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PS3-24 Structural profiles of cytokine families – Revisiting classification
Abstracts / Cytokine 52 (2010) 82–98 University of Turin, Turin, Italy, 3 Department of Clinical and Experimental Medicine, Second University of Naples, Napoli, 4 Institute of Medical Technology, University of Tampere, Tampere, Finland, 5 Department of Clinical and Biological Sciences, Division of Neurology, San Luigi Gonzaga School of Medicine, Orbassano, Turin, Italy The refractoriness of T cells to the inhibitory effect of Interferon (IFN)-c is mainly attributed to down-regulation of IFN-cR2 signalling chain. Here we show that IFN-cR2 accumulated on the cell surface of in vitro generated T helper (Th) 17 cells, as opposed to Th1 cells, and rendered them sensitive to IFN-c/STAT1 signaling. Of note, a more pronounced IFN-cR2 cell surface expression and sensitivity to IFN-c was observed in peripheral Th17 cells from multiple sclerosis patients than in Th17 from healthy subjects. Analysis of IFN-cR2 mRNA and protein in T cell clones from multiple sclerosis patients and polarized T cells from healthy subjects showed that IFN-cR2 was equally expressed in Th1 and Th17 cells, whereas mRNA for JAK2 was highly expressed in Th17 cells only. JAK2-deficient c2A cells displayed a rapid internalization and degradation of cell surface IFN-cR2, which was prevented by transduction of JAK2. In conclusion, these data identify JAK2 as a critical factor that stabilizes IFNcR2 surface expression in self-reactive Th17 cells and renders them sensitive to IFN-c.
doi:10.1016/j.cyto.2010.07.362
PS3-24 Structural profiles of cytokine families – Revisiting classification Awanti Sambarey, Nagasuma Chandra, Bioinformatics Centre, Indian Institute of Science, Bangalore, Karnataka, India Several attempts have been made to classify cytokines, such as those based on their order of discovery, function, role in inflammatory response, cell of origin and more recently, their shared structural homologies. We propose a detailed, unified schema for the classification of cytokines that takes into account sequence, structural and interacting-site properties of cytokines. Cytokines are initially clustered on the basis of their sequence similarity, which to a certain extent correlates with their biological function. These clusters are subsequently mapped onto fold-based clusters, generated as a result of structural super-positions of known and modeled cytokine structures. Identification of functional sites on cytokine structures and the resulting clustering pattern based on their site similarities may help fathom the specificity of interactions among cytokines and their receptors, which ultimately govern their functional effects. Such a comprehensive categorization of cytokines encompassing features of cytokine sequences, structures, interacting properties and functions provides a deeper insight into their biological activity. doi:10.1016/j.cyto.2010.07.363
PS3-25 Platelets are a source of macrophage-derived chemokine and thymus and activation-regulated chemokine in the newborn Trisha V. Macfarlane, Ruth H. Jones, Aled H. Bryant, Ina Laura Pieper, Gareth Morgan, Catherine A. Thornton, Institute of Life Science, School of Medicine, Swansea University, Swansea, Wales, UK Circulating macrophage-derived chemokine (MDC; CCL22) and thymus and activation-regulated chemokine (TARC; CCL17) levels are higher at birth than in adulthood and decline with increasing age in childhood. Variation in levels of these chemokines in the peri-natal period has implications for the early life origins of allergic disease. The main objective of this work was to identify the cellular source(s) of MDC and TARC in the fetal/neonatal circulation. IL-4- or IL-13-induced production of MDC and TARC by mononuclear cells (MNCs) from umbilical cord blood was not elevated compared to that from adult peripheral blood MNCs. Gestation-associated tissues only produced MDC and TARC on exposure to IL-4 or IL-13. As serum levels of both chemokines are higher than plasma levels, platelets were postulated as a source of these chemokines. Peripheral blood was collected from healthy adults, pregnant women (>36 weeks gestation), and newborns (umbilical cord blood). Serum and platelet-free