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Experimental allergic encephalomyelitis (EAE) can be induced in DA rats without adjuvant
P o s t e r A b s t r a c t s / J o u r n a l o f N e u r o i m m u n o l o g y 9 0 (1998) 1 3 - 1 0 5
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E x p e r i m e n t a l Allergie EneephalomyeUtis ( E A E ) c a n be Induced in D A R a t s WithoutAdjuvant Stoikovic. S. Stosic-Gmjicic, V. Badovinac, Z. Ramie, Universityof
Passive T r a n s f e r of Human M o n o d o n a l I g M Antibodies (m-IgM) Against Sulfatide (Sul) in C h r o n i c R e l a p s i n g E x p e r i m e n t a l Allergic
Belgrade, Yugoslavia
Ac[juvants generally induce a strong inflammatory response and exert nt,'.lerous immunomodulatory properties. The aim of this study was to ~stablish reproducible model of EAE that can be induced without the aid of any adjuvant, thus avoiding unwanted side effects. Rats of DA strain, when 2 and 12 months old. were i.d. injected with rat or guinea pig spinal cord homogenate (RSCH or GPSCH, 50% w/v) mixed with the equal volume of saline in a volume of 0.1 ml. There was a striking difference in the incidence and severity of EAE depending of the origin of encephalitogen and the age of animals. Clinical signs of EAE (mean score 2.5) appeared in the majority of rats injected with RSCH (76%), while GPSCH induced a mild disease (mean score 1.3) in only 10% of rats. The old animals were more susceptible to the induction of EAE with RSCH than young rats as judged from higher incidence (89% vs. 54%) and maximal clinical score. The mechanisms responsible for age-related differences in susceptibility to induction of EAE without adjuvant could not be ascribed to different immune response in the peripheral lymphoid tissue since the analysis of cellular composition as well as antigen specific proliferative response of draining lymph node cells revealed no difference between young and old rats.
Encephalomyelitis ( c r E A E ) E. Nardelli. A.P. Riviera, G. Andrighetto, M. Gobbo, A. Breadolan, G. Tridentc, T. Cestari, Universitd di Verona lntravenous(i.v.) treatment of DA rats at the acute onset of crEAE with antiSUL positive m-lgM (Nardelli et al. J.Nearoimmunol. 1995.63:29) recognized a large number of SUL positive cell in inflammatury areas. DA rats, sensitized with syngenic spinal cord. had i.v. injection with purified m-IgM from patient with m-IgM anti-SUL and from a patient with m-lgM and no antibodies against CNS antigens( total dose: 8mg per animal fractionated in four i.v. injections) when clearout paraplegia was evident. Clinical study, was unable to differentiate m-lgM treated or untreated animals. The rots were perfused with 4% paraformaldehyde/0,05% pycric acid in 0,1M PBS, cryopreserved with sucrose and stored ar -70°C.Lesional topography of inflammatory infiltrates prevalence was pofforrmed with commercially monoclonal antibodies (mAbs) against lymphocytes, macrophages, otigodendrocytes (i.e. mAbs against Gal-c, CNt~se. OLIGO, Rip), astrocytes in single as well as double immunofluorescence. Co-localization with m-lgM was obtained with antibody mixture of anti mouse -TRITC and anti human lgM -FITC.We found numerous m-lgM -engulfed macrophages as well as increased amount of SUL-positive oligedendrecytes in Iesional areas. Further studies are needed to determine whether primary SUL+oligodendrocyte proliferation is a significant in EAE.
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N F - k B Speeifie Inhibition Severely E n h a n e e s E x p e r i m e n t a l
I n t e r f e r e n c e w i t h C o s t i m u l a t i o n Alters Disease C o u r s e in T h e f l e r ' s Murine Encephalomyelitis V i r u s - i n d u c e d D e m y e l i n a t i n g Disease (TMEV-IDD) K.L. Neville, Northwestern University, USA, J.A. Bluestone, Universityof Chicago, USA, S.D. Miller, Northwestern University, USA
Autoimmune Encephalomyelitis M.P. Mycko, T. Berkowicz, MedicalAeademy ofLodz, Poland, B. Kaminska, Nencki Institute ofExperonentaI Biology, Poland, A. Glabinski, MedicalAcademy ofLodz, Poland, C.S. Raine, AlbertEinstetu College of Medictue, USA, K. Selal aj, Medical Academy of Lodz, Poland The transcription factor NF-kB/Rel family was found to be an critical intmcellular regulator of immune processes mediated by proinflammatory signals, including apoptosis. In the present study to evaluate the importance of NF-kB in the development of EAE we injected selective NF-kB activation inhibitor, gliotoxin, in concentration 1.25-5.0 mg/kg, to mice sensitized with PLP peptide 139-151. We formal that clinical scoring of the gliotoxin treated mice was significantly higher, in a dose dependant manner, than control EAE mice (3.8 versus 2.0, p<0.001), while the time of disease onset was not altered. At the pathologic level animals receiving NF-kI3 inhibitor showed more enhanced pathology of EAE including features typical for TNF activity like widening of periaxonal space. Gliotoxin did not changed electrophorctic ability of NF-kB DNA protein isolated from spleens and CNS confu'ming its action not by interfering NF-kB active complex structure. These data suggest that NF-kB inhibition leads to increased TNF induced pathology during effectory phase of EAE Interestingly, different NF-kB DNA binding complexes were present in both spleens and spinal cords in EAE and healthy mice suggesting than development of EAE involves changes in NF-kB composition. Thus, NF-Id3 might represent an attractive target for therapeutic interventions in EAE.
