blood Brain Barrier: Effect onAntigen Presentation A.M.Girvin.K.B.Gordon,L. Tan,NorthwesternUmversityMedicaISchool,USA, J. Welsh, TexasA& M University, USA, S.D. Miller, NorthwesternUniversityMedical

School, USA The influx of T lymphocytes into the CNS is an important pan of the pathology of various diseases which are associated with inflammation in the brain. The early phase of the inflammatory reaction involves the accumulation of activated, antigen specific T ceils. Inflammatory mediators produced by these T cells contribute to the influx of ,on-antigen specific leukocytes. The goal of this work is to understand the em'ly phase of CNS inflammatory reactions by studying the role of antigen specific T cells in the initiation of CNS inflammation. In order to understand the mechanism of T cefi infiltration into the CNS we have developed an antigen specific T cell infiltration assay. Because of the difficulties in physically tracking a small number of antigen specific T cells in a normal animal, we are applying transgenic strategies involving T cell receptor (TCR) transgenic animals and their respective antigen. TCR transgenic mice expressing Vctl I/V[33 T cell receptor specific for pigeon cytochrome c (PCC) residues 88-104 in the context of I-Ek MHC class 11 molecules were applied. Intraperitoneal and intraventncular injections of antigen were compared and it was established that intraventricalar injection of antigen results in a higher number of antigen specific T cell accumulating in the CNS. Our data suggest that: I.) The presence of antigen in the CNS induces strong infiltration of antigen specific T cells into the CNS; 2.) The antigen specific T cell infiltration assay will be suitable for finding new ways to intertere with T cell infiltration in the CNS.

Cells comprising the blood brain barrier (BBB), including astrocytcs and cerebrowlscular endothelial cells (CVE's), contribute to a relatively inflammation-l?ee environment during health, and also may play a role in the inflammation of the central nervous system ICNS) in response to infection or induced autoimmune disease. Previously. we have shown that IFN¥ activated SJL/J astrocytes upregulate MHC class II and B7-1 costimulatory molecules and are capable of processing and presenting antigen to both narye and memory T cells. Ille antigen presenting function and role of CVE's in regulating CNS inflammatory responses remains controversial We show that SJI.,/J CVE's upregutate MHC class 11 and B7-1 costimulatory molecules in response to [FNy. llowever, the addition of TNFct to SJLH CVE's, but not astrocytes. decreased the IFNy induced MHC class 11 expression and upregulated ICAM-I expression. The differential expression of MttC class 11 between CVE's and astrocytes is regulated at the level of the class I1 transactivator (CIITA). Also, we have lound that while CVE's constitutively express B7-1 on their surfaces, they are unable to [Unction as antigen presenting cells (APC's) when MHC class 11 is upregulated by IFNy. In Ihct. they appear to induce peptide-specitlc unresponsiveness. Our lindings suggest that in the absence of local inflammation associated with fNF(z production, CVE's may actively downregu[ate T cell responses in order to protect the CNS from inflammatory damage.

97 Encephalitogenie T Cell CC Chemokine Receptor Expression During the Pathogenesis of Autoimmune Encephalomyelitis

100 Lymphocyte Migration through CNS Endothelial Monolayers is Facilitated by an Endothelial Pertussis Toxin Sensitive Pathway

B.T. Fife, W.J, Karpus,Northwestern University, USA

L Greenwood. Inst. of Ophthalmology, London, UK, E-O. Couraud, 1net. Cochinde Gen Molec.,Paris, France, W. Calder, P. Adamson, Inst. of Ophthalmology. London, UK

Experimental autoimmune encephalomyelitis (EAE) is a CD4 ÷ T h l mediated demyetinating disease o f the C N S that serves as a model for the human disease multiple sclerosis. We have previously reported the important role of two CC chemokines, macrophage inflammatory protein l-ct (MIP-Ic0 and monocyte chemotactie protein-I ( M C P - I ) in the pathogenesis of EAE. MIP-I ct, but not MCP-t was found to correlate with the development of severe acute clinical disease. In the present study we investigated the expression of CC chemokine receptor ( CCR) expression on T cells and macrophages from the C N S , peripheral blood, and spleen during acute clinical disease and compared this expression to that on naive lymph node and splenic T cells. T cells from PLP139-151 primed mice showed CCR2 and CCR3 expression. CCRI expression on these T h y l . l + T cells was detected after in vitro antigen restimulation [)rior to adoptive transfer. F o l l o w i n g development o f acute EAE, Thy I.I + C D 4 T cells isolated from the C N S o f Thy 1.2 + recipients also showed CCR1 expression. F4/80 ÷ macrophages isolated from the spleen and C N S o f these animals d u r i n g acute E A E expressed CCR1, 2, 3, and 4. However, F4/80 + resident microgtia from the C N S o f na~'ve animals did not express CC chemokine receptors. Taken together, these data suggest a rote for chemokine receptor expression during the pathogenesis o f EAE. (Supported by N I H NS34510).

Endothelial cells (EC) of the CNS are fwraly bound together by complex fight jua,ctions which confers on the vasanlatum a unique anatomical barrier to migrating lymphocytes. Thus, for transvuscalur migration to occur at this site, lymphocytes must either breach the tight junctions or penetrate through the EC by other unresolved mechanisms. Although lymphocyte migration is largely governed by the activation state of the lymphocyte, it is increasingly clear that the EC plays an active part in the process of diapedesis. We have investigated the EC processes controlling lymphocyte migration across monolayers of mt brain EC. Endothdia derived from rat brain, aorta and high endothelial venule~ (HEV) were treated with pertussis toxin (PTX)which results in the ADP-ribosylation and inactivatien of specific EC G-protein alpha-subunita. Following extensive washing the monolayers were then used for lymphocyte adhesion attd migration assays. Pre-treatmant of CNS EC monolayers with PTX resulted in an 80% reduction of lymphocyte migration without affecting edhesian. A mutant form of PTX which is catalytically inactive had no significant effect on lymphocyte migration. PTX treatment of aortic and HEV EC resulted in a reduction in migration of 20% and 30% respectively. These data suggest that there is a PTX-sensitive mechanism operating within EC which facilif.ates lymphocyte migration and that brain EC supported lymphocyte diapdesis is more dependent on this pathway that, peripheral vascular endothelia_