- Email: [email protected]
Complement components in Alzheimer's disease brain
203
P05.09
P07.11 COMPI,JE~iBNT COMPON]EN'~ IN ~
'
S
D~uSEASE BRAIN
Vonrhuls R. ~z. Janssan L1, Zahn S.-S.. z, Efkelanbeom p.Z Graduate School Neurosdences Amsterdam, Research Institute Nonro~tancas Vrije Untversiteit, Depts Pathology ~ and Psychiat~ z, Amsterdam, The Netherlands, Introduction: In Alzheimer's disease (AD) patients, the complement components Clq, CA and C3 can be detected in amyloid (B/A4) plaques, one of the hallmarks of AD. In AD the formation of the lyric membrane attack complex (MAC) is supposed to be an Intermediate between the B/A4 depos/ts and the obu~vod neuroto0dcity. Matadals mid Metim~: We analyzed In an extensive histochemtcul study the presence of a number of complement components and regulatory proteins in AD temporal corte~ Clq, CA and C3 activation products, but not Clr, Cls and C1esterase inhibitor were colonallzed with B/A4 depmits. In addition, no late components C5, C7, C9, nor MAC and its inhibitor CD59 could be detected in ~/A4 positive plaques. O n the other hand, the MAC inltibitors clnsterin and vltronectln are present in both classical and diffuse plaques. CoadW[oa: The al~ence of the cytolyflc C5b-9 complex in AD brains suggests, that in AD complement does not function as the proposed i n 0 a m m t o i y medlator betwe¢~ B/A4 deposits and nettroflbrlllary changes. Probebly, in the brain the locally produced Clq and also C3 and the mul~ual Inhibitors cinstetin and viU-one~da serve another function than initiation or regulation of complement activation.
T Cell Response to Myelin Basic Protein in D R I $ Dw2 Positive MS patients: Role of DRB$*0101 and D R B I * I $ 0 1 as Restriction Elements for T Cell Lines. M. Vergalli*, H.F. Mc Farland*, R. M~Cin *^ Nenroimmonology Branch*, NINDS, Nil-I, Bethesda and Department of Neurology ^, University of T0bingen, TUbingen.
Introduction: Multiple Sclerosis (MS) is associated with the HLA DRI5 Dw2 haplotype in Caucasians. The mechanisms underlying this association are still unknown but it is currently befieved that MS-aasociatod HLA gone products are involved in the presentation of a putative autoantlgen related to the disease to encephalitogenic T cells. Myelin bns~ protein (MBP) is a candidate target of the autoimmune response in MS. Methods: MBP specific T c~ll lines (TCL) were generated from HLA DRI5 Dw2 poaltlve MS patients by limiting dilution tecnklue. Pepfide specificity was determined by proliferation or cytotoxicity assay using a panel of overlapping peptides spanning the sequence of human MBP. MHC restriction was evaluated using DR2-pnsitivo homozygous typing cells mul routine L fibreblast lines tranfncted with human HLA DR15 genes. Resells: A large number of diff¢~nt MBP peptldas were recognized by DR15 Dw2 restricted proliferative and cytotoxic TCL. Restriction of the TCL by either of the two polymosphic DR beta chains concomitantly expressed on DRI5 Dw2 positive cells (DR2a, encoded by DRB5*0101 and DR2b, encoded by DRBl*1501) has bsen detealnined. The data will be discnssed and related to the binding properties of MBP peptides to these HLA alleles (see abstract by Vogt et el.). Conelusinn: The understanding of biochemical and functional interaedons of antoantigenic peptides with diseaserelated HLA molecules provides a basis for highly specific immunotllerapies of autoimmunediseases.
W07.06
P01.16
ICAM EXPRESSION AND MICROGLIAL ACTIVATION IN ALZI-IEIMER SENILE PLAQUES: INDICATORS OF AN INFLAMMATORY RESPONSE.
Compartmentalizationof Disease Regulation: Acqutdng and Maintaining Resistance to EAE is Associated with Anterior Uveitls.
