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Myelin basic protein (MBP)-specific TGFβ-secreting CD4+ T-cell clones derived from orally tolerized animals suppress experimental autoimmune encephalomyelitis
205
WIZ.0T INTRATHECAL ADMINISTRATION OF SYNTHETIC PEPTIDE MBP75-g5 NEUTRALIZES MULTIPLE SCLEROSIS ANTI-MBP IN-VIVO
P17.16 THEILER'S VIRUS (TMEV) U P R E G ~ T E S THE EXPRF_~SION OF MHC CLASS I ON C E R E B R O V A S C ~ ENDOTHELIAL CELLS
K.G. Warren and I. Catz University of Alberta, Edmonton, CANADA
B.V. Sapatino and C.J.R. Welsh Department of Veterlnazy Anatomy and Public Health, Texas A&M University, College Station, Texas 77843-4458, USA.
The objective of this study was to determine whether synthetic peptide MBP75-95 will neutralize free (F) anti-MBP in CSF of MS patients after intrathecal inoculation. Matched pairs of MS patients with chronic progressive disease received 1, 2.5, 5 and 10 mg of either MBP35-58 or MBP75-95 dissolved in 5 ml of normal saline and injected into the CSF. Cerebrospinal fluid was sampled for anti-MBP monitoring before inoculation, every 30 minutes for 2 hours after injection, 24 hours later and then at weekly intervals until F anti-MBP levels returned to baseline value. Intrathecal administration of MBP75-95 produced neutralization of F antiMBP in a dose-dependent fasllion, whereas the control MBP35-58 failed to do so. With dosages of 5-10 mg of MBP75-95. the neutralization effect occurred within 30 minutes and persisted for penods up to 7 days. Since MS anti-MBP can be completely neutralized in vivo by MBP75-95 the epitope is most likely located in this area of the molecule. Additional Phase 1 reseamh is required prior to conducting Phase 2 trials for potential therapeutic benefit. If the autoimmune pathogenesis of MS is due to B cell and/or T lymphocyte mechaniams directed towards MBP in situ within the myelin sheath, it may be possible to stabilize this disease process by appropriate utilization of synthetic peptides.
Introduction: TMEV is a Plcornavlrus that persists in certain strains of mice and gives rise to demyelirmting disease which is sirnil~tr to multiple sclerosis. Both conditions are characterized by Immune cell Infiltration of the central nervous system (CNS]. We have been investigating i n t e r a c t i o n s b e t w e e n TMEV a n d cerebrovaacular endothelial cells [CVE]. Materials a n d Methods: CVE were isolated from the brains of SJL/J, CBA and BALB/c mice and cloned by the limiting dilution technique. The cells were characterized with respect to Factor VIII. ACE and uptake of acetylated LDL. CVE from all three strains of mice were Infected with TMEV and passaged six times and then examined for expresston of MHC Class I and II by flow cytometry. CVE from CBA and BALB/c mice expressed high levels of MHC Class I after infection with Thefler's virus (7S-94% of the cells were posttive for Class I c o m p a r e d to 10% in controls). Interestingly. SJL CVE did not express Class I. Co~n.~ TMEV infects CVE In vlvo and In vitro, In vitro infection of CBA and BALB/c CVE with TMEV, upregulated Class I expression which may allow these v/rally-infected cells to become, targets for cytotox~c T cells. Supported by NIH/NINDS #NS 29133.
P01.17
P17.17 MYOCARDIALINFARCTIONCAUSES REGIONAL REPRODUCIBLE BBB LEAKAGEVIA CYTOKINES.
