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Absence of caspase 8 (FLICE) confers resistance to CD95-mediated apoptosis in human fetal astroglia
14
Poster Abstracts / Journal of Neuroimmunology 90 (1998) 13-105
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Fas and Immune Co-StimulatoryMolecules are Expressed on Neuronal Cell Surface Membranes
CD40 E n g a g e m e n t Stimulates IL-12P70 P r o d u c t i o n by Human Mieroglial Cells: R e g u l a t i o n b y A u t o e r i n e T N F B. Becher~ J.P. Antel, M. Blain, MNI, McGill University, Canada
D.D. Aleman-Hoey, University of Vermont, USA, ILB. Birge, J.L. Kupperman, Rockefeller University, USA,M.K. Newell, University of Vermont, USA
The T cell co-stimulatory molecules B7.1 and B7.2 are up-regulated on B cells of patients with SLE. Due to the prevalence of neuropsychiatric manifestations in SLE, we tested the hypothesis that B7.1 and B7.2 may also be expressed on neuronal cells. We observed the expression of Fas, BT. 1 and B7.2 on PC12 cells and their clonally derived variants, Trk and v-Crk. We analyzed the expression of these molecules flow cytometrically, atter stimulation (S) or withdrawal (W) of NGF or EGF. NGFS down-regulates Fas in PCI2 and v-Crk cells, but up-regulates it in Trk cells. EGFS up-regulates Fas on v-Crk cells. NGFW and EGFW up-regulate B7.1 in Trk cells but it is down-regulated by NGFS in PCI2 cells. B7.2 is induced by EGFS in v-Crk and Trk cells. The significance of the induction of Fas and co-stimulatory molecules on neuronal cells and its relevance to SLE is under investigation.
IL-12 is critical role in the development of pro-inflammatory Thl cells which are considered cenh'al mediators of the target-directed auto-tmmane disorders EAE and MS. This study describes an immano-regulatory circuit which results in the production of IL-12 by human adult CNS-derived mieroglial cells, triggered upon cell contact with activated T cells, indicating that endogenous APC can drive the polarization of Thl cells. This response can be blocked by anti-CD154 mAbs. IL12 production could also be induced by engagement of CD40 on microglia by CD154:mCD8 fusion protein in a dose dependent manner. Previously, inhibition of TNF by soluble TNF-R:Fc fusion protein has been shown to inhibit development of EAE. To evaluate the role of TNF and LT in CD40/CDI54 interactions between activated T cells and microglia, we show that IL-12 production by microglia is inhibited by recombinant human TNF receptor (II)IgGl fusion protein (TNF-R:Fc). Upon stimulation with CDI54:mCD8, IL-12 production is preceded by TNF-ct. Inhibition of TNF-ct and/or IFN-T leads to reduced IL-12 levels. These results describe an immuno-regulatory circuit dependent on CD40 engagement leading to IL-12 production by human microglial ceils and its regulation by the Thl cytokines TNF and IFN-7.
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CTLA4 Engagement Can Have Both Positive and Negative Effects on Human T Call Activation
Absence o f Caspase 8 ( F L I C E ) Confers Resistance to CD95Mediated Apoptosis i n H u m a n FetalAstroglia
D.E. Anders on, HarvardMedical School, USA, K. B ieganowska, Brigham and Women's Hospital and HarvardMedical School, USA, A. B ar-Or, Brigham and Women's Hospital, USA, D.A. Hailer, Brigham and Women'sHospital and HarvardMedical School, USA
B. Becher, J.P. Antel, A. Ezra an, MNI, McGill University, Canada
There have been conflicting reports on the differences between B7-med~aed costimulation though the CD28 and CTLA4 reoeptom, though the majority of dam suggest the signaling through CD28 inchces a positive costimulatory signal wha'r.as CTLA4 sends an inhibitory signal. We used CHO cell transfectants expresshag MHC c1I (DR2) alone, or in conjunction with either human B7-1 or B7-2, Fab fi'~ments of blocking anti-CTLA4 mAl~, anda MBP-reactivehuman T cell clone to investigate therole of CTLA4 engagement in human T cell activation. Because enhanced T cell prdifemtion or ceil suwival can be atlributed to either positive signals which enhance cloual expansion or inhibition of activation-inducedceil death (AiCD), we compared the effects of blocking anti-CTLA4 Fab to a blocking anti-Fas mAb present dating T cell stimulation. T ceil proliferativeresponses andcell suwival wele enl'anced to the same degree in the presence of anti-Fas mAb or anti-CTLA4 Fab, andtho~e was no addtiveefix:t when both were added together. Whm there was no significant AICD, blockade of CTLA4 had either no ef~ct or inhibited T cell proliferation The results suggest tha CTLA4 engagement can have both positive and negative efficts on human T cell stimulation depending on the sUength of T cell activation, and that CTLA4 engagement may inlluenoe the Fas signaling pathway.