M.M. Verbeek. L Otte-H6ller, P. Weaseling, D.J. Ruiter. R.M.W. de Wual. Department of Pathology, University Hospital Nijmegen. Nijmegen, The Netherlands.
Core Verhagen and/run R. Cohen. Weizmann Institute of Science, Department Ceil Biology, PO Box 26, Rehovot, Israel.
Introduction. Some years aao , a local acute phase reaction was proposed as the triggering event in the pathogenesis of dementia of Alzheimer type (DAT). In this study, we provide evidence for an inflammatory response with eccompanying microgfi=l activation in senile plaques in DAT brains. Methods. Brain tissue cryceections from DAT patients and normal controls were immunohlstochemicelly analyzed for the expression of A~P, ICAM-I, ICAM-2, the lyacsomal entigan CD68 and the macrophegn differentiation marker 25F9. Resulls. In and around the classic, diffuse and burned-out types of senile plaques a marked diffuse or granular staining for ICAM-I was observed. Immunoprecipitetion analysis confirmed the presence of a 85 kd ICAM-I molecule in DAT frontal cortex. ICAM-2 expression was observed in microglial cells, especially those ac~umelating in sunlle plaques. CD68-stsining was observed in lysceomes in both resting end activated mieroglial cells, whereas MAb 25F9 only stained the, oflan e~wged, lysoecmes of activated microglial cells that weee found in nssoeiation with classic senile plaques. Candminm. The above presented data are in support of a cytokine-mediated pathogenesis of senile plaque formation, which includes enhanced production of ICAM-I. The association of ICAM-2- and 2.5F9-positive activated microglial cells with classic senile plaques only, suggests a secondary role for activated microglial cells in senile plaque formation.
Introduction. Our previous results demonstrated that in Lewis rats T-cell immunity to myelin basic protein (BP) may result in anterior uveitis (AU). In this study we examined whether AU was susceptible to tolerance induction. Methode & Reaults. Rats, recovered from a primary bout of EAE accompanied by AU, acquired resistance to a second induction of paralysis but were still susceptible to AU upon a challenge with BP/CFA. Moreover, AU was reinduced in BP/CFA immunized rats by a challenge with the BP-peptide 71-90. A challenge with the non disease-inducing BP-peptide 1-20 or CFA alone did not result in a recurrence of AU. In addition, immunization to BP/CFA resulted in a higher susceptibility to activated, BP-specific T cells with regard to development of AU: a BP-specific line that induced EAE without AU in naive animals, induced AU without EAE in BP/CFA immunized rats. Conduliona. These results demonstrate that limb paralysis and AU differ in their susceptibility to tolerance induction. These results, together with the previously reported differences in kinetics between EAE and AU, suggest that autoimmunity to BP is compartmentalized.
P06.23 THE ADHESION OF T LYMPHOCYTES TO HUMAN ENDOTHELIAL CELLS AND BRAIN PERICYTES IS DIFFERENTIALLY REGULATED.
P15.12 33~TF(TUMOR NECROSIS FACTOR) LEVELS IN THE SERUM AND CSF (CEREBROSPINAL FLUID) AFTER EAE (EXPERIMENTAL ALLERGIC ENCEI~IALOMYEUTIS) INDUCTION IN LEWIS RATS.
M.M. Verbeek, J.R. Westphal, D.J. Rulter, R.M.W. de WanL Department of Pathoiogy, University Hospital Nijmegen, Nijmegen, The Netherlands.
