RESUMEN
Ag encounter by naive CD8 T cells initiates a developmental program consisting of cellular proliferation, changes in gene expression, and the formation of effector and memory T cells. The strength and duration of TCR signaling are known to be important parameters regulating the differentiation of naive CD8 T cells, although the molecular signals arbitrating these processes remain poorly defined. The Ras-guanyl nucleotide exchange factor RasGRP1 has been shown to transduce TCR-mediated signals critically required for the maturation of developing thymocytes. To elucidate the role of RasGRP1 in CD8 T cell differentiation, in vitro and in vivo experiments were performed with 2C TCR transgenic CD8 T cells lacking RasGRP1. In this study, we report that RasGRP1 regulates the threshold of T cell activation and Ag-induced expansion, at least in part, through the regulation of IL-2 production. Moreover, RasGRP1(-/-) 2C CD8 T cells exhibit an anergic phenotype in response to cognate Ag stimulation that is partially reversible upon the addition of exogenous IL-2. By contrast, the capacity of IL-2/IL-2R interactions to mediate Ras activation and CD8 T cell expansion and differentiation appears to be largely RasGRP1-independent. Collectively, our results demonstrate that RasGRP1 plays a selective role in T cell signaling, controlling the initiation and duration of CD8 T cell immune responses.
Asunto(s)
Antígenos/inmunología , Linfocitos T CD8-positivos/inmunología , Factores de Intercambio de Guanina Nucleótido/fisiología , Animales , Diferenciación Celular/inmunología , Proliferación Celular , Factores de Intercambio de Guanina Nucleótido/deficiencia , Factores de Intercambio de Guanina Nucleótido/inmunología , Interleucina-2/farmacología , Activación de Linfocitos/inmunología , Ratones , Ratones Noqueados , Transducción de Señal/inmunología , TransgenesRESUMEN
Eosinophil degranulation is thought to play a pathophysiological role in asthma. Rab27A is a GTP-binding protein that is known to be essential for the degranulation of several leukocyte subsets and thus may be essential for eosinophil granule exocytosis. Here, we show that Rab27A mRNA and protein are expressed in human eosinophils. We have developed a novel assay to assess Rab27A activation and have found a similar activation pattern of this protein upon stimulation of eosinophils, neutrophils and NK cells suggesting a similar function in these cell types. Interestingly, Rab27A expression was elevated in eosinophils from asthmatic donors. Furthermore, eosinophils from eosinophilic donors displayed more rapid Rab27A activation kinetics than those from donors with lower eosinophil counts. Given that elevated blood eosinophil numbers correlate with increased priming of eosinophils, this pattern of Rab27A activation suggests differential protein expression in activated cells may allow eosinophils to degranulate more rapidly upon stimulation.
Asunto(s)
Degranulación de la Célula , Eosinofilia/enzimología , Eosinófilos/enzimología , Proteínas de Unión al GTP rab/biosíntesis , Asma/enzimología , Proteínas Bacterianas/inmunología , Activación Enzimática , Eosinofilia/sangre , Eosinófilos/inmunología , Exocitosis , Humanos , Células Asesinas Naturales/enzimología , Células Asesinas Naturales/inmunología , Neutrófilos/enzimología , Neutrófilos/inmunología , ARN Mensajero/biosíntesis , Proteínas de Unión al GTP rab/genética , Proteínas rab27 de Unión a GTPRESUMEN
Natural killer cells recognize and induce apoptosis in foreign, transformed or virus-infected cells through the release of perforin and granzymes from secretory lysosomes. Clinically, NK-cell mediated killing is a major limitation to successful allo- and xenotransplantation. The molecular mechanisms that regulate the fusion of granzyme B-containing secretory lysosomes to the plasma membrane in activated NK cells, prior to target cell killing, are not fully understood. Using the NK cell line YT-Indy as a model, we have investigated the expression of SNAP REceptors (SNAREs), both target (t-) and vesicular (v-) SNAREs, and their function in granzyme B-mediated target cell killing. Our data showed that YT-Indy cells express VAMP-7 and SNAP-23, but not VAMP-2. VAMP-7 was associated with granzyme B-containing lysosomal granules. Using VAMP-7 small interfering RNA (siRNA), we successfully knocked down the expression of VAMP-7 protein in YT-Indy to less than 10% of untreated cells in 24h. VAMP7-deficient YT-Indy cells activated via co-culture with Jurkat cells released <1ng/mL of granzyme B, compared to 1.5-2.5 microg/mL from controls. Using Jurkat cells as targets, we showed a 7-fold reduction in NK cell-mediated killing by VAMP-7 deficient YT-Indy cells. Our results show that VAMP-7 is a crucial component of granzyme B release and target cell killing in the NK cell line YT-Indy. Thus, targeting VAMP-7 expression specifically with siRNA, following transplantation, may be a viable strategy for preventing NK cell-mediated transplant rejection, in vivo.
