RESUMO
BACKGROUND: The study of autoinflammatory diseases has uncovered mechanisms underlying cytokine dysregulation and inflammation. METHODS: We analyzed the DNA of an index patient with early-onset systemic inflammation, cutaneous vasculopathy, and pulmonary inflammation. We sequenced a candidate gene, TMEM173, encoding the stimulator of interferon genes (STING), in this patient and in five unrelated children with similar clinical phenotypes. Four children were evaluated clinically and immunologically. With the STING ligand cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), we stimulated peripheral-blood mononuclear cells and fibroblasts from patients and controls, as well as commercially obtained endothelial cells, and then assayed transcription of IFNB1, the gene encoding interferon-ß, in the stimulated cells. We analyzed IFNB1 reporter levels in HEK293T cells cotransfected with mutant or nonmutant STING constructs. Mutant STING leads to increased phosphorylation of signal transducer and activator of transcription 1 (STAT1), so we tested the effect of Janus kinase (JAK) inhibitors on STAT1 phosphorylation in lymphocytes from the affected children and controls. RESULTS: We identified three mutations in exon 5 of TMEM173 in the six patients. Elevated transcription of IFNB1 and other gene targets of STING in peripheral-blood mononuclear cells from the patients indicated constitutive activation of the pathway that cannot be further up-regulated with stimulation. On stimulation with cGAMP, fibroblasts from the patients showed increased transcription of IFNB1 but not of the genes encoding interleukin-1 (IL1), interleukin-6 (IL6), or tumor necrosis factor (TNF). HEK293T cells transfected with mutant constructs show elevated IFNB1 reporter levels. STING is expressed in endothelial cells, and exposure of these cells to cGAMP resulted in endothelial activation and apoptosis. Constitutive up-regulation of phosphorylated STAT1 in patients' lymphocytes was reduced by JAK inhibitors. CONCLUSIONS: STING-associated vasculopathy with onset in infancy (SAVI) is an autoinflammatory disease caused by gain-of-function mutations in TMEM173. (Funded by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases; ClinicalTrials.gov number, NCT00059748.).
Assuntos
Inflamação/genética , Proteínas de Membrana/genética , Mutação , Dermatopatias Vasculares/genética , Idade de Início , Citocinas/genética , Citocinas/metabolismo , Feminino , Fibroblastos/metabolismo , Genes Dominantes , Humanos , Lactente , Recém-Nascido , Inflamação/metabolismo , Interferon gama/genética , Interferon gama/metabolismo , Janus Quinases/antagonistas & inibidores , Pneumopatias/genética , Masculino , Linhagem , Fosforilação , Fator de Transcrição STAT1/metabolismo , Análise de Sequência de DNA , Dermatopatias Vasculares/metabolismo , Síndrome , Transcrição Gênica , Regulação para CimaRESUMO
Several cytokines are involved in the complex processes ultimately leading to autoimmune diseases. In a preceding review, we have already discussed the role of the IL-12 and -17 families of cytokines. This review is focused on IL-15 and -18. Both these molecules have pro-inflammatory activity and act on many cell types and because of their broad spectrum of activity they play an important role in autoimmunity and disease pathogenesis. Their biological activity is ultimately regulated by the signalling cascades set into motion within their target cells. In this second review, we will, once again, describe the signal transduction pathways activated by these two cytokines and focus on how this relates to the pathogenesis of autoimmune diseases. We will also describe some of the therapeutic approaches that are being investigated to curtail the pro-inflammatory activities of these two molecules.
Assuntos
Autoimunidade/imunologia , Mediadores da Inflamação/imunologia , Interleucina-15/imunologia , Interleucina-18/imunologia , Doenças Autoimunes/imunologia , Humanos , Hipersensibilidade/imunologia , Inflamação/imunologia , Transdução de Sinais/imunologiaRESUMO
Autoimmune diseases such as rheumatoid arthritis are the consequence of a persistent imbalance between pro- and anti-inflammatory immune mechanisms leading to chronic inflammation. The action of several cytokines is at the basis of this complex process. This review is focused on the signalling events triggered by two major groups of cytokines, namely the IL-12 and IL-17 families, which in the past few years have been shown to have a prominent role in the pathogenesis of such diseases. In particular, we will focus on the signalling cascades set in motion by such cytokines and how this may relate to the pathogenesis of human immune and inflammatory disorders as knowledge of such cascades may help in the development of novel therapeutic approaches for such diseases.