CD95 (fas, Apo-l) has recently been shown to be expressed on oligodendrogliawithinthe CNS of patients with multiplesclerosis;httman oligodendrosliaare susceptibleto CD95 mediated cell death in vitro. Using flowcytometry, we have shown that human fetal CNSderivedastrogliaand their malignantcounterparts (U251-glinma)also express high levels of CD95 (fas, Apc.-l) on the cell surface. Using Annexin V stainingand PI-excinsionby flow cytometry, we show that U251 astrogliaand Jurkat T cells are susceptibleto CD95mediated apoptosis; fetal primary astroglia on the other hand are resistant to this deathpathway. Killing by C2-Ceramide confirmed that fetal astroglia carry the apoptotic signaling machinery. Ceramide formation is considered to occur downstream of proteolytic caspase activity. In an attempt to identify the differences in cell death signaling,we compared the presence of CD95-signalingcomponents in fetal astroglia to U251 cellsand Jurkat T cells. RNAseprotection assays revealedthat caspase 8 (FLICE)is absent in the fetal astroglia. Caspase 8 has been shown to be a crucial signaling component in the CD95 death pathway, indicatingthat its absence is responsible for the resistance of fetal astroglia to apoptosis. Althoughfetal astroglia express high levels of CD95 withoutsusceptibilityto apoptosis upon its engagement, the role of this molecule under non-pathologic conditions in the fetal CNS remains elusive. A possible role for CD95 in CNS-developmentis indicatedby the finding,that in contrast to fetal astroglia, mature adultastroglialoose CD95 expression.
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C T L A 4 Blockade Enhances B7-2 Dependent Th-2 Cytokine Production from Human MBP-reaetive T-cells A. Bar-Or. Brigham and Women'sHospital and Harvard Medical School, USA, D.E. Anderson, HarvardMedicalSchool, USA, K. Bieganowska, D.A. Hailer, Brigham and Women's'Hospital and HarvardMedicaI School, USA
Expression o f a B7-family Protein Distinct from BT. 1 and B7.2 L. Behrens. M. Kerschensteiner, T. Misgeld, Max-Planck [nstituteforNeurobiology, Martinsried, Germany, N. Goebels, Klinikum Grosshadern, Germany, H. Wekerle, R. Hohlfeld, Max-PlanckInstitute for Neurobiology, Martinsried, Germany
B7 molecules on antigen presenting cells (APCs) are necessary for activating naive T-cells r e s p o n d i n g to specific antigens. The selective effects o f B7-1 and B7-2 engagement o f their counterreceptors, C T L A - 4 and C D 2 8 , on T-cell cytokine production have not been elucidated in humans. Highly purified human C D 4 + T-cells were stimulated with M B P p 8 5 - 9 9 u s i n g C H O cell lines as A P C s expressing the appropriate M H C II (DRB 1* 1501) and either B7- I or B7-2 molecules with ~x-CTLA-4 or ct-CD28 Fabs. W e o b s e r v e d that B7-2 engagement o f CD28 in the absence o f a C T L A - 4 signal led to a dramatic enhancement o f antigen specific T-cell secretion o f the Th-2 cytokines, IL-4 and IL-5. Thus we demonstrate a dissociation in the cytokine profile o f e x vivo purified human M B P autoreactive T-cells upon selective costimulation with B7-1 vs. B7-2 molecules. The ability to generate Th-2 cytokines from human autoreactive T-cells, by modulating the costimulatory profile, may have important implications on therapeutic approaches to autoimmune diseases.
The B7 familyof costimulatorymolecules likely includesmembers distinct from B7.1 and B7.2. During an analysis of B7 expression in human muscle biopsy specimens we noted that in inflammatorymyopathy cases, but not in normal muscle, many fibers stronglyreact with anti-BB-I monoclonal antibodies. This mAb cross-reacts with B7,1 and as yet undefined eostimulatory molecule. Muscle fibers did not react with B7.1- or B7.2monospecific mAbs in any of the pathological specimens nor in normal muscle. Like muscle fibers in situ, human myoblasts in vitro express BB-I, but not B7,1 or B7.2, after stimulationwith IFN-T or TNF-a. The absence of B7. I and B7.2 was confirmed by RTPCR. The moleculedetected by anti-BB1 is functional, because CTLA4-Igand anti-BB1 mAb- but not anti-B7.1 or anti-B7.2 mAbs - completely inhibit Ag- presentation by cytokine induced myoblasts to HLA-DR matched Ag-specific CD4+ T cell lines. Our results demonstrate that in vivo and in vitro, human muscle cells can be induced to selectivelyexpress a functionalcostimulatorymolecule distinct from B7.1 and B7.2. This molecuIe may play an importantrole in the immunobiologyof muscle.
