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GPI-anchored molecules enhance proximal TCR signalling events
64
Signal transduction in lymphocytes
Reeuitaz Stimulation of human lymphocytes with monoclonal antibodies raised against the T-cell antigen receptor resulted in an immediate and transient activation of protein kinase C (PKC)-a and -8, whereas PKC isoenzymes PKC-6, -6 and -E were translocated with delay and remained bound to the plasma membrane up to 2 hours. Neutralization of PKC-a or 0 isoenzymes by specific antibodies introduced by electroporation resulted in a complete inhibition of interleukin-2 (IL-2) receptor expression. Introduction of anti-PKC-fi, -6 or -E antibodies led to a more then 70 per cent inhibition of IL-2 synthesis. Suppression of IL-2 synthesis was not enhanced by introducing combinations of several anti-PKC antibodies, suggesting that multiple signalllng pathways were involved in the regulation of IL-2 gene expression. Extensive control experiments have shown that neither electroporatfon nor introduction of control immunoglobulins influenced PKC activity and cellular functions of human T oeils. Conduslone: The data clearly show that different PKC isoenzymes regulate different cellular functions in stimulated lymphocytes. The results support the assumption that different T-cell receptor induced mechanisms underlie the activation of different PKC isoenzymes regulating different signal transduction pathways in activated lymphocytes.
P.1.02.28 L-I-I
Slgnal transduction by IFNa in human B cells
L. Hibbert, C. Brand I, L. Leadbeater ‘, H.C. Thomas, G.R. Foster. Department of Medicine, St Mary Hospital Medical School. England, ’ The Wellcome Foundation, England
Introduction: The type I interferons (12 subtypes of IFNa and IFNfi) are related cylokines with antiviral and immunomcdulatory activities. All of the subtypes have similar antiviral properties but subtle differences can be seen, for example in their immunomodulatory effects. This study was designed to investigate the effects of the type I ihterferons on 6 cell proliferation and signal transduction. Matariala and Methods: Human tonsillar B cells were isolated using Ficoll density gradient centrffugation followed by SRBC resetting to eliminate T cells. Resting B cells were isolated by further Percoll density gradient centrifugation. Proliferation assays and surface marker expression assays were carried out over three days. Activation of transcription factors was carded out using electrophoretic mobility shift assay (EMSA) with appropriate oligonucieotfde probes and tyrosine phosphoiylation of specific signal transduction molecules using immunoprecipitation and immunoblotting. Raauft.az It has been shown that all the IFNa subtypes can induce proliferation of human B cells, but that IFNa8 is aotive at much lower Concentrations (IOO-lOOO-fold).However, IFNaP and IFNa8 are able to increase MHC class I expression with similar doss response curves. One possible explanation for this is that the different intarferons act through different signalling pathways. Investigation of the STAT transcription factors revealed no differences between IFNa2 and IFNa8. Both were able to induce the binding activity of ISGF-3, composed of STAT-1 and STAT-P, but neither were able to induce activity of STAT-3,STAT-5or STAT-8. Conclusions: IFNa2 and IFNCZE can induce differential effects in human B cells, but the signal pathway responsible for these differences is not via the STATfamily of transcription factors. Other signal transduction pathways may be important, including those through the JAK family of tyrosine kinases, and these are at present under investigation.