Assuntos
Artrite Reumatoide/imunologia , Doenças Autoimunes/imunologia , Interleucinas/imunologia , Humanos , Interleucina-12/imunologia , Interleucina-17/imunologia , Transdução de Sinais/imunologiaRESUMO
Janus kinases comprise carboxyterminal kinase, pseudokinase, SH2-like, and N-terminal FERM domains. We identified three patient-derived mutations in the FERM domain of Jak3 and investigated the functional consequences of these mutations. These mutations inhibited receptor binding and also abrogated kinase activity, suggesting interactions between the FERM and kinase domains. In fact, the domains were found to physically associate, and coexpression of the FERM domain enhanced activity of the isolated kinase domain. Conversely, staurosporine, which alters kinase domain structure, disrupted receptor binding, even though the catalytic activity of Jak3 is dispensable for receptor binding. Thus, the Jak FERM domain appears to have two critical functions: receptor interaction and maintenance of kinase integrity.
Assuntos
Proteínas Tirosina Quinases/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Catálise , Inibidores Enzimáticos/farmacologia , Humanos , Subunidade gama Comum de Receptores de Interleucina , Janus Quinase 3 , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Pirimidinas/farmacologia , Receptores de Interleucina-7/genética , Receptores de Interleucina-7/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Estaurosporina/farmacologia , Quinases da Família src/antagonistas & inibidoresRESUMO
Differentiation of naive CD4(+) T cells into IFN-gamma-producing T helper 1 (T(H)1) cells is pivotal for protective immune responses against intracellular pathogens. T-bet, a recently discovered member of the T-box transcription factor family, has been reported to play a critical role in this process, promoting IFN-gamma production. Although terminal T(H)1 differentiation occurs over days, we now show that challenge of mice with a prototypical T(H)1-inducing stimulus, Toxoplasma gondii soluble extract, rapidly induced IFN-gamma and T-bet; T-bet induction was substantially lower in IFN-gamma-deficient mice. Naive T cells expressed little T-bet, but this transcription factor was induced markedly by the combination of IFN-gamma and cognate antigen. Human myeloid antigen-presenting cells showed T-bet induction after IFN-gamma stimulation alone, and this induction was antagonized by IL-4 and granulocyte/macrophage colony-stimulating factor. Although T-bet was induced rapidly and directly by IFN-gamma, it was not induced by IFN-alpha, lipopolysaccharide, or IL-1, indicating that this action of IFN-gamma was specific. Moreover, T-bet induction was dependent on Stat1 but not Stat4. These data argue for a model in which IFN-gamma gene regulation involves an autocrine loop, whereby the cytokine regulates a transcription factor that promotes its own production. These findings substantially alter the current view of T-bet in IFN-gamma regulation and promotion of cell-mediated immune responses.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Células Dendríticas/metabolismo , Interferon gama/fisiologia , Macrófagos/metabolismo , Fatores de Transcrição/biossíntese , Animais , Anticorpos Monoclonais/imunologia , Sequência de Bases , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/fisiologia , Células Cultivadas , Primers do DNA , Células Dendríticas/imunologia , Feminino , Humanos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas com Domínio T , Fatores de Transcrição/fisiologia , Regulação para CimaRESUMO
To study the role of cytokines that are relevant in cancer cachexia syndrome due to intracerebral tumours, mice were injected with human A431 epidermoid carcinoma, OVCAR3 ovarian carcinoma and GBLF glioma cells comparing intracerebral (i.c.) and systemic (i.p. or s.c.) routes of implantation. Anorexia and weight loss developed within 7-10 days in mice injected i.c. with A431 or OVCAR3 cells well before a large tumour developed, while i.c.-injected GBLF cells did not induce cachexia until day 20, when the tumour was large. By contrast, mice injected i.p. or s.c. developed tumours without evidence of anorexia. Thus, intracerebrally-growing A431 and OVCAR3 resulted in cancer cachexia independent of tumour mass, and we investigated their cytokine pattern. Serum levels of murine and human cytokines are not predictive of cancer cachexia development. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis revealed in the brain of i.c.-injected A431 tumour-bearing mice expression of human interleukin-(IL-)1alpha, IL-1beta and LIF in all samples and IL-6 in two of four samples while in i.c.-injected OVCAR3 tumour-bearing animals IL-6, and LIF were detected in all samples and tumour necrosis factor-alpha (TNFalpha) in two of four samples. Only LIF was expressed in brains of mice injected with GBLF cells. Murine IL-6 was increased only in the brains of A431-bearing mice. Only mice injected i.c. simultaneously with a monoclonal antibody (mAb) directed against the murine IL-6 receptor and OVCAR3 cells, but not those with mAb and A431 cells, showed a significant increase in survival time with a partial and temporary attenuation of cachexia symptoms. These results suggest that IL-6 in OVCAR3 model may be important cachectogenic factor when centrally released by even a limited number of tumour cells.