Poster Abstracts / Journal of Neuroimmunology 90 (1998) 13-105
53
56
Fas and Immune Co-StimulatoryMolecules are Expressed on Neuronal Cell Surface Membranes
CD40 E n g a g e m e n t Stimulates IL-12P70 P r o d u c t i o n by Human Mieroglial Cells: R e g u l a t i o n b y A u t o e r i n e T N F B. Becher~ J.P. Antel, M. Blain, MNI, McGill University, Canada
D.D. Aleman-Hoey, University of Vermont, USA, ILB. Birge, J.L. Kupperman, Rockefeller University, USA,M.K. Newell, University of Vermont, USA
The T cell co-stimulatory molecules B7.1 and B7.2 are up-regulated on B cells of patients with SLE. Due to the prevalence of neuropsychiatric manifestations in SLE, we tested the hypothesis that B7.1 and B7.2 may also be expressed on neuronal cells. We observed the expression of Fas, BT. 1 and B7.2 on PC12 cells and their clonally derived variants, Trk and v-Crk. We analyzed the expression of these molecules flow cytometrically, atter stimulation (S) or withdrawal (W) of NGF or EGF. NGFS down-regulates Fas in PCI2 and v-Crk cells, but up-regulates it in Trk cells. EGFS up-regulates Fas on v-Crk cells. NGFW and EGFW up-regulate B7.1 in Trk cells but it is down-regulated by NGFS in PCI2 cells. B7.2 is induced by EGFS in v-Crk and Trk cells. The significance of the induction of Fas and co-stimulatory molecules on neuronal cells and its relevance to SLE is under investigation.
IL-12 is critical role in the development of pro-inflammatory Thl cells which are considered cenh'al mediators of the target-directed auto-tmmane disorders EAE and MS. This study describes an immano-regulatory circuit which results in the production of IL-12 by human adult CNS-derived mieroglial cells, triggered upon cell contact with activated T cells, indicating that endogenous APC can drive the polarization of Thl cells. This response can be blocked by anti-CD154 mAbs. IL12 production could also be induced by engagement of CD40 on microglia by CD154:mCD8 fusion protein in a dose dependent manner. Previously, inhibition of TNF by soluble TNF-R:Fc fusion protein has been shown to inhibit development of EAE. To evaluate the role of TNF and LT in CD40/CDI54 interactions between activated T cells and microglia, we show that IL-12 production by microglia is inhibited by recombinant human TNF receptor (II)IgGl fusion protein (TNF-R:Fc). Upon stimulation with CDI54:mCD8, IL-12 production is preceded by TNF-ct. Inhibition of TNF-ct and/or IFN-T leads to reduced IL-12 levels. These results describe an immuno-regulatory circuit dependent on CD40 engagement leading to IL-12 production by human microglial ceils and its regulation by the Thl cytokines TNF and IFN-7.
54
57
CTLA4 Engagement Can Have Both Positive and Negative Effects on Human T Call Activation
Absence o f Caspase 8 ( F L I C E ) Confers Resistance to CD95Mediated Apoptosis i n H u m a n FetalAstroglia
D.E. Anders on, HarvardMedical School, USA, K. B ieganowska, Brigham and Women's Hospital and HarvardMedical School, USA, A. B ar-Or, Brigham and Women's Hospital, USA, D.A. Hailer, Brigham and Women'sHospital and HarvardMedical School, USA
B. Becher, J.P. Antel, A. Ezra an, MNI, McGill University, Canada
There have been conflicting reports on the differences between B7-med~aed costimulation though the CD28 and CTLA4 reoeptom, though the majority of dam suggest the signaling through CD28 inchces a positive costimulatory signal wha'r.as CTLA4 sends an inhibitory signal. We used CHO cell transfectants expresshag MHC c1I (DR2) alone, or in conjunction with either human B7-1 or B7-2, Fab fi'~ments of blocking anti-CTLA4 mAl~, anda MBP-reactivehuman T cell clone to investigate therole of CTLA4 engagement in human T cell activation. Because enhanced T cell prdifemtion or ceil suwival can be atlributed to either positive signals which enhance cloual expansion or inhibition of activation-inducedceil death (AiCD), we compared the effects of blocking anti-CTLA4 Fab to a blocking anti-Fas mAb present dating T cell stimulation. T ceil proliferativeresponses andcell suwival wele enl'anced to the same degree in the presence of anti-Fas mAb or anti-CTLA4 Fab, andtho~e was no addtiveefix:t when both were added together. Whm there was no significant AICD, blockade of CTLA4 had either no ef~ct or inhibited T cell proliferation The results suggest tha CTLA4 engagement can have both positive and negative efficts on human T cell stimulation depending on the sUength of T cell activation, and that CTLA4 engagement may inlluenoe the Fas signaling pathway.