plasma (PFP) were obtained by centrifugation, and platelets were isolated; all were stored at 20 °C. MDC and TARC were measured using specific ELISAs; platelets were lysed via two freeze-thaw-sonication cycles prior to analysis and values were corrected for platelet volume in whole blood. MDC and TARC could be detected in serum, plasma and isolated platelets from all donors; serum levels were higher than plasma levels in all three study groups; levels were highest in samples prepared from umbilical cord blood. Lysed isolated platelets (purity > 94% by flow cytometry) had similar levels of MDC and TARC in all groups; when corrected for the number of platelets present in whole blood, chemokine levels associated with umbilical cord blood were elevated compared to the other groups. These results indicate that higher levels of TARC and
87
MDC at birth reflect higher number of platelets per millilitre of blood at this stage of development. doi:10.1016/j.cyto.2010.07.364
Cytokines and Cell Death PS3-26 Phosphorylation of PACT at specific serines in response to stress signals regulates PKR activation by reducing PACT’s interaction with TRBP Madhurima Singh 1, David Castillo 1, Chandrashekhar V. Patel 2, Rekha C. Patel 1, 1 Department of Biological Sciences, University of South Carolina, Columbia, SC USA, 2 Department of Cell, Developmental Biology, and Anatomy, University of South Carolina Medical School, Columbia, SC USA PKR is an interferon (IFN)-induced serine-threonine protein kinase, which plays a crucial role in IFN’s antiviral and antiproliferative actions. PACT activates PKR by direct protein-protein interaction in response to cellular stress. The human TAR (trans-activating region)-binding protein (TRBP), which is homologous to PACT, also interacts with PKR leading to an inhibition of PKR activity. The focus of this study was to investigate the interplay between TRBP and PACT proteins on the regulation of PKR activation in response to stress signals. Our results demonstrate that TRBPPACT interaction controls PACT activation of PKR. Previously, it has been shown that phosphorylation of serines 246 and 287 of PACT is required for PKR activation in response to stress signals. We have extended these studies to investigate the effect of PACT phosphorylation on TRBP-PACT interactions and PKR activation. Yeast two hybrid assays as well as co-immunoprecipitation data demonstrates that PACT-TRBP interaction is significantly weakened due to phosphorylation of PACT on serine 246 and serine 287 in response to cellular stress. In contrast to this, PKR-PACT interaction is significantly strengthened by phosphorylation at these sites. In apoptosis assays, an overexpression of TRBP efficiently inhibits wtPACT-induced apoptosis but does not inhibit apoptosis induced by the phosphomimetic mutant of PACT (S246D, S287D). Thus, our results demonstrate that stress-induced phosphorylation at serines 246 and 287 decreases PACT’s interaction with TRBP while increases its interaction with PKR and thus serves a dual function in stress-dependent activation of PKR. doi:10.1016/j.cyto.2010.07.365
PS3-28 IL-21 induces apoptosis of splenic conventional dendritic cells through STAT3 and BIM and can be rescued by GM-CSF Edwin C.K. Wan, Warren J. Leonard, Laboratory of Molecular Immunology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA IL-21 is a type I cytokine with pleiotropic actions. The IL-21 receptor Pis composed of IL-21R and the common cytokine receptor c chain (cc), mutation of which causes X-linked severe combined immunodeficiency (XSCID) in humans. The effects of IL-21 on T and B cells have been extensively studied, but little is known about the roles of IL-21 on dendritic cells. We show here that IL-21 induces apoptosis of splenic conventional dendritic cells (cDCs). GM-CSF inhibits IL-21-induced apoptosis as well as Bim and Il21r gene expression. IL-21-induced apoptosis of DCs is abolished in Bim KO mice, indicating that BIM is critical for IL-21-induced apoptosis of cDCs. IL21 activates mainly STAT1 and STAT3 in cDCs. Using KO mice, we found that STAT3 but not STAT1 is essential for IL-21-induced apoptosis. Interestingly, microarray analysis showed that GM-CSF inhibited expression of only 33 out of 209 IL-21-induced genes, suggesting that GM-CSF does not globally inhibits the effects of IL-21 on splenic DCs even though it diminishes IL-21 receptor expression. These findings elucidate how GM-CSF can modulate the actions of IL-21 on DCs, with potential implications for modulating the immune responses. doi:10.1016/j.cyto.2010.07.367