Assuntos
Neoplasias Encefálicas/metabolismo , Caquexia/metabolismo , Citocinas/fisiologia , Neoplasias/metabolismo , Animais , Anorexia/metabolismo , Peso Corporal , Encéfalo/metabolismo , Sistema Nervoso Central/metabolismo , Citocinas/sangue , Ensaio de Imunoadsorção Enzimática , Comportamento Alimentar , Feminino , Inibidores do Crescimento/sangue , Inibidores do Crescimento/metabolismo , Humanos , Interleucina-1/sangue , Interleucina-1/metabolismo , Interleucina-6/sangue , Interleucina-6/metabolismo , Fator Inibidor de Leucemia , Linfocinas/sangue , Linfocinas/metabolismo , Camundongos , Camundongos Nus , Transplante de Neoplasias , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Discovered during the past ten years, Janus kinases and signal transducers and activators of transcription have emerged as critical elements in cytokine signaling and immunoregulation. Recently, knockout mice for all the members of these families have been generated, with remarkably specific outcomes. Equally exciting is the discovery of a new class of inhibitors, the suppressor of cytokine signaling family. The phenotypes of mice deficient in these molecules are also striking, underscoring the importance of negative regulation in cytokine signaling.
Assuntos
Receptores de Citocinas/classificação , Receptores de Citocinas/fisiologia , Transdução de Sinais/imunologia , Animais , HumanosRESUMO
Autocrine activation of APC by IL-12 has recently been revealed; we demonstrate here that inducible expression of Stat4 in APC is central to this process. Stat4 is induced in dendritic cells (DC) in a maturation-dependent manner and in macrophages in an activation-dependent manner. Stat4 levels directly correlate with IL-12-dependent IFN-gamma production by APC as well as IFN-gamma production by DC during Ag presentation. The Th2 cytokines IL-4 and IL-10 suppress Stat4 induction in DC and macrophages when present during maturation and activation, respectively, diminishing IFN-gamma production. In contrast, IL-4 has no effect on Stat4 levels in mature DC and actually augments IFN-gamma production by DC during Ag presentation, indicating that IL-4 acts differently in a spatiotemporal manner. The functional importance of Stat4 is evident in Stat4(-/-) DC and macrophages, which fail to produce IFN-gamma. Furthermore, Stat4(-/-) macrophages are defective in NO production in response to IL-12 and are susceptible to TOXOPLASMA: Autocrine IL-12 signaling is required for high-level IFN-gamma production by APC at critical stages in both innate and adaptive immunity, and the control of Stat4 expression is likely an important regulator of this process.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/fisiologia , Células Dendríticas/metabolismo , Macrófagos/metabolismo , Transativadores/biossíntese , Transativadores/fisiologia , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Comunicação Autócrina/genética , Comunicação Autócrina/imunologia , Antígenos CD8/biossíntese , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Cultivadas , Citocinas/fisiologia , Proteínas de Ligação a DNA/genética , Células Dendríticas/citologia , Células Dendríticas/imunologia , Imunidade Celular/genética , Imunidade Inata/genética , Injeções Intravenosas , Interferon gama/biossíntese , Interleucina-12/fisiologia , Lipopolissacarídeos/administração & dosagem , Ativação de Macrófagos/genética , Macrófagos/imunologia , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Transcrição STAT4 , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Células Th2/imunologia , Células Th2/metabolismo , Toxoplasma/imunologia , Toxoplasma/patogenicidade , Transativadores/genética , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