MYELIN ASSOCIATED GLYCOPROTEIN: A TARGET AUTOANTIGEN IN EXPERIMENTAL AUTOIMMUNE ENCEPHALITIS S. Wssrth, R. Seitz, J. Mslotka, K. Dornmsir, Lassmann 1, H. Weksrle and C. Linington. Max-Planck Institute for Psychiatry, Martinsriad; Vienna, Vienna
T. Berger 1, H. 1 University of
Autoimmuna T- and B- cell responses against myelin associated glycoprotain (MAG) in patients with multiple sclerosis have been described by several groups. As the molecular basis of multiple sclerosis is still unknown, wa investigated the autoimmune response against MAC in the Lewis rat using synthetic rat MAC peptides and a panel of 6 recombinant rat ft~ion proteins. The sequences of these MAC fusion proteins represent the full length isoforms (S° + LMAC), the extracallular part and pairs of overlapping IgG domains (2 + 3, 3 + 4, 4 + 5). After subcloning these sequences in front of the His s coding sequence of the pQE12 vector, the fusion proteins were expressed in E. coil and purified by Ni-chelate chromatography, preparative SDS-PAGE and subsequent electroelution. Two diffarsnt T-cell epitopes were identified in the sxtracellular region corresponding to MPI.1 (aa 20-34) and MP3 (as 119-137) and one intracallular in the unique carboxy-terminal part of S-MAG corresponding to MP9 (aa 561-582). Adoptive transfer of these specific CD4 + T-cell lines in Lewis rats induced an inflammatory response in the CNS and PNS. These results identify MAC as an additional myelin autoantigen that could be involved in the immunopathoganesis of MS.
Y.D. van der WeftI, M.J.L. de Jongste2, G.J. ter Horstt Dept. of Biological Psychiatry and 2 Cardiology/Thoraxce~tre, Un/versityand AcademicHospitalGroninge~tt,Oostersinge159postbus 30.0019700 RB Groningem Introduction: It is known that myocardial infarction (MI) is accompanied by inflammation in the coronary vessels. It is hypothesized that the inflammatory proce~ becomes systemicand reaches the brain where it may have adveaseeffects on Central Nervous Systemfamctioning.Materials and Methods: MaleWistsr rats receiveda myocardialinfarctionthroughligationof part of the left veatricularwall. Between two and fourteendays after MI the rats were perfused. Resulls: Evidence for inflammationin the brain came from inmlt~2-cy~L.,~alcat~ ¢ m of ICAMpositive small vessels in certain areas of the brain, includingthe prefrontalcortex, the entorhinal cortex, the somatosansory cortex, the reticular formation and the bulints olfaetorius. Staining for Albumin revealed that specifically around the lCAM-posltive vessels there had been extntvasation of serum proteins. This indicates leakage of the Blood-Braln-Berrier(BBB), pmhably caused by reactive, cytokine-produeing leucocytes adhering to the vessel wall. In order to assess whether the inflammationrather than the operation resulted in leakageof the BBB, we injected TNF in the tail artery of Male Wister rats. The same procedureswere used, and the stainingof ICAM-positivevesselsand Albumin-positivepatches were observedin the same places in the brain. Conclusion: Myocardialinfarction results in a systemic inflammJationwhich causes leucocytes to adhere to the vessel walls in certain well-defined areas in the brain. The nonspecific immune overreaction cause~ extravasationof blood constituents and may result in loss of function of the specific brain areas.
