- Email: [email protected]
Phenotypic and functional characterization of rat lipid-bound MBP specific T-cell lines
181
P17.10 TEIE ROLE OF CELL ADEIESION MOLECULES IN LYMPHOCYTE ACTIVATION AND HOMING, Di Manro. M . E . J G leanle, M J . =. & Alger, A . 1, . . 'tmmunotog~ Keseas~ ufonp ~:CID), university ot Mancaester, Manchester M 13 9PT, U.K.:-'Tenovus Research LaBoratory, Smithampton SO9 4XY UK. lntrodoetion: Resting lymphccytes preferentially recirculate via the blood and lymphaties,..migr.~ing @ ~ [~mpfi. nodes . ~ . spleen for efficient ..a.ntigen encounter. Ponow'ing activation, lympbccy~. display distinct migration pamways ("homing') ~ to their into of activation. Activated lymphocyt~ also migrate to. non-.lymphoid organs and sites of inflamn'~tiun, enstiring efficient antigen elimination. The rrrrrrrrr~or mechanisms involved are, as yet, unclear but appoat to be altered upon lymphocyte activation and by the acuon of cytokines on endothelial ceils. Materials and Methods: Rat ~ h . o c y t e . s were activated in vitro using a CD2/¢~I'CR bi~ecifie .agtibefly (usAe) consisting of ehemi.cal,ly crosslinked I~b: f r ~ (no Pc region), m^wgeneratod blasts were usea in an in vitro mooe£ of'lymphocyte adhesion to aria migration across HEV. Adhesion blocking exj~'iments were carried out by pre-mcubating the lynwhocytes (4"C; 30rain') wfth mAbs agalust CDI la, CDfS, ICAM-1, VLA-4, CD'4, CD8, MHC Class II and CD45 (control). R e s u l t : The BsAb stimniated the proliferation of mlsep..era~d l y . ~ h node cells (LNC.) "m. a dose- and t ~ F l m M c n t manner. The "BsAb dla not stimulate proliIermion of M~hly purified'popal.ations of T cells - indicatinR a role for ~,'~__~_,~,'y celia or factors in its action. Inclusion of antibodies againsfCD 1 la and CD 18 we~ f.cund to inhibi(BsAb:.in@ced T cell proliferation in a d u s e - d ~ d ~ t manner, while also inbibitmLcel[ 91uste"+r'+mg.A .g.r~ter . l ~ n t a g e ot activat~ than of rest'rag lympbocytos ~ to high ['~domelial cells (HEC). None of the antibodies lisfed abbve blocked lymphocy'~e adhesion to HEC. Conclualan: Crosalinked antibodies to the aSTCR and CD2 are not sufficient to induce T cell proliferation. LFA-1 recq~ition between T lymphocytes and accessory cells ts also .Rquired. The re~,ul~ting systems involveTd in lymphocyte adhesion to and migration across H ~ are, as yet, unclear, but die receptor mechanisms do appear to be altered upon lymphocyte activation.
P03.12 CHANGES IN CELLUIJ~ DISPOSITION OF LIPOCORTIN-1 (ANNEXIN1) IN RAT PRIMARY .a~TROCYTE CULTURE8 AND C$ GLIOMA CELI..S AFTER EXPOSURE TO DEXAMETH/~ONE. J.D. McLeod and C. BoRon.
Phan~scology Dept., School of Pben'nacy & Pharmacology, University of Bath, Clsverton Down, Bath BA2 7AY, England. Introducllon: Astmcytes have been implicated in the immunological processes underlying the pathogenesis of Multiple Sclerosis (MS). The severity of MS can be regulated by glucocorticold steroids, an effect which may be mediated by the induction of an intracallulsr protein, lipocodin-1 (LC-1). The current study investigated whether astrocytes respond to dexamethasone (Dex.) by Increasing LC-1 expression. Methods: Pdmary astmcyte cultures and C6 glioma cells were exposed to Dex. (10nM-100nM) for different time pedods. LC-1 was extracted from separate call fractions; cell-suffsca, membrane and intracellular. The LC-1 content associated with each fraction was detected using electrophoresis and immunoblotting techniques. Results: In pdmary sstrocyte cultures, Dex. induced a nse in intraonllular LC-1 which was translocsted to the call membrane. Changes in intracallular and membrane-associated LC-1 also occurred in C6 cells. However, unlike primary cultures, LC-1 was further extemalised to the cell-surface. Conclusion: The involvemen~ of LC-1 in the actions of glucocorticoids on rat astrocytes may be responsible, in part, for the therapeutic effects of steroids in animal and human demyelinating diseases.