Mutations of the Janus kinase 3 (JAK3) have been previously described to cause an autosomal recessive variant of severe combined immunodeficiency (SCID) usually characterized by the near absence of T and NK cells, but preserved numbers of B lymphocytes (T-B+SCID). We now report a family whose JAK3 mutations are associated with the persistence of circulating T cells, resulting in previously undescribed clinical presentations, ranging from a nearly unaffected 18-year-old subject to an 8-year-old sibling with a severe lymphoproliferative disorder. Both siblings were found to be compound heterozygotes for the same deleterious JAK3 mutations: an A96G initiation start site mutation, resulting in a dysfunctional, truncated protein product and a G2775(+3)C mutation in the splice donor site sequence of intron 18, resulting in a splicing defect and a predicted premature stop. These mutations were compatible with minimal amounts of functional JAK3 expression, leading to defective cytokine-dependent signaling. Activated T cells in these patients failed to express Fas ligand (FasL) in response to IL-2, which may explain the accumulation of T cells with an activated phenotype and a skewed T cell receptor (TcR) Vbeta family distribution. We speculate that residual JAK3 activity accounted for the maturation of thymocytes, but was insufficient to sustain IL-2-mediated homeostasis of peripheral T cells via Fas/FasL interactions. These data demonstrate that the clinical spectrum of JAK3 deficiency is quite broad and includes immunodeficient patients with accumulation of activated T cells, and indicate an essential role for JAK3 in the homeostasis of peripheral T cells in humans.
Assuntos
Proteínas Tirosina Quinases/deficiência , Adolescente , Sequência de Aminoácidos , Linfócitos B/metabolismo , Linhagem Celular Transformada , Criança , DNA Complementar , Proteína Ligante Fas , Feminino , Expressão Gênica , Humanos , Imunofenotipagem , Interleucina-2/metabolismo , Janus Quinase 3 , Masculino , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , Mutação , Linhagem , Proteínas Tirosina Quinases/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores de Interleucina-2/metabolismo , Análise de Sequência de DNA , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/imunologia , Imunodeficiência Combinada Severa/patologia , Transdução de Sinais , Linfócitos T , Regulação para CimaRESUMO
Two subunits of the IL-12 receptor (IL-12R), IL-12R beta 1 and IL-12R beta 2, have been identified and cloned. Previous studies demonstrated that the IL-12R beta 1 subunit was required for mouse T and NK cells to respond to IL-12 in vivo. To investigate the role of IL-12R beta 2 in IL-12 signaling, we have generated IL-12R beta 2-deficient (IL-12R beta 2(-/-)) mice by targeted mutation in embryonic stem (ES) cells. Although Con A-activated splenocytes from IL-12R beta 2(-/-) mice still bind IL-12 with both high and low affinity, no IL-12-induced biological functions can be detected. Con A-activated splenocytes of IL-12R beta 2(-/-) mice failed to produce IFN-gamma or proliferate in response to IL-12 stimulation. NK lytic activity of IL-12R beta 2(-/-) splenocytes was not induced when incubated with IL-12. IL-12R beta 2(-/-) splenocytes were deficient in IFN-gamma secretion when stimulated with either Con A or anti-CD3 mAb in vitro. Furthermore, IL-12R beta 2(-/-) mice were deficient in vivo in their ability to produce IFN-gamma following endotoxin administration and to generate a type 1 cytokine response. IL-12-mediated signal transduction was also defective as measured by phosphorylation of STAT4. These results demonstrate that although mouse IL-12R beta 1 is the subunit primarily responsible for binding IL-12, IL-12R beta 2 plays an essential role in mediating the biological functions of IL-12 in mice.