W15.02
P08.20
MYELIN BASIC PROTEIN (MBP)-SPECIFIC TGFp-SECRETING CIM+ T-CELL CLONES DERIVED FROM ORALLY TOLERIZED A N I M A L S SUPPRESS E X P E R I M E N T A L A U T O I M M U N E ENCEPHALOMYELITIS. Youhai Chen. Viiav Kuchroo. Howar~l L. Weiner, Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115 USA Oral administration of MBP generates T cells capable of suppressing EAE which secrete regulatory cytokines TGFI3, IL-4, and IL-10 but not IFNy following specific antigen stimulation. In bulk culture, TGPI3 was produced by both CD4+ and CD8+ T cells whereas IL4 and I L l 0 were produced primarily by CD4+ T cells. SJ'L mice were fed five times with 0.5 mg MBP, boosted intrat~ritoneally with MBP in CFA and T cell lines derived from mesenteric lymph nodes. T cell clones were then generated by single cell clonin~g of the lines. Both CD4+ and CDa+ clones were established and detailed analysis of CD4+ clones undertaken. Clones were tested for epitope specificity, MHC restriction and cytokine production. Using overlapping 20 mer peptides, CD4+ clones react to two major MBP determinants, 84-102 and 71-90. All clones are IA s restricted. On cytokine analysis the clones fell into two categories: (1) TGFI3hi IL-4/IL-10Io T ceils which produced large amounts of T G I ~ mtd small amounts of IL-4/IL-10; and (2) TGFp Io ILo 4/IL-10tniT cells. Injection of these clones into SJL mice immunized for EAE with MBP or PLP suppressed the incidence and severity of disease. These clones will further allow characterization of regulatory cells generated by oral tolerance and TGFphi IL-4/IL-10io cells may represent a unique mucosal type TH2 cell. (Supported by NIH grant NS-29352.1
GLUCOCORTICOIDS ACCELERATE ANTI T-CELL RECEPTOR STIMULATED GROWTH OF RAT SPLEEN T-CELLS G.J. Wieoers. M.S. Labeur, I.E.M, Stec, F. Holsboar, J.M.H.M. Reul Max-Planck-lnstltuta of Psychiatry, Clinical Institute, Department of Neuroendocrinology, Munich. Germany Introduction: Glucocorticoids are widely used as immuncaupprassive agents. However, glucocorticoids seem to affect cellular and humorsi immunity in a distinct manner (i.e. inhibition vs stimulation). Our objective is to study how glucocorticoids regulate these principal tools of the immune system. Materials and Methods: In this study, we investigated glucocorticoid effects on anti T-cell receptor (TCR) stimulated spleen lymphocyte mitogenasis. Results: The natural glucocorticoid corticosterone (CORT), under specific conditions, appeared to exert tamporstiy distinct effects on anti*TCR induced splenocyte proliferation. Early lymphocyte proliferation (2-3 days] was strongly potantinted by CORT which preceded an inhibited cell growth at 5-7 days of culture. To produce such initially stimulated proliferation, CORT needed to be present at the time of TCR stimulation. Conclusion: Thus, glucocorticoids appear to accelerate anti-TCR induced T-cell proliferation. These data add a novel stimulatory component to the inhibitory properties of glucocorticolds and expand their regulatory role in cellular immunity.
WIZ.0T INTRATHECAL ADMINISTRATION OF SYNTHETIC PEPTIDE MBP75-g5 NEUTRALIZES MULTIPLE SCLEROSIS ANTI-MBP IN-VIVO
P17.16 THEILER'S VIRUS (TMEV) U P R E G ~ T E S THE EXPRF_~SION OF MHC CLASS I ON C E R E B R O V A S C ~ ENDOTHELIAL CELLS
K.G. Warren and I. Catz University of Alberta, Edmonton, CANADA
B.V. Sapatino and C.J.R. Welsh Department of Veterlnazy Anatomy and Public Health, Texas A&M University, College Station, Texas 77843-4458, USA.
The objective of this study was to determine whether synthetic peptide MBP75-95 will neutralize free (F) anti-MBP in CSF of MS patients after intrathecal inoculation. Matched pairs of MS patients with chronic progressive disease received 1, 2.5, 5 and 10 mg of either MBP35-58 or MBP75-95 dissolved in 5 ml of normal saline and injected into the CSF. Cerebrospinal fluid was sampled for anti-MBP monitoring before inoculation, every 30 minutes for 2 hours after injection, 24 hours later and then at weekly intervals until F anti-MBP levels returned to baseline value. Intrathecal administration of MBP75-95 produced neutralization of F antiMBP in a dose-dependent fasllion, whereas the control MBP35-58 failed to do so. With dosages of 5-10 mg of MBP75-95. the neutralization effect occurred within 30 minutes and persisted for penods up to 7 days. Since MS anti-MBP can be completely neutralized in vivo by MBP75-95 the epitope is most likely located in this area of the molecule. Additional Phase 1 reseamh is required prior to conducting Phase 2 trials for potential therapeutic benefit. If the autoimmune pathogenesis of MS is due to B cell and/or T lymphocyte mechaniams directed towards MBP in situ within the myelin sheath, it may be possible to stabilize this disease process by appropriate utilization of synthetic peptides.