P10.04
P17.11
PHENOI'YPIC A%D FIN('TI()NAI. CHARA('I'ERI/~110 ~', OF RAI" LIPID-BO! ND MBP SPE(!I FIC [-('El.l. L IN VS.
INCREASED SOLUBLE VCAM-I LEVELS IN CEREBROSPINAL FLUID OF PATIENTS WITH MULTIPLE SCLEROSIS.
[3 Ma.~anti. ',I, \'~rz~lh. ,~. ~'a]l~:iJ.+. : ,=:.=;. ~
S.A.McMillsn I, J.P.Dougls# A.G.Droogeu 2 and S.A.Hawkins 2 Regional Immunology Laboratory, Belfast City HespiteP and Neurology Department, Royal Vietoda HospiteF, Belfast, Northern Ireland.
.~-~ii+ ~ ..~.,::d ......
[,lf+'o(lu(+ion ':+ :t D72VlOt,~ ~ttrcl} ,~ e h,+,+., cl,,e: +c' + ' + +' - : ' .:.++x'+T~. +~+"I t].+v ['~+~;+!,+t;,:+ ~'~,i ',i:, ,.. ?. :: ,~ i
:*:+ + x !"
+lCx+x Hr+t+++tt Nho+ t+PII Itltd~ geller+~ted t~om l x-~+l++tl~,~+~ ,.-++]Jccte(t at the tdt):+til.+H r++~,p,,:tsd t,? [ _ B - ' ~ F P b!tt +~+,:t tJ [. +'~[l~']: ~+IPl|lod~, ~: o ; I + : +o ]+,~tt+i ~+]l,tthCtCltL+ the illllltt~lh~etii~ l>r,>l)ert+e+ ,st ;ill+ almmet+ u,+' geJierated + +-NI~'++: -1~+¢I++C t+ce+! IHte~ fit+n+ [,~-M~+l ) ltttrt+tllVZ+d HIIIIIIlJ- -+eltUIg 5Pl<+;tt+t;tlCle,tl c+qb~ u+ ]inlitffl~2 dfll+tl~m ac~o:!tmg re, +~-t? t'reQtlJ+!c +` ,+( \+F+P ~[!J~.'][i~ i~IgC!II;'+F'~. resulls: the ,m+til~:rati,.',~':~p.+n:,e , f th,: :n,O,,J~ty or" I[tCs+
{m,~s v+a~ lughJ,. ~p~ctfiu Io LB-MB[~ (A~ d~s~ O i u~ lid, :~ztJl I,) c~,s~reaol~n~+ to I,F-\,IISP .gtltomated ¢ ' ~ , t l t l o n ! t l ¢ l r l ¢ anaIy~in ~,t: these Iinc~ ~!, ,,~ z!(l :¢ ( ' D I "
]~}!erlcfl'}"Dc
!,F~-NI}~P ..:;3eclt}c fir*e:~[! ]Itle~ did nc,i tee.Dot/} h~
t~,J ilIIlllUJlc~'d~qllilld,*llepltope or" '~!BP !p~F+t:de -2-S:% ',.,~ ~he IAiRTI, h.g,'L.'i~.l:'.:,'f Ih,: t,cwi, :at. ,~[tcle+t+ th+ b]lc~ >[~ecilic ;:r th,~ ";~l[lt+ tl'~Jlll) i~' :~,th It- told Lt +NIE:+ did r.+.,pund tu thl+ [),:[)+tdd ('OIK'lUXiOll Ii;~> tlll+t~talil Pitt,: +pccl|Jctt~. ]]ldl+tltcN I}tilt ~lt~+' wi~en +mi,eddcd t;I [iI'+dm md~:ce, a n~. x~r ?all,;r+l of, -+¢[[ ['c,-,poil>,:probabP< bccut:sc ;+["(Ii[]'~F,2Ii T c[~:i~2]! ~?'t+.2.:~SlT[g ~;;ttl pIcSellt,Jl],,ll i:} :\P("
Introduction: Increased levels of circulating adhesion molecules have beea found in many inflammatory diseases. As multiple sclerosis (MS) is an inflammatory disorder levels of these molecules may give a valuable insight into the vascular pathogenesis and therefore diagnusia of this disease. Materials and Methods: Serum and corebruspinal fluid levels o f soluble ICAM1, sE-selectin (ELAM) aad sVCAM-1 wore determined by ELISA in patients prior to a confirmed neurological diagnosis. 