Assuntos
Interleucina-12/metabolismo , Receptores de Interleucina/deficiência , Receptores de Interleucina/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Animais , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Concanavalina A/farmacologia , Proteínas de Ligação a DNA/metabolismo , Feminino , Marcação de Genes , Tolerância Imunológica/genética , Interferon gama/biossíntese , Interferon gama/metabolismo , Interleucina-12/farmacologia , Ativação Linfocitária/genética , Subpopulações de Linfócitos/imunologia , Subpopulações de Linfócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Fosforilação , Isoformas de Proteínas/deficiência , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Receptores de Interleucina/fisiologia , Receptores de Interleucina-12 , Fator de Transcrição STAT4 , Baço/citologia , Baço/imunologia , Baço/metabolismo , Células Th1/imunologia , Células Th1/metabolismo , Transativadores/metabolismoRESUMO
Cytokines have critical functions in regulating immune responses. A large number of these factors bind related receptors termed the Type I and Type II families of cytokine receptors. These receptors activate Janus kinases (Jaks) and Stat family of transcription factors. The essential and specific function of Jaks and Stats is particularly well illustrated by human and mouse mutations. The possibility that these molecules could be targeted to produce novel immunosuppressive compounds is considered in this review.
Assuntos
Proteínas de Ligação a DNA/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Imunossupressores/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Transativadores/antagonistas & inibidores , Animais , Proteínas de Ligação a DNA/imunologia , Humanos , Camundongos , Proteínas Tirosina Quinases/imunologia , Transativadores/imunologiaRESUMO
Interleukin-12 (IL-12) is a key immunoregulatory cytokine that promotes Th1 differentiation and cell-mediated immune responses. The transcription factor STAT4 (signal transducer and activator of transcription 4) is an important element in mediating IL-12 signals, as evidenced by the fact that STAT4(-/-) mice display impaired responsiveness to IL-12 and deficient Th1 differentiation. STAT4 is inducibly phosphorylated on tyrosine and serine in response to IL-12, but the kinase(s) responsible for the latter event is unknown. Here we show that IL-12 induces STAT4 phosphorylation on serine 721 and that mutation of serine 721 interferes with STAT4 transcriptional activity. In addition, we show that mutation of tyrosine 693 abrogates IL-12-induced STAT4 tyrosine phosphorylation and transcriptional activity. Although the site surrounding serine 721 is an optimum consensus sequence for mitogen-activated family of protein kinases (MAPKs)-mediated phosphorylation, we demonstrate that IL-12 does not induce extracellular signal-regulated kinase (ERK) or c-Jun N-terminal kinase (JNK) activation in T and natural killer (NK) cells and that IL-12-induced STAT4 transcriptional activity is not affected by these kinases. Rather, we show that IL-12 induces p38 activation. Moreover, we demonstrate that p38alpha and its upstream activator, MKK6, phosphorylate STAT4 on serine 721, and are required for STAT4 full transcriptional activity induced by IL-12, establishing the MKK6/p38alpha/STAT4 pathway as an important mediator of IL-12 actions. (Blood. 2000;96:1844-1852)
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Interleucina-12/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Serina/metabolismo , Transativadores/metabolismo , Células 3T3 , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Linhagem Celular , Proteínas de Ligação a DNA/genética , Humanos , Isoenzimas/metabolismo , Células Jurkat , MAP Quinase Quinase 4 , MAP Quinase Quinase 6 , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Mutação , Fosforilação/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fator de Transcrição STAT4 , Serina/genética , Transdução de Sinais/efeitos dos fármacos , Transativadores/genética , Ativação Transcricional/efeitos dos fármacos , Transfecção , Tirosina/metabolismo , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Interleukin (IL)-2, a critical cytokine with indispensable functions in regulating lymphoid homeostasis, induces the activation of several biochemical pathways. Precisely how these pathways are linked and how they relate to the biological action of IL-2 is incompletely understood. We previously identified SHP-2 (Src homology 2 domain containing phosphatase 2) as an important intermediate in IL-2-dependent MAPK activation and showed its association with a 98-kDa phosphoprotein in response to IL-2. Here, we demonstrate that Gab2, a recently identified adapter molecule, is the major SHP-2 and phosphatidylinositol 3'-kinase-associated 98-kDa protein in normal, IL-2-activated lymphocytes. We further demonstrate that phosphorylation of both Gab2 and SHP-2 is largely dependent upon tyrosine 338 of the IL-2 receptor beta chain. Gab2 can be a substrate of all the three major classes of non-receptor tyrosine kinases associated with the IL-2R, but in terms of IL-2 signaling, JAK3 but not Lck or Syk is essential for Gab2 phosphorylation. We also demonstrate that only IL-2 and IL-15, but not other gammac cytokines induce Gab2 phosphorylation; the ability to phosphorylate Gab2 correlates with Shc phosphorylation and ERK1/ERK2 activation. Finally, we also show that Gab2 levels are regulated by T cell activation, and resting T cells express little Gab2. Therefore, up-regulation and activation of Gab2 may be important in linking the IL-2 receptor to activation of MAPK and may be an important means of achieving specificity in cytokine signaling.