Asunto(s)
Apoptosis/fisiología , Granzimas/metabolismo , Células Asesinas Naturales/metabolismo , Proteínas R-SNARE/metabolismo , Proteínas SNARE/metabolismo , Línea Celular , Humanos , Células JurkatRESUMEN
Eosinophil granules store a vast array of cytokines and chemokines, many of which possess opposing activities. Specific stimuli can induce the release of entire granules or selective mediators through a process termed piecemeal degranulation. Until recently, the mechanisms that governed the decision to opt for either of these processes were unknown. Recent research has identified a mechanism through which differential secretion occurs during piecemeal degranulation. Eotaxin stimulation of eosinophils induces the selective mobilization of the granule-stored alpha chain of the interleukin-4 (IL-4) receptor into secretory vesicles. This process selectively recruits IL-4 to these vesicles and facilitates its differential secretion. There is also recent evidence for a mechanism of differential mobilization and membrane fusion of secretory vesicles versus granules. These two compartments possess a different set of SNARE and Rab molecules as vesicle fusion and transport-docking proteins, respectively. This presumably allows differential regulation of the processes of mobilization and plasma membrane fusion. These findings provide a model to explain the mechanism by which eosinophils, and likely many other cell types, differentially secrete cytokines and chemokines.
Asunto(s)
Citocinas/metabolismo , Eosinófilos/metabolismo , Animales , Anticuerpos/farmacología , Antígenos CD/inmunología , Antígenos CD/fisiología , Quimiocina CCL11 , Quimiocina CCL5/metabolismo , Quimiocinas/metabolismo , Quimiocinas CC/metabolismo , Gránulos Citoplasmáticos/química , Gránulos Citoplasmáticos/metabolismo , Peroxidasa del Eosinófilo/metabolismo , Eosinófilos/efectos de los fármacos , Exocitosis/fisiología , Humanos , Interferón gamma/farmacología , Interleucinas/metabolismo , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/fisiología , Transporte de Proteínas , Receptores Inmunológicos/inmunología , Receptores Inmunológicos/fisiología , Proteínas SNARE/fisiología , Transducción de Señal/fisiología , Tetraspanina 29RESUMEN
RasGRP1 links TCR signaling to Ras in T cells, while both RasGRP1 and RasGRP3 link BCR signaling to Ras in B cells. T cells deficient in RasGRP1 have defective proliferative responses as do B cells deficient in both RasGRP1 and RasGRP3, confirming the importance of Ras activation in lymphocyte proliferation. While aged Rasgrp1-/- mice develop late-onset autoimmunity characterized by splenomegaly and the presence of anti-nuclear antibodies (ANA), the additional loss of RasGRP3 expression inhibits this phenotype. We show here that the autoimmunity in Rasgrp1-/- mice is T cell dependent. Compared to wildtype, Rasgrp1-/- T cells induce greater in vitro B cell proliferation that is due, at least in part, to increased production of interleukin-4 (IL-4). Rasgrp1 Rasgrp3 double mutant B cells are less responsive to this T cell stimulation. The reduced double mutant B cell proliferative response was paralleled by decreased induction of cyclin D2 upon stimulation with IL-4 and anti-IgM. Taken together these results suggest that double mutant mice fail to generate autoimmunity due to their decreased B cell cyclin D2 accumulation, and thus proliferation, in response to the elevated levels of IL-4 produced by mutant T cells.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/metabolismo , Linfocitos/inmunología , Linfocitos/metabolismo , Animales , Autoinmunidad , Linfocitos B/citología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Proliferación Celular , Factores de Intercambio de Guanina Nucleótido/deficiencia , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/inmunología , Técnicas In Vitro , Interleucina-4/biosíntesis , Activación de Linfocitos , Linfocitos/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Desnudos , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de SeñalRESUMEN
The RasGRPs are a family of Ras activators that possess diacylglycerol-binding C1 domains. In T cells, RasGRP1 links TCR signaling to Ras. B cells coexpress RasGRP1 and RasGRP3. Using Rasgrp1 and Rasgrp3 single and double null mutant mice, we analyzed the role of these proteins in signaling to Ras and Erk in B cells. RasGRP1 and RasGRP3 both contribute to BCR-induced Ras activation, although RasGRP3 alone is responsible for maintaining basal Ras-GTP levels in unstimulated cells. Surprisingly, RasGRP-mediated Ras activation is not essential for B cell development because this process occurs normally in double-mutant mice. However, RasGRP-deficient mice do exhibit humoral defects. Loss of RasGRP3 led to isotype-specific deficiencies in Ab induction in immunized young mice. As reported previously, older Rasgrp1-/- mice develop splenomegaly and antinuclear Abs as a result of a T cell defect. We find that such mice have elevated serum Ig levels of several isotypes. In contrast, Rasgrp3-/- mice exhibit hypogammaglobulinemia and show no signs of splenomegaly or autoimmunity. Double-mutant mice exhibit intermediate serum Ab titers, albeit higher than wild-type mice. Remarkably, double-mutant mice exhibit no signs of autoimmunity or splenomegaly. B cell proliferation induced by BCR ligation with or without IL-4 was found to be RasGRP1- and RasGRP3-dependent. However, the RasGRPs are not required for B cell proliferation per se, because LPS-induced proliferation is unaffected in double-mutant mice.