45 patiente were later dlasnused as MS, 23 as inflammatory (IND) and 37 as non-inflammatory neurological disease (NIND). Results: Significantly higher levels of sVCAM in CSF (mean values 18.1 ng/ml : 12.1 ng/ml; p = 0,001) and lower levels of sELAM in serum (mean values 45.5 ng/mh 55.0 ng/ml; p=0.03) were detected in MS patients compared with the non MS group. In the MS patients there was a c o n ~ t i n n between levels of sICAM in the CSF and serum and betwee~ sICAM and sVCAM in the CSF. There was also an association between pusitivity for oligoclonal bands and high CSF levels of slCAM and sVCAM in die MS group (p=0.01 and 0.004 respectively), this association was not seen with other indicators of local CNS immunogiobulin production. Conclusion: Our findings suggests that an interaction between brain endothelium and lymphocytes may be an important factor in the pathogenesis of MS.
P10.05
P14.10
T CELLS AND MICROGUA IN CHRONIC RELAPSING EAE: EVIDENCL THAT T CELLS ARE CD45RC- AND THAT MICROGLIA PROLIFERATE
ACUTE PHASE REACTANTS (APR) IN SERA FROM OLDER PERSONS WITH DOWN SYNDROME (DS) AND AL.ZHEIMER DISEASE (AD),
P.A. McCombe, J. de Jersey, M.P. Ponder Dept. of M~llclne, The Unlverolty of Queensland, Brisbane, Australia Introduction: CD45RC expression by T cells correlates with Thl-like function, but is lost on activation. The majority of the T cells in the spinal cord in Lewis rats with acute EAE are CD45RC-. We have now assessed CD45RC expression by T cells in the apinal cord during chronic relapsing EAE (CR-EAE). We also studied mlcrogMa In CR-EAE. Msterlele and methods: CR-EAE was induced by inoculation of Lewis rats with guinea pig spinal cord and vestment with low dose cyclosporin A (CsA). Cells extracted from the spinei ~ were labelled with monoclonal antibodies to cell surface mad
P.D. Mehta, T. Pirttila, S.P. Mehta, A.J. Dalton, M, Percy, H. Frey H.M. WisniewskL Institute for Basic Research, Staten Island, NY, USA; Tarnpere University Hospital. Tampero. Finland. and University of Toronto, Toronto, Canada. I ~ - ~ Persons with DS (40 years and older) develop brain lesions similar to AD. APR ere components in senile plaques and their levels are increased in sera from a subset of patients with AD. The aim was to compare the levels of APR in sera from DS and AD patients. Mstodall II1¢1 M~hod~ We measured the concentrations of C-reactiva protein (CRP), al-antitrypsin (AT), and a2-macroglobulin (a2-M) in sera from 40 DS and agematched controls, and 22 patients with probal:~,eAD and controls. using enzyme linked immunoserbent assay. ~ Levels of CRP, AT, and ++2-M were sigrffeantly higher in DS sere than in controls. Levels of CRP were signifeantly decreased in AD then in DS and controls. AT arid a2-M concentrations were similar in AD and controls. Co4ndullen: A lack of increase in APR in AD compared to DS sara suggest that the mechanisms involved in the d~ease process in DS and AD are d~erent.