Assuntos
Interleucina-15/metabolismo , Interleucina-2/metabolismo , Ativação Linfocitária , Fosfoproteínas/biossíntese , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal , Sítios de Ligação , Linhagem Celular , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Receptores de Interleucina-2/metabolismoRESUMO
The biological activities of IL-12 are mediated through a specific, high-affinity receptor composed of IL-12 receptor(R)beta1 and IL-12Rbeta2 subunits that exist primarily on T and NK cells. Remarkably, the expression of IL-12Rbeta2 on CD4(+) T cells in mouse and humans appears to be differentially regulated by IFN-gamma and IFN-alpha, respectively. Using an antibody specific for the human IL-12Rbeta2 subunit, the effect of IFN-gamma, IFN-alpha, IL-12 and IL-2 on the regulation of IL-12R expression and IL-12 responsiveness of human T and NK cells was assessed. The presence of IFN-alpha or IFN-gamma in cultures enhanced IL-12Rbeta2 expression of CD4(+) and CD8(+) T cells. The enhancing effect of IFN-alpha and IFN-gamma was independent of endogenous IL-12. Furthermore, the clearest effects of IFN-alpha and IFN-gamma on IL-12Rbeta2 expression on T cells were seen by abrograting the inhibition induced by the presence of IL-4 in cultures. In contrast to T cells, IFN-alpha and IFN-gamma had little effect on regulating IL-12Rbeta2 expression on human NK cells. Taken together, these data show that there is differential regulation of IL-12Rbeta2 expression by IFN-alpha and IFN-gamma on human T and NK cells.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Citocinas/imunologia , Células Matadoras Naturais/imunologia , Receptores de Interleucina/imunologia , Animais , Citocinas/farmacologia , Humanos , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos , Fosforilação , Receptores de Interleucina/biossíntese , Receptores de Interleucina-12 , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologiaRESUMO
Interleukin-2 (IL-2) activates several different families of tyrosine kinases, but precisely how these kinases interact is not completely understood. We therefore investigated the functional relationships among Jak3, Lck, and Syk in IL-2 signaling. We first observed that in the absence of Jak3, both Lck and Syk had the capacity to phosphorylate Stat3 and Stat5a. However, neither supported IL-2-induced STAT activation, nor did dominant negative alleles of these kinases inhibit. Moreover, pharmacological abrogation of Lck activity did not inhibit IL-2-mediated phosphorylation of Jak3 and Stat5a. Importantly, ligand-dependent Syk activation was dependent on the presence of catalytically active Jak3, whereas Lck activation was not. Interestingly, Syk functioned as a direct substrate of Jak1 but not Jak3. Additionally, Jak3 phosphorylated Jak1, whereas the reverse was not the case. Taken together, our data support a model in which Lck functions in parallel with Jak3, while Syk functions as a downstream element of Jaks in IL-2 signaling. Jak3 may regulate Syk catalytic activity indirectly via Jak1. However, IL-2-mediated Jak3/Stat activation is not dependent on Lck or Syk. While the essential roles of Jak1 and Jak3 in signaling by gammac-utilizing cytokines are clear, it will be important to dissect the exact contributions of Lck and Syk in mediating the effects of IL-2 and related cytokines.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Interleucina-2/fisiologia , Proteínas do Leite , Proteínas Tirosina Quinases/fisiologia , Transdução de Sinais , Transativadores/fisiologia , Animais , Linhagem Celular , Ativação Enzimática , Precursores Enzimáticos/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Janus Quinase 3 , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/fisiologia , Fosforilação , Receptores de Interleucina-2/fisiologia , Fator de Transcrição STAT5 , Quinase Syk , Proteínas Supressoras de TumorRESUMO