P.1.02.29
Lowering cellular cholesterol Inhibits signal transduction via giycosyl phosphatldyl-lnosltoI(GPl)-anchored protelns
T.M. Stulnig ‘, M. Berger ‘, H. Stockinger *, V. Horej#, W. Waldh&usl ’ ’ De@. of fnlemal Medicine Ill, Unfversily of Vienna, Vienna, Au&f&, */nsl. of /mmuno/osv; Universify of Vienna, Vienna, Austria, 3Czech Acad. Sciences, Prague, Czech Republic Introduction: Glycosyl phosphatkfylinositol(GPl)-anchored proteins can deliver costimulatory signals to lymphocytes, but the exact signal transduction pathway is not yet characterized. GPI-anchored proteins have no transmembrane or cytoplasmic domain but are attached to the outer leaflet of the plasma membrane by a phospholipid moiety. Since GPI-anchored proteins are clustered in distinct membrane domains formed by an unique lipid composition requiring cholesterol, we studied the influence of metabolic alterations in cellular cholesterol content on the signal transduction via GPI-anchored proteins human T-cells. Yatarlala and Methods: Jurkat T-calls were grown in serum-free medium in presence of inhibitors of cholesterol synthesis, i.e. lovastatin. an inhibitor of 3-hydroxy-3-methyfglutaryl coenzyrne A reductase, or squalestatin, an inhibitor of squalene synthase. Calcium response was assessed by flow-cytometric determination of INDO-I fluorescence ratio. Detergent-insotuble complexes were analyzed by gel filtration and western blotting. Results: Lowering cholesterol suppressed calcium response via GPIanchored proteins CD59 and CD48 by about % whereas stimulation via the transmembrane CD3 complex was essentially unaffected. The decrease in overall calcium response via GPI-anchored proteins was reflected by an inhibi-
23 June 1997 - Poster presentations tion oftherelease of calcium from intracellular stores. Cell surface expression of GPI-anchored proteins was not changed quantitatively by the treatment, neither was the pattern of immunofluorescence in microscopic examination. In addffon, the distrtbution of GPl-anchored proteins in detergent-insoluble complexes was not altered. Conoluslon: We conclude that cellular cholesterol Is an important prereq uisite for signal transduction via GPI-anchored proteins beyond formation of membrane domains. This work was supported by the Austrian Science Foundation (Pll404Med).
) P.l.02.30 1 GPl-anchored molecules enhance proximal TCR slgnalllng events Paola Romagnoli, Claude Bron. institute of Biochemistry U&e&y Laosanne, 1066 Epalinges, Switzerland
of
Introduction: Previous reports suggested that T lymphocyte activation through GPI-anchored molecules is dependent on the surface expression of the T cell receptor (TCR). Here we show that stimulation of the TCR in five mutant cell lines which have deficiencies in GPI biosynthesis, fails to induce tyrosine phosphorylation of the TCR-< chain and ZAPJO, indicating that early events in TCR siganl transduction are affected in these mutants. Materials and Methods: The cell lines used in this study are T cell lymphoma and spontaneous Thy-l 1~ variants derived from them. These GPl-pmcessing mutants belong to five distinct classes of complementation defined by discrete steps of GPI-biosynthesis. Tyrosine phosphorylation events upon TCR engage ment have been analysed by western blot and in vitro kinasa assays. Ca++ mobilization was assessed by flow cytometry. Intracellular localization of Src kinases has been studied by subcellular fractionation and confocal microscopy. Raaultr:The roleof GPI-anchored molecules in TCR signal transduction was examined by analysing the intracellular signal transduction pathway following TCr stimulation in cell lines deficient in GPI-biosynthesis. In these cell lines Ca++ mobilisation was found to be severely compromised, despite a normal stoichimetty of the subunit of the TCR complex and comparable expression levels of the Src kinase fyn and Ick and of the phosphatase CD45. Analysis of more proximal TCR-signal transduction events revealed a defect in the tyrosine phosphorylation of the TCR-r chain and ZAP-70. This impaired phosphorylation was caused by a decreased accumulation of kinase-active fyn and Ick in engaged TCR complexes. Dlacuealon: We report here that proximal TCR-signaling events are enhanced by the presence of GPI-anchored molecules. Given the importance of the GPI anchor in signal transduction via GPI-anchored proteins, it is most probably its loss that affects TCR signaling. Specific microdomains enriched in GPI-anchored molecules and active Src kinases are found on the plasma membrane. Thus the lipid anchor could contribute to the formation of specific phosphatase-depletad or kinase rich microdomains on the membrane, where upon activation component of the T cell signaling machinery are preferentially located. Defects in the GPI-biosynthesis pathway would hinder the formation of such domains.