325 MagneticResonanceImagingof PLP-lndueedExperimental Allergic Encephalomyelitisin LewisRat I.I.S. ~ IntitutPhysique B olog que, Straa'bourg,France, J. Steibel, lnstitut Physique Biologique, Strasbourg, France, C. Klinguer, E. Trifilieff, Centre de Igeurochirnie, Strasbourg, France, M. Mohr, Institut d'Anatomie P athologique, Strasbourg, France, P. Poulct, lnst~tutde Physique Biologique, Strasbourg, France An in vivo magnetic resonance (MR) imaging study was realized on experimental allergic encephalomyelitis (EAE) induced in Lewis rats through proteolipid protein (PLP). PLP was solubilized in water or in an aqueous solution of 1% 10 tridecyl ether (TDE), a non-ionic detergent used in membrane protein research. All 16 rats immunized with 500 /ag of TDEsolubilized PLP developed clinical signs and MR abnormalities fully comparable to those observed in MBP-induced EAE. Total paraplegia was observed in 12.5% of rats, mild or moderate paraparesis in 68.8% of rats and tail paralysis in the remaining 18.7% of rats. Whereas only 37.5% of the 8 rats immunized with 500 p.g of water-solubilized PLP developed minor clinical signs (tail weakness or paralysis). Our observations confirm that the difficulties encountered when trying to induce EAE by means of PLP arise ti-om the highly hydrophobic nature of this protein. Accordingly, if a reproducible model is to be developed, it seems more judicious to use non-ionic detergents in both the extraction and solubilization phases of PLP preparation, as this would allow maximal solubilization of the protein while avoiding aggregates, which may otherwise form in either of the PLP preparation phases.
Theiler's Virus-Induced Demyelinating Disease is a relevant model for the autoimmune disease multiple sclerosis (MS). In feetiun of SJL miee with TMEV presents a clinical disease characterized by spastic paralysis, chronic disease progression, and mononuelear cell infiltrate into the CNS. While initial demyelination in TMEV-IDD is mediated by vires-specific CD4+ T cells, reactivity to a myelin antigen, proteolipid protein epitope 139-151, arises 50-55 days post infection, demonstrating an autoimmune component in this virally induced disease. Administration of molecules which interfere with proper T cell costimulation such as ctB7-1, ctB7-2, or CTLA-41g can affect disease progression in certain experimental autoimmune diseases. Treatment of TMEV infected SJL mice with costimulatory antagonists at the time of infection indicates that administration ofcLBT- l/aB7-2 in combination, or CTLA-41g increases disease severity and delayed type hypersensitivity (DTH) responses to viral and myelin antigens. However, the same treatment begun 25 days post infection indicates CTLA-4Ig treatment may decrease disease severity and DTH reactivity to viral and myelin antigens, suggesting that costimutatory molecules play a critical role in both viral disease and the progression of TMEV-IDD into an autoimmune disease. (This work supported by NIH grant NS-35814)
328 Interleukin-4 and Interleukin-10 Mediated Inhibition of Interferongamma Induction of CD40 Expression V.T. Nguyen. E.N. Benveniste, University o/Alabama at Birmingham, USA
Micreglia are one of the major glial cell types within the central nervous system, and can function as immune effector cells upon activation. CIMO is a cell surface receptor belonging to the TNF-recoptor family that plays a critical role in the regulation of immune responses. In this study, we investigated the expression of CD40 on microglia, and the role of IL-4 and IL-10 in regulating CD40 expression. Microglia constitutively express very low levels of CD40, and IFNq, enhances CD40 mRNA and protein expression in these coils. IFN-T induction of ~ expression is dependeat on STAT-Ict activation by tyrosine phosphorylation; as such, microglin from STAT-la deficient mice a ~ refractory to IFN-T induced CIMO expression. IL-4 and IL-10 inhibit IFN-y induced CD40 protein and mRNA expression by suppression of CD40 transcription. The CD40 promoter contains at least three potential STAT binding sites that we befieve piny an important role in modulating CD40 expression. We are currently investigating the roles of S T A T - I ~ , STAT-3, and STAT-6 in the modulation of CD40 mRNA expression.