Glucocorticoids are widely used in the therapy of inflammatory, autoimmune, and allergic diseases. As the end-effectors of the hypothalamic-pituitary-adrenal axis, endogenous glucocorticoids also play an important role in suppressing innate and cellular immune responses. Previous studies have indicated that glucocorticoids inhibit Th1 and enhance Th2 cytokine secretion. IL-12 promotes Th1 cell-mediated immunity, while IL-4 stimulates Th2 humoral-mediated immunity. Here, we examined the regulatory effect of glucocorticoids on key elements of IL-12 and IL-4 signaling. We first investigated the effect of dexamethasone on IL-12-inducible genes and showed that dexamethasone inhibited IL-12-induced IFN-gamma secretion and IFN regulatory factor-1 expression in both NK and T cells. This occurred even though the level of expression of IL-12 receptors and IL-12-induced Janus kinase phosphorylation remained unaltered. However, dexamethasone markedly inhibited IL-12-induced phosphorylation of Stat4 without altering its expression. This was specific, as IL-4-induced Stat6 phosphorylation was not affected, and mediated by the glucocorticoid receptor, as it was antagonized by the glucocorticoid receptor antagonist RU486. Moreover, transfection experiments showed that dexamethasone reduced responsiveness to IL-12 through the inhibition of Stat4-dependent IFN regulatory factor-1 promoter activity. We conclude that blocking IL-12-induced Stat4 phosphorylation, without altering IL-4-induced Stat6 phosphorylation, appears to be a new suppressive action of glucocorticoids on the Th1 cellular immune response and may help explain the glucocorticoid-induced shift toward the Th2 humoral immune response.
Assuntos
Proteínas de Ligação a DNA/antagonistas & inibidores , Dexametasona/farmacologia , Imunossupressores/farmacologia , Interleucina-12/fisiologia , Proteínas Proto-Oncogênicas , Linfócitos T/efeitos dos fármacos , Células Th1/efeitos dos fármacos , Transativadores/antagonistas & inibidores , Células 3T3 , Animais , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Fator Regulador 1 de Interferon , Interferon gama/antagonistas & inibidores , Interferon gama/biossíntese , Interleucina-12/antagonistas & inibidores , Interleucina-12/metabolismo , Janus Quinase 2 , Camundongos , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/biossíntese , Fosfoproteínas/genética , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/imunologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Proteínas/antagonistas & inibidores , Proteínas/metabolismo , Receptores de Interleucina/metabolismo , Receptores de Interleucina-12 , Fator de Transcrição STAT4 , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Linfócitos T/metabolismo , TYK2 Quinase , Células Th1/imunologia , Transativadores/metabolismo , TransfecçãoRESUMO
IFNgamma, once called the macrophage-activating factor, stimulates many genes in macrophages, ultimately leading to the elicitation of innate immunity. IFNgamma's functions depend on the activation of STAT1, which stimulates transcription of IFNgamma-inducible genes through the GAS element. The IFN consensus sequence binding protein (icsbgamma or IFN regulatory factor 8), encoding a transcription factor of the IFN regulatory factor family, is one of such IFNgamma-inducible genes in macrophages. We found that macrophages from ICSBP-/- mice were defective in inducing some IFNgamma-responsive genes, even though they were capable of activating STAT1 in response to IFNgamma. Accordingly, IFNgamma activation of luciferase reporters fused to the GAS element was severely impaired in ICSBP-/- macrophages, but transfection of ICSBP resulted in marked stimulation of these reporters. Consistent with its role in activating IFNgamma-responsive promoters, ICSBP stimulated reporter activity in a GAS-specific manner, even in the absence of IFNgamma treatment, and in STAT1 negative cells. Indicative of a mechanism for this stimulation, DNA affinity binding assays revealed that endogenous ICSBP was recruited to a multiprotein complex that bound to GAS. These results suggest that ICSBP, when induced by IFNgamma through STAT1, in turn generates a second wave of transcription from GAS-containing promoters, thereby contributing to the elicitation of IFNgamma's unique activities in immune cells.