( P.1.02.31 1 Inactlvatlon of heterotrimerlc 612 proteins In human T-cells results In reduced IL 2 productlon E. Lippert, Y. Jacques, S. Hermouet. INSERM U 463 “Interactions ligands-&epteurs en immunologic et cancbtvlqie”, Groupe Wcepteurs et Cytokines”, lnstitut de Biologic, 9 Quai Moncousu, 44035 Nanfes, France Introduction: We used T-lymphocytes from pedpheral blood and Jurkat cells to investigate the functions of heterotdmerfc Gi proteins in human T-cell activation. Msterlalaand Methods:GI protein function was inhibited either by pertussis toxin (PT) (blood lymphocytes) or by stable expression of a dominant negative mutated form of the a subunit of GE (Gui2-G204A) proteins (Jurkat cells). In each case, we studied cell proliferation, IL 2 production, CD3 expression and CD89 up-regulation after stimulation by Leuw A or CD3 cross-linking. Raaults:In blood T-lymphocytes stimulated by Leuco A or CD3 cross-linking and grown in vitro for 7 days, PT treatment resulted in 50% inhibition of pmliferation. PT did not affect the up-regulation of IL 2 receptor (I chain expression, nor the transduction of IL 2 proliferation signals. In contrast, in purified T-cells exposed to PT, IL 2 production measured within 48 hours of CD3 cross-linking was decreased at least 80%. Addition of exogenous IL 2 prevented the inhibition of activated T-cell proliferation by PT. further suggesting that Gi proteins may modulate the proliferation of activated T-cells by regulating IL 2 production. In Jurkat cells expressing GaQ-G204A and stimulated by Leuco A or CD3 cross-linking, CD3 expression and activation were not altered, but up-regulation of CD89 was completely abolished, and IL 2 transcription and secretion were inhibited 80% compared to cells transfected with vector alone. Conclualon: Our data indicate that, in human T-cells, rnetabdic pathways controlled by GQ proteins regulate IL 2 production after TCR activation, resulting in modulation of activated T-cell proliferation.
Signal transduction in lymphocytes
Reeuitaz Stimulation of human lymphocytes with monoclonal antibodies raised against the T-cell antigen receptor resulted in an immediate and transient activation of protein kinase C (PKC)-a and -8, whereas PKC isoenzymes PKC-6, -6 and -E were translocated with delay and remained bound to the plasma membrane up to 2 hours. Neutralization of PKC-a or 0 isoenzymes by specific antibodies introduced by electroporation resulted in a complete inhibition of interleukin-2 (IL-2) receptor expression. Introduction of anti-PKC-fi, -6 or -E antibodies led to a more then 70 per cent inhibition of IL-2 synthesis. Suppression of IL-2 synthesis was not enhanced by introducing combinations of several anti-PKC antibodies, suggesting that multiple signalllng pathways were involved in the regulation of IL-2 gene expression. Extensive control experiments have shown that neither electroporatfon nor introduction of control immunoglobulins influenced PKC activity and cellular functions of human T oeils. Conduslone: The data clearly show that different PKC isoenzymes regulate different cellular functions in stimulated lymphocytes. The results support the assumption that different T-cell receptor induced mechanisms underlie the activation of different PKC isoenzymes regulating different signal transduction pathways in activated lymphocytes.