P17.10 TEIE ROLE OF CELL ADEIESION MOLECULES IN LYMPHOCYTE ACTIVATION AND HOMING, Di Manro. M . E . J G leanle, M J . =. & Alger, A . 1, . . 'tmmunotog~ Keseas~ ufonp ~:CID), university ot Mancaester, Manchester M 13 9PT, U.K.:-'Tenovus Research LaBoratory, Smithampton SO9 4XY UK. lntrodoetion: Resting lymphccytes preferentially recirculate via the blood and lymphaties,..migr.~ing @ ~ [~mpfi. nodes . ~ . spleen for efficient ..a.ntigen encounter. Ponow'ing activation, lympbccy~. display distinct migration pamways ("homing') ~ to their into of activation. Activated lymphocyt~ also migrate to. non-.lymphoid organs and sites of inflamn'~tiun, enstiring efficient antigen elimination. The rrrrrrrrr~or mechanisms involved are, as yet, unclear but appoat to be altered upon lymphocyte activation and by the acuon of cytokines on endothelial ceils. Materials and Methods: Rat ~ h . o c y t e . s were activated in vitro using a CD2/¢~I'CR bi~ecifie .agtibefly (usAe) consisting of ehemi.cal,ly crosslinked I~b: f r ~ (no Pc region), m^wgeneratod blasts were usea in an in vitro mooe£ of'lymphocyte adhesion to aria migration across HEV. Adhesion blocking exj~'iments were carried out by pre-mcubating the lynwhocytes (4"C; 30rain') wfth mAbs agalust CDI la, CDfS, ICAM-1, VLA-4, CD'4, CD8, MHC Class II and CD45 (control). R e s u l t : The BsAb stimniated the proliferation of mlsep..era~d l y . ~ h node cells (LNC.) "m. a dose- and t ~ F l m M c n t manner. The "BsAb dla not stimulate proliIermion of M~hly purified'popal.ations of T cells - indicatinR a role for ~,'~__~_,~,'y celia or factors in its action. Inclusion of antibodies againsfCD 1 la and CD 18 we~ f.cund to inhibi(BsAb:.in@ced T cell proliferation in a d u s e - d ~ d ~ t manner, while also inbibitmLcel[ 91uste"+r'+mg.A .g.r~ter . l ~ n t a g e ot activat~ than of rest'rag lympbocytos ~ to high ['~domelial cells (HEC). None of the antibodies lisfed abbve blocked lymphocy'~e adhesion to HEC. Conclualan: Crosalinked antibodies to the aSTCR and CD2 are not sufficient to induce T cell proliferation. LFA-1 recq~ition between T lymphocytes and accessory cells ts also .Rquired. The re~,ul~ting systems involveTd in lymphocyte adhesion to and migration across H ~ are, as yet, unclear, but die receptor mechanisms do appear to be altered upon lymphocyte activation.
P03.12 CHANGES IN CELLUIJ~ DISPOSITION OF LIPOCORTIN-1 (ANNEXIN1) IN RAT PRIMARY .a~TROCYTE CULTURE8 AND C$ GLIOMA CELI..S AFTER EXPOSURE TO DEXAMETH/~ONE. J.D. McLeod and C. BoRon.
Phan~scology Dept., School of Pben'nacy & Pharmacology, University of Bath, Clsverton Down, Bath BA2 7AY, England. Introducllon: Astmcytes have been implicated in the immunological processes underlying the pathogenesis of Multiple Sclerosis (MS). The severity of MS can be regulated by glucocorticold steroids, an effect which may be mediated by the induction of an intracallulsr protein, lipocodin-1 (LC-1). The current study investigated whether astrocytes respond to dexamethasone (Dex.) by Increasing LC-1 expression. Methods: Pdmary astmcyte cultures and C6 glioma cells were exposed to Dex. (10nM-100nM) for different time pedods. LC-1 was extracted from separate call fractions; cell-suffsca, membrane and intracellular. The LC-1 content associated with each fraction was detected using electrophoresis and immunoblotting techniques. Results: In pdmary sstrocyte cultures, Dex. induced a nse in intraonllular LC-1 which was translocsted to the call membrane. Changes in intracallular and membrane-associated LC-1 also occurred in C6 cells. However, unlike primary cultures, LC-1 was further extemalised to the cell-surface. Conclusion: The involvemen~ of LC-1 in the actions of glucocorticoids on rat astrocytes may be responsible, in part, for the therapeutic effects of steroids in animal and human demyelinating diseases.