PS3-29 A defect in store-operated calcium entry sensitizes T cells to Type I interferoninduced apoptosis Chanyu Yue, Jonathan Soboloff, Ana M. Gamero, Department of Biochemistry, Temple University School of Medicine, Philadelphia, PA, USA Type I interferons (IFN-a/b) are a family of pleiotropic cytokines known to modulate T cell responses. IFN-a/b inhibits the proliferation of activated T cells by delaying their entry into the cell cycle. IFN-a/b supports the survival of memory CD8+ T
doi:10.1016/j.cyto.2010.07.362
PS3-24 Structural profiles of cytokine families – Revisiting classification Awanti Sambarey, Nagasuma Chandra, Bioinformatics Centre, Indian Institute of Science, Bangalore, Karnataka, India Several attempts have been made to classify cytokines, such as those based on their order of discovery, function, role in inflammatory response, cell of origin and more recently, their shared structural homologies. We propose a detailed, unified schema for the classification of cytokines that takes into account sequence, structural and interacting-site properties of cytokines. Cytokines are initially clustered on the basis of their sequence similarity, which to a certain extent correlates with their biological function. These clusters are subsequently mapped onto fold-based clusters, generated as a result of structural super-positions of known and modeled cytokine structures. Identification of functional sites on cytokine structures and the resulting clustering pattern based on their site similarities may help fathom the specificity of interactions among cytokines and their receptors, which ultimately govern their functional effects. Such a comprehensive categorization of cytokines encompassing features of cytokine sequences, structures, interacting properties and functions provides a deeper insight into their biological activity. doi:10.1016/j.cyto.2010.07.363
PS3-25 Platelets are a source of macrophage-derived chemokine and thymus and activation-regulated chemokine in the newborn Trisha V. Macfarlane, Ruth H. Jones, Aled H. Bryant, Ina Laura Pieper, Gareth Morgan, Catherine A. Thornton, Institute of Life Science, School of Medicine, Swansea University, Swansea, Wales, UK Circulating macrophage-derived chemokine (MDC; CCL22) and thymus and activation-regulated chemokine (TARC; CCL17) levels are higher at birth than in adulthood and decline with increasing age in childhood. Variation in levels of these chemokines in the peri-natal period has implications for the early life origins of allergic disease. The main objective of this work was to identify the cellular source(s) of MDC and TARC in the fetal/neonatal circulation. IL-4- or IL-13-induced production of MDC and TARC by mononuclear cells (MNCs) from umbilical cord blood was not elevated compared to that from adult peripheral blood MNCs. Gestation-associated tissues only produced MDC and TARC on exposure to IL-4 or IL-13. As serum levels of both chemokines are higher than plasma levels, platelets were postulated as a source of these chemokines. Peripheral blood was collected from healthy adults, pregnant women (>36 weeks gestation), and newborns (umbilical cord blood). Serum and platelet-free plasma (PFP) were obtained by centrifugation, and platelets were isolated; all were stored at 20 °C. MDC and TARC were measured using specific ELISAs; platelets were lysed via two freeze-thaw-sonication cycles prior to analysis and values were corrected for platelet volume in whole blood. MDC and TARC could be detected in serum, plasma and isolated platelets from all donors; serum levels were higher than plasma levels in all three study groups; levels were highest in samples prepared from umbilical cord blood. Lysed isolated platelets (purity > 94% by flow cytometry) had similar levels of MDC and TARC in all groups; when corrected for the number of platelets present in whole blood, chemokine levels associated with umbilical cord blood were elevated compared to the other groups. These results indicate that higher levels of TARC and