P.1.02.28 L-I-I
Slgnal transduction by IFNa in human B cells
L. Hibbert, C. Brand I, L. Leadbeater ‘, H.C. Thomas, G.R. Foster. Department of Medicine, St Mary Hospital Medical School. England, ’ The Wellcome Foundation, England
Introduction: The type I interferons (12 subtypes of IFNa and IFNfi) are related cylokines with antiviral and immunomcdulatory activities. All of the subtypes have similar antiviral properties but subtle differences can be seen, for example in their immunomodulatory effects. This study was designed to investigate the effects of the type I ihterferons on 6 cell proliferation and signal transduction. Matariala and Methods: Human tonsillar B cells were isolated using Ficoll density gradient centrffugation followed by SRBC resetting to eliminate T cells. Resting B cells were isolated by further Percoll density gradient centrifugation. Proliferation assays and surface marker expression assays were carried out over three days. Activation of transcription factors was carded out using electrophoretic mobility shift assay (EMSA) with appropriate oligonucieotfde probes and tyrosine phosphoiylation of specific signal transduction molecules using immunoprecipitation and immunoblotting. Raauft.az It has been shown that all the IFNa subtypes can induce proliferation of human B cells, but that IFNa8 is aotive at much lower Concentrations (IOO-lOOO-fold).However, IFNaP and IFNa8 are able to increase MHC class I expression with similar doss response curves. One possible explanation for this is that the different intarferons act through different signalling pathways. Investigation of the STAT transcription factors revealed no differences between IFNa2 and IFNa8. Both were able to induce the binding activity of ISGF-3, composed of STAT-1 and STAT-P, but neither were able to induce activity of STAT-3,STAT-5or STAT-8. Conclusions: IFNa2 and IFNCZE can induce differential effects in human B cells, but the signal pathway responsible for these differences is not via the STATfamily of transcription factors. Other signal transduction pathways may be important, including those through the JAK family of tyrosine kinases, and these are at present under investigation.
P.1.02.29
Lowering cellular cholesterol Inhibits signal transduction via giycosyl phosphatldyl-lnosltoI(GPl)-anchored protelns
T.M. Stulnig ‘, M. Berger ‘, H. Stockinger *, V. Horej#, W. Waldh&usl ’ ’ De@. of fnlemal Medicine Ill, Unfversily of Vienna, Vienna, Au&f&, */nsl. of /mmuno/osv; Universify of Vienna, Vienna, Austria, 3Czech Acad. Sciences, Prague, Czech Republic Introduction: Glycosyl phosphatkfylinositol(GPl)-anchored proteins can deliver costimulatory signals to lymphocytes, but the exact signal transduction pathway is not yet characterized. GPI-anchored proteins have no transmembrane or cytoplasmic domain but are attached to the outer leaflet of the plasma membrane by a phospholipid moiety. Since GPI-anchored proteins are clustered in distinct membrane domains formed by an unique lipid composition requiring cholesterol, we studied the influence of metabolic alterations in cellular cholesterol content on the signal transduction via GPI-anchored proteins human T-cells. Yatarlala and Methods: Jurkat T-calls were grown in serum-free medium in presence of inhibitors of cholesterol synthesis, i.e. lovastatin. an inhibitor of 3-hydroxy-3-methyfglutaryl coenzyrne A reductase, or squalestatin, an inhibitor of squalene synthase. Calcium response was assessed by flow-cytometric determination of INDO-I fluorescence ratio. Detergent-insotuble complexes were analyzed by gel filtration and western blotting. Results: Lowering cholesterol suppressed calcium response via GPIanchored proteins CD59 and CD48 by about % whereas stimulation via the transmembrane CD3 complex was essentially unaffected. The decrease in overall calcium response via GPI-anchored proteins was reflected by an inhibi-
23 June 1997 - Poster presentations tion oftherelease of calcium from intracellular stores. Cell surface expression of GPI-anchored proteins was not changed quantitatively by the treatment, neither was the pattern of immunofluorescence in microscopic examination. In addffon, the distrtbution of GPl-anchored proteins in detergent-insoluble complexes was not altered. Conoluslon: We conclude that cellular cholesterol Is an important prereq uisite for signal transduction via GPI-anchored proteins beyond formation of membrane domains. This work was supported by the Austrian Science Foundation (Pll404Med).