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Interferon gama/metabolismo , Macrófagos/metabolismo , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Animais , Linhagem Celular , Genes Reporter , Humanos , Fatores Reguladores de Interferon , Interferon gama/química , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT1 , Ativação Transcricional/genética , TransfecçãoAssuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Citocinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Quinases Ativadas por Mitógeno , Transativadores/metabolismo , Animais , Evolução Biológica , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Proteínas de Ligação a DNA/antagonistas & inibidores , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , Neoplasias/etiologia , Receptores de Citocinas/metabolismo , Transdução de Sinais , Transativadores/antagonistas & inibidoresRESUMO
Autosomal dominant periodic fever syndromes are characterized by unexplained episodes of fever and severe localized inflammation. In seven affected families, we found six different missense mutations of the 55 kDa tumor necrosis factor receptor (TNFR1), five of which disrupt conserved extracellular disulfide bonds. Soluble plasma TNFR1 levels in patients were approximately half normal. Leukocytes bearing a C52F mutation showed increased membrane TNFR1 and reduced receptor cleavage following stimulation. We propose that the autoinflammatory phenotype results from impaired downregulation of membrane TNFR1 and diminished shedding of potentially antagonistic soluble receptor. TNFR1-associated periodic syndromes (TRAPS) establish an important class of mutations in TNF receptors. Detailed analysis of one such mutation suggests impaired cytokine receptor clearance as a novel mechanism of disease.
Assuntos
Antígenos CD/genética , Febre Familiar do Mediterrâneo/genética , Mutação em Linhagem Germinativa/genética , Receptores do Fator de Necrose Tumoral/genética , Sequência de Aminoácidos , Antígenos CD/biossíntese , Antígenos CD/sangue , Antígenos CD/metabolismo , Análise Mutacional de DNA/métodos , Feminino , Genes Dominantes/genética , Humanos , Leucócitos/metabolismo , Masculino , Dados de Sequência Molecular , Linhagem , Receptores do Fator de Necrose Tumoral/biossíntese , Receptores do Fator de Necrose Tumoral/sangue , Receptores do Fator de Necrose Tumoral/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral , SíndromeRESUMO
Binding of IL-2 to its receptor activates several biochemical pathways, including JAK-STAT, Ras-mitogen-activated protein kinase, and phosphatidylinositol 3'-kinase (PI 3'-kinase) pathways. Recently, it has been shown that the SH2-containing phosphatase, SHP-2, becomes phosphorylated in response to IL-2 stimulation, associates with PI3'-kinase and Grb2, and can exert a positive regulatory role in IL-2 signaling. We now report the identification of a prominent 98-kDa protein (p98) found to be phosphorylated in response to IL-2 stimulation and coprecipitated with SHP-2, the p85 subunit of PI 3'-kinase and Grb2. Interestingly, whereas IL-4 is known to activate PI 3'-kinase, we did not observe any p98 phosphorylation in response to IL-4 stimulation. p98 can form a multipartite complex with all these proteins as immunodepleting with anti-p85 antiserum substantially reduced the amount of p98 immunoprecipitated by SHP-2 and Grb2; the converse was also true. Furthermore, phosphorylation of p98 did not occur in cells lacking JAK3, suggesting that it may be a JAK substrate. Finally, deglycosylation of p98 did not alter its migration, suggesting p98 is not a member of the recently described SHP substrate/signal-regulatory proteins family of transmembrane glycoproteins. Thus p98 is a prominent IL-2-dependent substrate that associates with multiple proteins involved in IL-2 signaling and may play an important role in coupling the different signal transduction pathways activated by IL-2.