Villarroya H., Violleeu K., Ben Younen-Chennoufi A., Baumaxm N. INSERM U.134, Hospital Salpetriero, Paris, France
Introduction. T cell migration through the cerebral micmvasrulatuse is a key phenomenom in multiple sclerosis (MS). In thin study, we analysed the interactions betwean T cells and the cells constituting the cerebral microvasculatore, i.e. endothelial cells (EC) and pedcytus (HBP). Methods. Using in vitro adhesion assays we investigated the molecular medumistm involved in the interactions between T cells and EC or HBP. Results. T ceil adhesion to unstimuisted EC or HBP was low (3-4%), Wheo EC or HBP were induced to express ICAM-I and VCAM-I by treatment with TNFct, T cell edheainn increased three-fold. Activation of T cell integdns with phorhei ester or Mn +÷ resulted in a 4 or 10-fold increase in T cell adhesion, respectively. The adhesion of Mn**-treated T cells to TNFot-stimulated EC or HBP was further investigated by inhibition studies using antibodies to adhesion molecules on either call type. T cell adhesion to HBP was dominated by VLA4/VCAM-1 interactions, whereas VLA-4/VCAM-I and LFA-1/ICAM-I interactions equaliy contributed to the adhesion of T cells to EC. Conclusions. This study identifies the molecules mediating the suo~mive phases of T cell migration into the brain, and shows that T cell adhesion to either EC or HBP is differentially regulated. These molecules may serve as a target to block T ceil infiltration from the microvasculature into the brain, as occnsrin 8 in MS.
EAE is the best characterized demyelinating disease of the CNS in animals. EAE is an antlgen-speeific, T-cell m e d i a l ~ ! , a u t o i m m u n e disease. Cytokines m a y play k e y roles in the regulation of myelin autoimmttne responses. Soluble TNF activity, assessed b y L 929 fibroblast bioasuay, was determined in aerom and CSF samples frotr Lewis rats after EAE induction with pttrJfied guinea pig myelin. Results were expressed in U/ml in reference to the cytotoxie activity of a standardized (Gelxzyme) preparation of recombinant h u m a n TNFcL The data obtained s h o w e d that EAE induction results in detectable circtdatin S levels of biologically active T N F ~ S e r u m T N F a m e d i a n v a l u e was h i g h e r in d a y s 11-13 and w a s well correlated with encephalitogenlc symptoms. We have also investigated the localization of T N F expression in the CNS. TNFct immtmoreecqivity was localized primarily in neuronal cells in the cerebral cortex. On day 10 postimmunization, we showed TN'Fc¢ exprosalon by activated monocytes and macrophagea around the perivascular lesions, specially i~ the spinal cord. Between days 12-15, astmeytes in the cerebellum 8ranular layer and spinal cord, as well as radial gilt, showed T N F a production as demonstrated by double labeling,
P05.09
P07.11 COMPI,JE~iBNT COMPON]EN'~ IN ~
'
S
D~uSEASE BRAIN
Vonrhuls R. ~z. Janssan L1, Zahn S.-S.. z, Efkelanbeom p.Z Graduate School Neurosdences Amsterdam, Research Institute Nonro~tancas Vrije Untversiteit, Depts Pathology ~ and Psychiat~ z, Amsterdam, The Netherlands, Introduction: In Alzheimer's disease (AD) patients, the complement components Clq, CA and C3 can be detected in amyloid (B/A4) plaques, one of the hallmarks of AD. In AD the formation of the lyric membrane attack complex (MAC) is supposed to be an Intermediate between the B/A4 depos/ts and the obu~vod neuroto0dcity. Matadals mid Metim~: We analyzed In an extensive histochemtcul study the presence of a number of complement components and regulatory proteins in AD temporal corte~ Clq, CA and C3 activation products, but not Clr, Cls and C1esterase inhibitor were colonallzed with B/A4 depmits. In addition, no late components C5, C7, C9, nor MAC and its inhibitor CD59 could be detected in ~/A4 positive plaques. O n the other hand, the MAC inltibitors clnsterin and vltronectln are present in both classical and diffuse plaques. CoadW[oa: The al~ence of the cytolyflc C5b-9 complex in AD brains suggests, that in AD complement does not function as the proposed i n 0 a m m t o i y medlator betwe¢~ B/A4 deposits and nettroflbrlllary changes. Probebly, in the brain the locally produced Clq and also C3 and the mul~ual Inhibitors cinstetin and viU-one~da serve another function than initiation or regulation of complement activation.
T Cell Response to Myelin Basic Protein in D R I $ Dw2 Positive MS patients: Role of DRB$*0101 and D R B I * I $ 0 1 as Restriction Elements for T Cell Lines. M. Vergalli*, H.F. Mc Farland*, R. M~Cin *^ Nenroimmonology Branch*, NINDS, Nil-I, Bethesda and Department of Neurology ^, University of T0bingen, TUbingen.