Introduction: TMEV is a Plcornavlrus that persists in certain strains of mice and gives rise to demyelirmting disease which is sirnil~tr to multiple sclerosis. Both conditions are characterized by Immune cell Infiltration of the central nervous system (CNS]. We have been investigating i n t e r a c t i o n s b e t w e e n TMEV a n d cerebrovaacular endothelial cells [CVE]. Materials a n d Methods: CVE were isolated from the brains of SJL/J, CBA and BALB/c mice and cloned by the limiting dilution technique. The cells were characterized with respect to Factor VIII. ACE and uptake of acetylated LDL. CVE from all three strains of mice were Infected with TMEV and passaged six times and then examined for expresston of MHC Class I and II by flow cytometry. CVE from CBA and BALB/c mice expressed high levels of MHC Class I after infection with Thefler's virus (7S-94% of the cells were posttive for Class I c o m p a r e d to 10% in controls). Interestingly. SJL CVE did not express Class I. Co~n.~ TMEV infects CVE In vlvo and In vitro, In vitro infection of CBA and BALB/c CVE with TMEV, upregulated Class I expression which may allow these v/rally-infected cells to become, targets for cytotox~c T cells. Supported by NIH/NINDS #NS 29133.
P01.17
P17.17 MYOCARDIALINFARCTIONCAUSES REGIONAL REPRODUCIBLE BBB LEAKAGEVIA CYTOKINES.
MYELIN ASSOCIATED GLYCOPROTEIN: A TARGET AUTOANTIGEN IN EXPERIMENTAL AUTOIMMUNE ENCEPHALITIS S. Wssrth, R. Seitz, J. Mslotka, K. Dornmsir, Lassmann 1, H. Weksrle and C. Linington. Max-Planck Institute for Psychiatry, Martinsriad; Vienna, Vienna
T. Berger 1, H. 1 University of
Autoimmuna T- and B- cell responses against myelin associated glycoprotain (MAG) in patients with multiple sclerosis have been described by several groups. As the molecular basis of multiple sclerosis is still unknown, wa investigated the autoimmune response against MAC in the Lewis rat using synthetic rat MAC peptides and a panel of 6 recombinant rat ft~ion proteins. The sequences of these MAC fusion proteins represent the full length isoforms (S° + LMAC), the extracallular part and pairs of overlapping IgG domains (2 + 3, 3 + 4, 4 + 5). After subcloning these sequences in front of the His s coding sequence of the pQE12 vector, the fusion proteins were expressed in E. coil and purified by Ni-chelate chromatography, preparative SDS-PAGE and subsequent electroelution. Two diffarsnt T-cell epitopes were identified in the sxtracellular region corresponding to MPI.1 (aa 20-34) and MP3 (as 119-137) and one intracallular in the unique carboxy-terminal part of S-MAG corresponding to MP9 (aa 561-582). Adoptive transfer of these specific CD4 + T-cell lines in Lewis rats induced an inflammatory response in the CNS and PNS. These results identify MAC as an additional myelin autoantigen that could be involved in the immunopathoganesis of MS.
Y.D. van der WeftI, M.J.L. de Jongste2, G.J. ter Horstt Dept. of Biological Psychiatry and 2 Cardiology/Thoraxce~tre, Un/versityand AcademicHospitalGroninge~tt,Oostersinge159postbus 30.0019700 RB Groningem Introduction: It is known that myocardial infarction (MI) is accompanied by inflammation in the coronary vessels. It is hypothesized that the inflammatory proce~ becomes systemicand reaches the brain where it may have adveaseeffects on Central Nervous Systemfamctioning.Materials and Methods: MaleWistsr rats receiveda myocardialinfarctionthroughligationof part of the left veatricularwall. Between two and fourteendays after MI the rats were perfused. Resulls: Evidence for inflammationin the brain came from inmlt~2-cy~L.,~alcat~ ¢ m of ICAMpositive small vessels in certain areas of the brain, includingthe prefrontalcortex, the entorhinal cortex, the somatosansory cortex, the reticular formation and the bulints olfaetorius. Staining for Albumin revealed that specifically around the lCAM-posltive vessels there had been extntvasation of serum proteins. This indicates leakage of the Blood-Braln-Berrier(BBB), pmhably caused by reactive, cytokine-produeing leucocytes adhering to the vessel wall. In order to assess whether the inflammationrather than the operation resulted in leakageof the BBB, we injected TNF in the tail artery of Male Wister rats. The same procedureswere used, and the stainingof ICAM-positivevesselsand Albumin-positivepatches were observedin the same places in the brain. Conclusion: Myocardialinfarction results in a systemic inflammJationwhich causes leucocytes to adhere to the vessel walls in certain well-defined areas in the brain. The nonspecific immune overreaction cause~ extravasationof blood constituents and may result in loss of function of the specific brain areas.