87
MDC at birth reflect higher number of platelets per millilitre of blood at this stage of development. doi:10.1016/j.cyto.2010.07.364
Cytokines and Cell Death PS3-26 Phosphorylation of PACT at specific serines in response to stress signals regulates PKR activation by reducing PACT’s interaction with TRBP Madhurima Singh 1, David Castillo 1, Chandrashekhar V. Patel 2, Rekha C. Patel 1, 1 Department of Biological Sciences, University of South Carolina, Columbia, SC USA, 2 Department of Cell, Developmental Biology, and Anatomy, University of South Carolina Medical School, Columbia, SC USA PKR is an interferon (IFN)-induced serine-threonine protein kinase, which plays a crucial role in IFN’s antiviral and antiproliferative actions. PACT activates PKR by direct protein-protein interaction in response to cellular stress. The human TAR (trans-activating region)-binding protein (TRBP), which is homologous to PACT, also interacts with PKR leading to an inhibition of PKR activity. The focus of this study was to investigate the interplay between TRBP and PACT proteins on the regulation of PKR activation in response to stress signals. Our results demonstrate that TRBPPACT interaction controls PACT activation of PKR. Previously, it has been shown that phosphorylation of serines 246 and 287 of PACT is required for PKR activation in response to stress signals. We have extended these studies to investigate the effect of PACT phosphorylation on TRBP-PACT interactions and PKR activation. Yeast two hybrid assays as well as co-immunoprecipitation data demonstrates that PACT-TRBP interaction is significantly weakened due to phosphorylation of PACT on serine 246 and serine 287 in response to cellular stress. In contrast to this, PKR-PACT interaction is significantly strengthened by phosphorylation at these sites. In apoptosis assays, an overexpression of TRBP efficiently inhibits wtPACT-induced apoptosis but does not inhibit apoptosis induced by the phosphomimetic mutant of PACT (S246D, S287D). Thus, our results demonstrate that stress-induced phosphorylation at serines 246 and 287 decreases PACT’s interaction with TRBP while increases its interaction with PKR and thus serves a dual function in stress-dependent activation of PKR. doi:10.1016/j.cyto.2010.07.365
PS3-28 IL-21 induces apoptosis of splenic conventional dendritic cells through STAT3 and BIM and can be rescued by GM-CSF Edwin C.K. Wan, Warren J. Leonard, Laboratory of Molecular Immunology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA IL-21 is a type I cytokine with pleiotropic actions. The IL-21 receptor Pis composed of IL-21R and the common cytokine receptor c chain (cc), mutation of which causes X-linked severe combined immunodeficiency (XSCID) in humans. The effects of IL-21 on T and B cells have been extensively studied, but little is known about the roles of IL-21 on dendritic cells. We show here that IL-21 induces apoptosis of splenic conventional dendritic cells (cDCs). GM-CSF inhibits IL-21-induced apoptosis as well as Bim and Il21r gene expression. IL-21-induced apoptosis of DCs is abolished in Bim KO mice, indicating that BIM is critical for IL-21-induced apoptosis of cDCs. IL21 activates mainly STAT1 and STAT3 in cDCs. Using KO mice, we found that STAT3 but not STAT1 is essential for IL-21-induced apoptosis. Interestingly, microarray analysis showed that GM-CSF inhibited expression of only 33 out of 209 IL-21-induced genes, suggesting that GM-CSF does not globally inhibits the effects of IL-21 on splenic DCs even though it diminishes IL-21 receptor expression. These findings elucidate how GM-CSF can modulate the actions of IL-21 on DCs, with potential implications for modulating the immune responses. doi:10.1016/j.cyto.2010.07.367
PS3-29 A defect in store-operated calcium entry sensitizes T cells to Type I interferoninduced apoptosis Chanyu Yue, Jonathan Soboloff, Ana M. Gamero, Department of Biochemistry, Temple University School of Medicine, Philadelphia, PA, USA Type I interferons (IFN-a/b) are a family of pleiotropic cytokines known to modulate T cell responses. IFN-a/b inhibits the proliferation of activated T cells by delaying their entry into the cell cycle. IFN-a/b supports the survival of memory CD8+ T