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E x p e r i m e n t a l Allergie EneephalomyeUtis ( E A E ) c a n be Induced in D A R a t s WithoutAdjuvant Stoikovic. S. Stosic-Gmjicic, V. Badovinac, Z. Ramie, Universityof
Passive T r a n s f e r of Human M o n o d o n a l I g M Antibodies (m-IgM) Against Sulfatide (Sul) in C h r o n i c R e l a p s i n g E x p e r i m e n t a l Allergic
Belgrade, Yugoslavia
Ac[juvants generally induce a strong inflammatory response and exert nt,'.lerous immunomodulatory properties. The aim of this study was to ~stablish reproducible model of EAE that can be induced without the aid of any adjuvant, thus avoiding unwanted side effects. Rats of DA strain, when 2 and 12 months old. were i.d. injected with rat or guinea pig spinal cord homogenate (RSCH or GPSCH, 50% w/v) mixed with the equal volume of saline in a volume of 0.1 ml. There was a striking difference in the incidence and severity of EAE depending of the origin of encephalitogen and the age of animals. Clinical signs of EAE (mean score 2.5) appeared in the majority of rats injected with RSCH (76%), while GPSCH induced a mild disease (mean score 1.3) in only 10% of rats. The old animals were more susceptible to the induction of EAE with RSCH than young rats as judged from higher incidence (89% vs. 54%) and maximal clinical score. The mechanisms responsible for age-related differences in susceptibility to induction of EAE without adjuvant could not be ascribed to different immune response in the peripheral lymphoid tissue since the analysis of cellular composition as well as antigen specific proliferative response of draining lymph node cells revealed no difference between young and old rats.
Encephalomyelitis ( c r E A E ) E. Nardelli. A.P. Riviera, G. Andrighetto, M. Gobbo, A. Breadolan, G. Tridentc, T. Cestari, Universitd di Verona lntravenous(i.v.) treatment of DA rats at the acute onset of crEAE with antiSUL positive m-lgM (Nardelli et al. J.Nearoimmunol. 1995.63:29) recognized a large number of SUL positive cell in inflammatury areas. DA rats, sensitized with syngenic spinal cord. had i.v. injection with purified m-IgM from patient with m-IgM anti-SUL and from a patient with m-lgM and no antibodies against CNS antigens( total dose: 8mg per animal fractionated in four i.v. injections) when clearout paraplegia was evident. Clinical study, was unable to differentiate m-lgM treated or untreated animals. The rots were perfused with 4% paraformaldehyde/0,05% pycric acid in 0,1M PBS, cryopreserved with sucrose and stored ar -70°C.Lesional topography of inflammatory infiltrates prevalence was pofforrmed with commercially monoclonal antibodies (mAbs) against lymphocytes, macrophages, otigodendrocytes (i.e. mAbs against Gal-c, CNt~se. OLIGO, Rip), astrocytes in single as well as double immunofluorescence. Co-localization with m-lgM was obtained with antibody mixture of anti mouse -TRITC and anti human lgM -FITC.We found numerous m-lgM -engulfed macrophages as well as increased amount of SUL-positive oligedendrecytes in Iesional areas. Further studies are needed to determine whether primary SUL+oligodendrocyte proliferation is a significant in EAE.
324
327
N F - k B Speeifie Inhibition Severely E n h a n e e s E x p e r i m e n t a l
I n t e r f e r e n c e w i t h C o s t i m u l a t i o n Alters Disease C o u r s e in T h e f l e r ' s Murine Encephalomyelitis V i r u s - i n d u c e d D e m y e l i n a t i n g Disease (TMEV-IDD) K.L. Neville, Northwestern University, USA, J.A. Bluestone, Universityof Chicago, USA, S.D. Miller, Northwestern University, USA
Autoimmune Encephalomyelitis M.P. Mycko, T. Berkowicz, MedicalAeademy ofLodz, Poland, B. Kaminska, Nencki Institute ofExperonentaI Biology, Poland, A. Glabinski, MedicalAcademy ofLodz, Poland, C.S. Raine, AlbertEinstetu College of Medictue, USA, K. Selal aj, Medical Academy of Lodz, Poland The transcription factor NF-kB/Rel family was found to be an critical intmcellular regulator of immune processes mediated by proinflammatory signals, including apoptosis. In the present study to evaluate the importance of NF-kB in the development of EAE we injected selective NF-kB activation inhibitor, gliotoxin, in concentration 1.25-5.0 mg/kg, to mice sensitized with PLP peptide 139-151. We formal that clinical scoring of the gliotoxin treated mice was significantly higher, in a dose dependant manner, than control EAE mice (3.8 versus 2.0, p<0.001), while the time of disease onset was not altered. At the pathologic level animals receiving NF-kI3 inhibitor showed more enhanced pathology of EAE including features typical for TNF activity like widening of periaxonal space. Gliotoxin did not changed electrophorctic ability of NF-kB DNA protein isolated from spleens and CNS confu'ming its action not by interfering NF-kB active complex structure. These data suggest that NF-kB inhibition leads to increased TNF induced pathology during effectory phase of EAE Interestingly, different NF-kB DNA binding complexes were present in both spleens and spinal cords in EAE and healthy mice suggesting than development of EAE involves changes in NF-kB composition. Thus, NF-Id3 might represent an attractive target for therapeutic interventions in EAE.