) P.l.02.30 1 GPl-anchored molecules enhance proximal TCR slgnalllng events Paola Romagnoli, Claude Bron. institute of Biochemistry U&e&y Laosanne, 1066 Epalinges, Switzerland
of
Introduction: Previous reports suggested that T lymphocyte activation through GPI-anchored molecules is dependent on the surface expression of the T cell receptor (TCR). Here we show that stimulation of the TCR in five mutant cell lines which have deficiencies in GPI biosynthesis, fails to induce tyrosine phosphorylation of the TCR-< chain and ZAPJO, indicating that early events in TCR siganl transduction are affected in these mutants. Materials and Methods: The cell lines used in this study are T cell lymphoma and spontaneous Thy-l 1~ variants derived from them. These GPl-pmcessing mutants belong to five distinct classes of complementation defined by discrete steps of GPI-biosynthesis. Tyrosine phosphorylation events upon TCR engage ment have been analysed by western blot and in vitro kinasa assays. Ca++ mobilization was assessed by flow cytometry. Intracellular localization of Src kinases has been studied by subcellular fractionation and confocal microscopy. Raaultr:The roleof GPI-anchored molecules in TCR signal transduction was examined by analysing the intracellular signal transduction pathway following TCr stimulation in cell lines deficient in GPI-biosynthesis. In these cell lines Ca++ mobilisation was found to be severely compromised, despite a normal stoichimetty of the subunit of the TCR complex and comparable expression levels of the Src kinase fyn and Ick and of the phosphatase CD45. Analysis of more proximal TCR-signal transduction events revealed a defect in the tyrosine phosphorylation of the TCR-r chain and ZAP-70. This impaired phosphorylation was caused by a decreased accumulation of kinase-active fyn and Ick in engaged TCR complexes. Dlacuealon: We report here that proximal TCR-signaling events are enhanced by the presence of GPI-anchored molecules. Given the importance of the GPI anchor in signal transduction via GPI-anchored proteins, it is most probably its loss that affects TCR signaling. Specific microdomains enriched in GPI-anchored molecules and active Src kinases are found on the plasma membrane. Thus the lipid anchor could contribute to the formation of specific phosphatase-depletad or kinase rich microdomains on the membrane, where upon activation component of the T cell signaling machinery are preferentially located. Defects in the GPI-biosynthesis pathway would hinder the formation of such domains.
( P.1.02.31 1 Inactlvatlon of heterotrimerlc 612 proteins In human T-cells results In reduced IL 2 productlon E. Lippert, Y. Jacques, S. Hermouet. INSERM U 463 “Interactions ligands-&epteurs en immunologic et cancbtvlqie”, Groupe Wcepteurs et Cytokines”, lnstitut de Biologic, 9 Quai Moncousu, 44035 Nanfes, France Introduction: We used T-lymphocytes from pedpheral blood and Jurkat cells to investigate the functions of heterotdmerfc Gi proteins in human T-cell activation. Msterlalaand Methods:GI protein function was inhibited either by pertussis toxin (PT) (blood lymphocytes) or by stable expression of a dominant negative mutated form of the a subunit of GE (Gui2-G204A) proteins (Jurkat cells). In each case, we studied cell proliferation, IL 2 production, CD3 expression and CD89 up-regulation after stimulation by Leuw A or CD3 cross-linking. Raaults:In blood T-lymphocytes stimulated by Leuco A or CD3 cross-linking and grown in vitro for 7 days, PT treatment resulted in 50% inhibition of pmliferation. PT did not affect the up-regulation of IL 2 receptor (I chain expression, nor the transduction of IL 2 proliferation signals. In contrast, in purified T-cells exposed to PT, IL 2 production measured within 48 hours of CD3 cross-linking was decreased at least 80%. Addition of exogenous IL 2 prevented the inhibition of activated T-cell proliferation by PT. further suggesting that Gi proteins may modulate the proliferation of activated T-cells by regulating IL 2 production. In Jurkat cells expressing GaQ-G204A and stimulated by Leuco A or CD3 cross-linking, CD3 expression and activation were not altered, but up-regulation of CD89 was completely abolished, and IL 2 transcription and secretion were inhibited 80% compared to cells transfected with vector alone. Conclualon: Our data indicate that, in human T-cells, rnetabdic pathways controlled by GQ proteins regulate IL 2 production after TCR activation, resulting in modulation of activated T-cell proliferation.