P10.04
P17.11
PHENOI'YPIC A%D FIN('TI()NAI. CHARA('I'ERI/~110 ~', OF RAI" LIPID-BO! ND MBP SPE(!I FIC [-('El.l. L IN VS.
INCREASED SOLUBLE VCAM-I LEVELS IN CEREBROSPINAL FLUID OF PATIENTS WITH MULTIPLE SCLEROSIS.
[3 Ma.~anti. ',I, \'~rz~lh. ,~. ~'a]l~:iJ.+. : ,=:.=;. ~
S.A.McMillsn I, J.P.Dougls# A.G.Droogeu 2 and S.A.Hawkins 2 Regional Immunology Laboratory, Belfast City HespiteP and Neurology Department, Royal Vietoda HospiteF, Belfast, Northern Ireland.
.~-~ii+ ~ ..~.,::d ......
[,lf+'o(lu(+ion ':+ :t D72VlOt,~ ~ttrcl} ,~ e h,+,+., cl,,e: +c' + ' + +' - : ' .:.++x'+T~. +~+"I t].+v ['~+~;+!,+t;,:+ ~'~,i ',i:, ,.. ?. :: ,~ i
:*:+ + x !"
+lCx+x Hr+t+++tt Nho+ t+PII Itltd~ geller+~ted t~om l x-~+l++tl~,~+~ ,.-++]Jccte(t at the tdt):+til.+H r++~,p,,:tsd t,? [ _ B - ' ~ F P b!tt +~+,:t tJ [. +'~[l~']: ~+IPl|lod~, ~: o ; I + : +o ]+,~tt+i ~+]l,tthCtCltL+ the illllltt~lh~etii~ l>r,>l)ert+e+ ,st ;ill+ almmet+ u,+' geJierated + +-NI~'++: -1~+¢I++C t+ce+! IHte~ fit+n+ [,~-M~+l ) ltttrt+tllVZ+d HIIIIIIlJ- -+eltUIg 5Pl<+;tt+t;tlCle,tl c+qb~ u+ ]inlitffl~2 dfll+tl~m ac~o:!tmg re, +~-t? t'reQtlJ+!c +` ,+( \+F+P ~[!J~.'][i~ i~IgC!II;'+F'~. resulls: the ,m+til~:rati,.',~':~p.+n:,e , f th,: :n,O,,J~ty or" I[tCs+
{m,~s v+a~ lughJ,. ~p~ctfiu Io LB-MB[~ (A~ d~s~ O i u~ lid, :~ztJl I,) c~,s~reaol~n~+ to I,F-\,IISP .gtltomated ¢ ' ~ , t l t l o n ! t l ¢ l r l ¢ anaIy~in ~,t: these Iinc~ ~!, ,,~ z!(l :¢ ( ' D I "
]~}!erlcfl'}"Dc
!,F~-NI}~P ..:;3eclt}c fir*e:~[! ]Itle~ did nc,i tee.Dot/} h~
t~,J ilIIlllUJlc~'d~qllilld,*llepltope or" '~!BP !p~F+t:de -2-S:% ',.,~ ~he IAiRTI, h.g,'L.'i~.l:'.:,'f Ih,: t,cwi, :at. ,~[tcle+t+ th+ b]lc~ >[~ecilic ;:r th,~ ";~l[lt+ tl'~Jlll) i~' :~,th It- told Lt +NIE:+ did r.+.,pund tu thl+ [),:[)+tdd ('OIK'lUXiOll Ii;~> tlll+t~talil Pitt,: +pccl|Jctt~. ]]ldl+tltcN I}tilt ~lt~+' wi~en +mi,eddcd t;I [iI'+dm md~:ce, a n~. x~r ?all,;r+l of, -+¢[[ ['c,-,poil>,:probabP< bccut:sc ;+["(Ii[]'~F,2Ii T c[~:i~2]! ~?'t+.2.:~SlT[g ~;;ttl pIcSellt,Jl],,ll i:} :\P("
Introduction: Increased levels of circulating adhesion molecules have beea found in many inflammatory diseases. As multiple sclerosis (MS) is an inflammatory disorder levels of these molecules may give a valuable insight into the vascular pathogenesis and therefore diagnusia of this disease. Materials and Methods: Serum and corebruspinal fluid levels o f soluble ICAM1, sE-selectin (ELAM) aad sVCAM-1 wore determined by ELISA in patients prior to a confirmed neurological diagnosis. 