W15.02
P08.20
MYELIN BASIC PROTEIN (MBP)-SPECIFIC TGFp-SECRETING CIM+ T-CELL CLONES DERIVED FROM ORALLY TOLERIZED A N I M A L S SUPPRESS E X P E R I M E N T A L A U T O I M M U N E ENCEPHALOMYELITIS. Youhai Chen. Viiav Kuchroo. Howar~l L. Weiner, Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115 USA Oral administration of MBP generates T cells capable of suppressing EAE which secrete regulatory cytokines TGFI3, IL-4, and IL-10 but not IFNy following specific antigen stimulation. In bulk culture, TGPI3 was produced by both CD4+ and CD8+ T cells whereas IL4 and I L l 0 were produced primarily by CD4+ T cells. SJ'L mice were fed five times with 0.5 mg MBP, boosted intrat~ritoneally with MBP in CFA and T cell lines derived from mesenteric lymph nodes. T cell clones were then generated by single cell clonin~g of the lines. Both CD4+ and CDa+ clones were established and detailed analysis of CD4+ clones undertaken. Clones were tested for epitope specificity, MHC restriction and cytokine production. Using overlapping 20 mer peptides, CD4+ clones react to two major MBP determinants, 84-102 and 71-90. All clones are IA s restricted. On cytokine analysis the clones fell into two categories: (1) TGFI3hi IL-4/IL-10Io T ceils which produced large amounts of T G I ~ mtd small amounts of IL-4/IL-10; and (2) TGFp Io ILo 4/IL-10tniT cells. Injection of these clones into SJL mice immunized for EAE with MBP or PLP suppressed the incidence and severity of disease. These clones will further allow characterization of regulatory cells generated by oral tolerance and TGFphi IL-4/IL-10io cells may represent a unique mucosal type TH2 cell. (Supported by NIH grant NS-29352.1
GLUCOCORTICOIDS ACCELERATE ANTI T-CELL RECEPTOR STIMULATED GROWTH OF RAT SPLEEN T-CELLS G.J. Wieoers. M.S. Labeur, I.E.M, Stec, F. Holsboar, J.M.H.M. Reul Max-Planck-lnstltuta of Psychiatry, Clinical Institute, Department of Neuroendocrinology, Munich. Germany Introduction: Glucocorticoids are widely used as immuncaupprassive agents. However, glucocorticoids seem to affect cellular and humorsi immunity in a distinct manner (i.e. inhibition vs stimulation). Our objective is to study how glucocorticoids regulate these principal tools of the immune system. Materials and Methods: In this study, we investigated glucocorticoid effects on anti T-cell receptor (TCR) stimulated spleen lymphocyte mitogenasis. Results: The natural glucocorticoid corticosterone (CORT), under specific conditions, appeared to exert tamporstiy distinct effects on anti*TCR induced splenocyte proliferation. Early lymphocyte proliferation (2-3 days] was strongly potantinted by CORT which preceded an inhibited cell growth at 5-7 days of culture. To produce such initially stimulated proliferation, CORT needed to be present at the time of TCR stimulation. Conclusion: Thus, glucocorticoids appear to accelerate anti-TCR induced T-cell proliferation. These data add a novel stimulatory component to the inhibitory properties of glucocorticolds and expand their regulatory role in cellular immunity.