325 MagneticResonanceImagingof PLP-lndueedExperimental Allergic Encephalomyelitisin LewisRat I.I.S. ~ IntitutPhysique B olog que, Straa'bourg,France, J. Steibel, lnstitut Physique Biologique, Strasbourg, France, C. Klinguer, E. Trifilieff, Centre de Igeurochirnie, Strasbourg, France, M. Mohr, Institut d'Anatomie P athologique, Strasbourg, France, P. Poulct, lnst~tutde Physique Biologique, Strasbourg, France An in vivo magnetic resonance (MR) imaging study was realized on experimental allergic encephalomyelitis (EAE) induced in Lewis rats through proteolipid protein (PLP). PLP was solubilized in water or in an aqueous solution of 1% 10 tridecyl ether (TDE), a non-ionic detergent used in membrane protein research. All 16 rats immunized with 500 /ag of TDEsolubilized PLP developed clinical signs and MR abnormalities fully comparable to those observed in MBP-induced EAE. Total paraplegia was observed in 12.5% of rats, mild or moderate paraparesis in 68.8% of rats and tail paralysis in the remaining 18.7% of rats. Whereas only 37.5% of the 8 rats immunized with 500 p.g of water-solubilized PLP developed minor clinical signs (tail weakness or paralysis). Our observations confirm that the difficulties encountered when trying to induce EAE by means of PLP arise ti-om the highly hydrophobic nature of this protein. Accordingly, if a reproducible model is to be developed, it seems more judicious to use non-ionic detergents in both the extraction and solubilization phases of PLP preparation, as this would allow maximal solubilization of the protein while avoiding aggregates, which may otherwise form in either of the PLP preparation phases.
Theiler's Virus-Induced Demyelinating Disease is a relevant model for the autoimmune disease multiple sclerosis (MS). In feetiun of SJL miee with TMEV presents a clinical disease characterized by spastic paralysis, chronic disease progression, and mononuelear cell infiltrate into the CNS. While initial demyelination in TMEV-IDD is mediated by vires-specific CD4+ T cells, reactivity to a myelin antigen, proteolipid protein epitope 139-151, arises 50-55 days post infection, demonstrating an autoimmune component in this virally induced disease. Administration of molecules which interfere with proper T cell costimulation such as ctB7-1, ctB7-2, or CTLA-41g can affect disease progression in certain experimental autoimmune diseases. Treatment of TMEV infected SJL mice with costimulatory antagonists at the time of infection indicates that administration ofcLBT- l/aB7-2 in combination, or CTLA-41g increases disease severity and delayed type hypersensitivity (DTH) responses to viral and myelin antigens. However, the same treatment begun 25 days post infection indicates CTLA-4Ig treatment may decrease disease severity and DTH reactivity to viral and myelin antigens, suggesting that costimutatory molecules play a critical role in both viral disease and the progression of TMEV-IDD into an autoimmune disease. (This work supported by NIH grant NS-35814)
328 Interleukin-4 and Interleukin-10 Mediated Inhibition of Interferongamma Induction of CD40 Expression V.T. Nguyen. E.N. Benveniste, University o/Alabama at Birmingham, USA
Micreglia are one of the major glial cell types within the central nervous system, and can function as immune effector cells upon activation. CIMO is a cell surface receptor belonging to the TNF-recoptor family that plays a critical role in the regulation of immune responses. In this study, we investigated the expression of CD40 on microglia, and the role of IL-4 and IL-10 in regulating CD40 expression. Microglia constitutively express very low levels of CD40, and IFNq, enhances CD40 mRNA and protein expression in these coils. IFN-T induction of ~ expression is dependeat on STAT-Ict activation by tyrosine phosphorylation; as such, microglin from STAT-la deficient mice a ~ refractory to IFN-T induced CIMO expression. IL-4 and IL-10 inhibit IFN-y induced CD40 protein and mRNA expression by suppression of CD40 transcription. The CD40 promoter contains at least three potential STAT binding sites that we befieve piny an important role in modulating CD40 expression. We are currently investigating the roles of S T A T - I ~ , STAT-3, and STAT-6 in the modulation of CD40 mRNA expression.