45 patiente were later dlasnused as MS, 23 as inflammatory (IND) and 37 as non-inflammatory neurological disease (NIND). Results: Significantly higher levels of sVCAM in CSF (mean values 18.1 ng/ml : 12.1 ng/ml; p = 0,001) and lower levels of sELAM in serum (mean values 45.5 ng/mh 55.0 ng/ml; p=0.03) were detected in MS patients compared with the non MS group. In the MS patients there was a c o n ~ t i n n between levels of sICAM in the CSF and serum and betwee~ sICAM and sVCAM in the CSF. There was also an association between pusitivity for oligoclonal bands and high CSF levels of slCAM and sVCAM in die MS group (p=0.01 and 0.004 respectively), this association was not seen with other indicators of local CNS immunogiobulin production. Conclusion: Our findings suggests that an interaction between brain endothelium and lymphocytes may be an important factor in the pathogenesis of MS.
P10.05
P14.10
T CELLS AND MICROGUA IN CHRONIC RELAPSING EAE: EVIDENCL THAT T CELLS ARE CD45RC- AND THAT MICROGLIA PROLIFERATE
ACUTE PHASE REACTANTS (APR) IN SERA FROM OLDER PERSONS WITH DOWN SYNDROME (DS) AND AL.ZHEIMER DISEASE (AD),
P.A. McCombe, J. de Jersey, M.P. Ponder Dept. of M~llclne, The Unlverolty of Queensland, Brisbane, Australia Introduction: CD45RC expression by T cells correlates with Thl-like function, but is lost on activation. The majority of the T cells in the spinal cord in Lewis rats with acute EAE are CD45RC-. We have now assessed CD45RC expression by T cells in the apinal cord during chronic relapsing EAE (CR-EAE). We also studied mlcrogMa In CR-EAE. Msterlele and methods: CR-EAE was induced by inoculation of Lewis rats with guinea pig spinal cord and vestment with low dose cyclosporin A (CsA). Cells extracted from the spinei ~ were labelled with monoclonal antibodies to cell surface mad
P.D. Mehta, T. Pirttila, S.P. Mehta, A.J. Dalton, M, Percy, H. Frey H.M. WisniewskL Institute for Basic Research, Staten Island, NY, USA; Tarnpere University Hospital. Tampero. Finland. and University of Toronto, Toronto, Canada. I ~ - ~ Persons with DS (40 years and older) develop brain lesions similar to AD. APR ere components in senile plaques and their levels are increased in sera from a subset of patients with AD. The aim was to compare the levels of APR in sera from DS and AD patients. Mstodall II1¢1 M~hod~ We measured the concentrations of C-reactiva protein (CRP), al-antitrypsin (AT), and a2-macroglobulin (a2-M) in sera from 40 DS and agematched controls, and 22 patients with probal:~,eAD and controls. using enzyme linked immunoserbent assay. ~ Levels of CRP, AT, and ++2-M were sigrffeantly higher in DS sere than in controls. Levels of CRP were signifeantly decreased in AD then in DS and controls. AT arid a2-M concentrations were similar in AD and controls. Co4ndullen: A lack of increase in APR in AD compared to DS sara suggest that the mechanisms involved in the d~ease process in DS and AD are d~erent.