Introduction: Multiple Sclerosis (MS) is associated with the HLA DRI5 Dw2 haplotype in Caucasians. The mechanisms underlying this association are still unknown but it is currently befieved that MS-aasociatod HLA gone products are involved in the presentation of a putative autoantlgen related to the disease to encephalitogenic T cells. Myelin bns~ protein (MBP) is a candidate target of the autoimmune response in MS. Methods: MBP specific T c~ll lines (TCL) were generated from HLA DRI5 Dw2 poaltlve MS patients by limiting dilution tecnklue. Pepfide specificity was determined by proliferation or cytotoxicity assay using a panel of overlapping peptides spanning the sequence of human MBP. MHC restriction was evaluated using DR2-pnsitivo homozygous typing cells mul routine L fibreblast lines tranfncted with human HLA DR15 genes. Resells: A large number of diff¢~nt MBP peptldas were recognized by DR15 Dw2 restricted proliferative and cytotoxic TCL. Restriction of the TCL by either of the two polymosphic DR beta chains concomitantly expressed on DRI5 Dw2 positive cells (DR2a, encoded by DRB5*0101 and DR2b, encoded by DRBl*1501) has bsen detealnined. The data will be discnssed and related to the binding properties of MBP peptides to these HLA alleles (see abstract by Vogt et el.). Conelusinn: The understanding of biochemical and functional interaedons of antoantigenic peptides with diseaserelated HLA molecules provides a basis for highly specific immunotllerapies of autoimmunediseases.
W07.06
P01.16
ICAM EXPRESSION AND MICROGLIAL ACTIVATION IN ALZI-IEIMER SENILE PLAQUES: INDICATORS OF AN INFLAMMATORY RESPONSE.
Compartmentalizationof Disease Regulation: Acqutdng and Maintaining Resistance to EAE is Associated with Anterior Uveitls.
M.M. Verbeek. L Otte-H6ller, P. Weaseling, D.J. Ruiter. R.M.W. de Wual. Department of Pathology, University Hospital Nijmegen. Nijmegen, The Netherlands.
Core Verhagen and/run R. Cohen. Weizmann Institute of Science, Department Ceil Biology, PO Box 26, Rehovot, Israel.
Introduction. Some years aao , a local acute phase reaction was proposed as the triggering event in the pathogenesis of dementia of Alzheimer type (DAT). In this study, we provide evidence for an inflammatory response with eccompanying microgfi=l activation in senile plaques in DAT brains. Methods. Brain tissue cryceections from DAT patients and normal controls were immunohlstochemicelly analyzed for the expression of A~P, ICAM-I, ICAM-2, the lyacsomal entigan CD68 and the macrophegn differentiation marker 25F9. Resulls. In and around the classic, diffuse and burned-out types of senile plaques a marked diffuse or granular staining for ICAM-I was observed. Immunoprecipitetion analysis confirmed the presence of a 85 kd ICAM-I molecule in DAT frontal cortex. ICAM-2 expression was observed in microglial cells, especially those ac~umelating in sunlle plaques. CD68-stsining was observed in lysceomes in both resting end activated mieroglial cells, whereas MAb 25F9 only stained the, oflan e~wged, lysoecmes of activated microglial cells that weee found in nssoeiation with classic senile plaques. Candminm. The above presented data are in support of a cytokine-mediated pathogenesis of senile plaque formation, which includes enhanced production of ICAM-I. The association of ICAM-2- and 2.5F9-positive activated microglial cells with classic senile plaques only, suggests a secondary role for activated microglial cells in senile plaque formation.