CD95 (fas, Apo-l) has recently been shown to be expressed on oligodendrogliawithinthe CNS of patients with multiplesclerosis;httman oligodendrosliaare susceptibleto CD95 mediated cell death in vitro. Using flowcytometry, we have shown that human fetal CNSderivedastrogliaand their malignantcounterparts (U251-glinma)also express high levels of CD95 (fas, Apc.-l) on the cell surface. Using Annexin V stainingand PI-excinsionby flow cytometry, we show that U251 astrogliaand Jurkat T cells are susceptibleto CD95mediated apoptosis; fetal primary astroglia on the other hand are resistant to this deathpathway. Killing by C2-Ceramide confirmed that fetal astroglia carry the apoptotic signaling machinery. Ceramide formation is considered to occur downstream of proteolytic caspase activity. In an attempt to identify the differences in cell death signaling,we compared the presence of CD95-signalingcomponents in fetal astroglia to U251 cellsand Jurkat T cells. RNAseprotection assays revealedthat caspase 8 (FLICE)is absent in the fetal astroglia. Caspase 8 has been shown to be a crucial signaling component in the CD95 death pathway, indicatingthat its absence is responsible for the resistance of fetal astroglia to apoptosis. Althoughfetal astroglia express high levels of CD95 withoutsusceptibilityto apoptosis upon its engagement, the role of this molecule under non-pathologic conditions in the fetal CNS remains elusive. A possible role for CD95 in CNS-developmentis indicatedby the finding,that in contrast to fetal astroglia, mature adultastroglialoose CD95 expression.
55
58
C T L A 4 Blockade Enhances B7-2 Dependent Th-2 Cytokine Production from Human MBP-reaetive T-cells A. Bar-Or. Brigham and Women'sHospital and Harvard Medical School, USA, D.E. Anderson, HarvardMedicalSchool, USA, K. Bieganowska, D.A. Hailer, Brigham and Women's'Hospital and HarvardMedicaI School, USA
Expression o f a B7-family Protein Distinct from BT. 1 and B7.2 L. Behrens. M. Kerschensteiner, T. Misgeld, Max-Planck [nstituteforNeurobiology, Martinsried, Germany, N. Goebels, Klinikum Grosshadern, Germany, H. Wekerle, R. Hohlfeld, Max-PlanckInstitute for Neurobiology, Martinsried, Germany
B7 molecules on antigen presenting cells (APCs) are necessary for activating naive T-cells r e s p o n d i n g to specific antigens. The selective effects o f B7-1 and B7-2 engagement o f their counterreceptors, C T L A - 4 and C D 2 8 , on T-cell cytokine production have not been elucidated in humans. Highly purified human C D 4 + T-cells were stimulated with M B P p 8 5 - 9 9 u s i n g C H O cell lines as A P C s expressing the appropriate M H C II (DRB 1* 1501) and either B7- I or B7-2 molecules with ~x-CTLA-4 or ct-CD28 Fabs. W e o b s e r v e d that B7-2 engagement o f CD28 in the absence o f a C T L A - 4 signal led to a dramatic enhancement o f antigen specific T-cell secretion o f the Th-2 cytokines, IL-4 and IL-5. Thus we demonstrate a dissociation in the cytokine profile o f e x vivo purified human M B P autoreactive T-cells upon selective costimulation with B7-1 vs. B7-2 molecules. The ability to generate Th-2 cytokines from human autoreactive T-cells, by modulating the costimulatory profile, may have important implications on therapeutic approaches to autoimmune diseases.
The B7 familyof costimulatorymolecules likely includesmembers distinct from B7.1 and B7.2. During an analysis of B7 expression in human muscle biopsy specimens we noted that in inflammatorymyopathy cases, but not in normal muscle, many fibers stronglyreact with anti-BB-I monoclonal antibodies. This mAb cross-reacts with B7,1 and as yet undefined eostimulatory molecule. Muscle fibers did not react with B7.1- or B7.2monospecific mAbs in any of the pathological specimens nor in normal muscle. Like muscle fibers in situ, human myoblasts in vitro express BB-I, but not B7,1 or B7.2, after stimulationwith IFN-T or TNF-a. The absence of B7. I and B7.2 was confirmed by RTPCR. The moleculedetected by anti-BB1 is functional, because CTLA4-Igand anti-BB1 mAb- but not anti-B7.1 or anti-B7.2 mAbs - completely inhibit Ag- presentation by cytokine induced myoblasts to HLA-DR matched Ag-specific CD4+ T cell lines. Our results demonstrate that in vivo and in vitro, human muscle cells can be induced to selectivelyexpress a functionalcostimulatorymolecule distinct from B7.1 and B7.2. This molecuIe may play an importantrole in the immunobiologyof muscle.