Introduction. Our previous results demonstrated that in Lewis rats T-cell immunity to myelin basic protein (BP) may result in anterior uveitis (AU). In this study we examined whether AU was susceptible to tolerance induction. Methode & Reaults. Rats, recovered from a primary bout of EAE accompanied by AU, acquired resistance to a second induction of paralysis but were still susceptible to AU upon a challenge with BP/CFA. Moreover, AU was reinduced in BP/CFA immunized rats by a challenge with the BP-peptide 71-90. A challenge with the non disease-inducing BP-peptide 1-20 or CFA alone did not result in a recurrence of AU. In addition, immunization to BP/CFA resulted in a higher susceptibility to activated, BP-specific T cells with regard to development of AU: a BP-specific line that induced EAE without AU in naive animals, induced AU without EAE in BP/CFA immunized rats. Conduliona. These results demonstrate that limb paralysis and AU differ in their susceptibility to tolerance induction. These results, together with the previously reported differences in kinetics between EAE and AU, suggest that autoimmunity to BP is compartmentalized.
P06.23 THE ADHESION OF T LYMPHOCYTES TO HUMAN ENDOTHELIAL CELLS AND BRAIN PERICYTES IS DIFFERENTIALLY REGULATED.
P15.12 33~TF(TUMOR NECROSIS FACTOR) LEVELS IN THE SERUM AND CSF (CEREBROSPINAL FLUID) AFTER EAE (EXPERIMENTAL ALLERGIC ENCEI~IALOMYEUTIS) INDUCTION IN LEWIS RATS.
M.M. Verbeek, J.R. Westphal, D.J. Rulter, R.M.W. de WanL Department of Pathoiogy, University Hospital Nijmegen, Nijmegen, The Netherlands.
Villarroya H., Violleeu K., Ben Younen-Chennoufi A., Baumaxm N. INSERM U.134, Hospital Salpetriero, Paris, France
Introduction. T cell migration through the cerebral micmvasrulatuse is a key phenomenom in multiple sclerosis (MS). In thin study, we analysed the interactions betwean T cells and the cells constituting the cerebral microvasculatore, i.e. endothelial cells (EC) and pedcytus (HBP). Methods. Using in vitro adhesion assays we investigated the molecular medumistm involved in the interactions between T cells and EC or HBP. Results. T ceil adhesion to unstimuisted EC or HBP was low (3-4%), Wheo EC or HBP were induced to express ICAM-I and VCAM-I by treatment with TNFct, T cell edheainn increased three-fold. Activation of T cell integdns with phorhei ester or Mn +÷ resulted in a 4 or 10-fold increase in T cell adhesion, respectively. The adhesion of Mn**-treated T cells to TNFot-stimulated EC or HBP was further investigated by inhibition studies using antibodies to adhesion molecules on either call type. T cell adhesion to HBP was dominated by VLA4/VCAM-1 interactions, whereas VLA-4/VCAM-I and LFA-1/ICAM-I interactions equaliy contributed to the adhesion of T cells to EC. Conclusions. This study identifies the molecules mediating the suo~mive phases of T cell migration into the brain, and shows that T cell adhesion to either EC or HBP is differentially regulated. These molecules may serve as a target to block T ceil infiltration from the microvasculature into the brain, as occnsrin 8 in MS.
EAE is the best characterized demyelinating disease of the CNS in animals. EAE is an antlgen-speeific, T-cell m e d i a l ~ ! , a u t o i m m u n e disease. Cytokines m a y play k e y roles in the regulation of myelin autoimmttne responses. Soluble TNF activity, assessed b y L 929 fibroblast bioasuay, was determined in aerom and CSF samples frotr Lewis rats after EAE induction with pttrJfied guinea pig myelin. Results were expressed in U/ml in reference to the cytotoxie activity of a standardized (Gelxzyme) preparation of recombinant h u m a n TNFcL The data obtained s h o w e d that EAE induction results in detectable circtdatin S levels of biologically active T N F ~ S e r u m T N F a m e d i a n v a l u e was h i g h e r in d a y s 11-13 and w a s well correlated with encephalitogenlc symptoms. We have also investigated the localization of T N F expression in the CNS. TNFct immtmoreecqivity was localized primarily in neuronal cells in the cerebral cortex. On day 10 postimmunization, we showed TN'Fc¢ exprosalon by activated monocytes and macrophagea around the perivascular lesions, specially i~ the spinal cord. Between days 12-15, astmeytes in the cerebellum 8ranular layer and spinal cord, as well as radial gilt, showed T N F a production as demonstrated by double labeling,