RESUMO
In mast cells (MCs), the TEC family kinase (TFK) BTK constitutes a central regulator of antigen (Ag)-triggered, FcεRI-mediated PLCγ phosphorylation, Ca2+ mobilization, degranulation, and pro-inflammatory cytokine production. Less is known about the function of BTK in the context of stem cell factor (SCF)-induced KIT signaling. In bone marrow-derived MCs (BMMCs), Ag stimulation caused intense phosphorylation of BTK at Y551 in its active center and at Y223 in its SH3-domain, whereas in response to SCF only Y223 was significantly phosphorylated. Further data using the TFK inhibitor Ibrutinib indicated that BTK Y223 is phosphorylated by a non-BTK TFK upon SCF stimulation. In line, SCF-induced PLCγ1 phosphorylation was stronger attenuated by Ibrutinib than by BTK deficiency. Subsequent pharmacological analysis of PLCγ function revealed a total block of SCF-induced Ca2+ mobilization by PLC inhibition, whereas only the sustained phase of Ca2+ flux was curtailed in Ag-stimulated BMMCs. Despite this severe stimulus-dependent difference in inducing Ca2+ mobilization, PLCγ inhibition suppressed Ag- and SCF-induced degranulation and pro-inflammatory cytokine production to comparable extents, suggesting involvement of additional TFK(s) or PLCγ-dependent signaling components. In addition to PLCγ, the MAPKs p38 and JNK were activated by Ag in a BTK-dependent manner; this was not observed upon SCF stimulation. Hence, FcεRI and KIT employ different mechanisms for activating PLCγ, p38, and JNK, which might strengthen their cooperation regarding pro-inflammatory MC effector functions. Importantly, our data clearly demonstrate that analyzing BTK Y223 phosphorylation is not sufficient to prove BTK activation.
Assuntos
Tirosina Quinase da Agamaglobulinemia/metabolismo , Citocinas/metabolismo , Mastócitos/metabolismo , Fosfolipase C gama/metabolismo , Adenina/análogos & derivados , Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Animais , Cálcio/metabolismo , Células Cultivadas , Citocinas/genética , Feminino , MAP Quinase Quinase 4/metabolismo , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Piperidinas , Proteínas Proto-Oncogênicas c-kit/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Receptores de IgE/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Antigen (Ag)-mediated crosslinking of IgE-loaded high-affinity receptors for IgE (FcεRI) on mast cells (MCs) triggers activation of proinflammatory effector functions relevant for IgE-associated allergic disorders. The cytosolic tyrosine kinase BTK and the SH2-containing inositol-5'-phosphatase SHIP1 are central positive and negative regulators of Ag-triggered MC activation, respectively, contrarily controlling Ca2+ mobilisation, degranulation, and cytokine production. Using genetic and pharmacological techniques, we examined whether BTK activation in Ship1-/- MCs is mandatory for the manifestation of the well-known hyperactive phenotype of Ship1-/- MCs. We demonstrate the prominence of BTK for the Ship1-/- phenotype in a manner strictly dependent on the strength of the initial Ag stimulus; particular importance for BTK was identified in Ship1-/- bone marrow-derived MCs in response to stimulation with suboptimal Ag concentrations. With respect to MAPK activation, BTK showed particular importance at suboptimal Ag concentrations, allowing for an analogous-to-digital switch resulting in full activation of ERK1/2 already at low Ag concentrations. Our data allow for a more precise definition of the role of BTK in FcεRI-mediated signal transduction and effector function in MCs. Moreover, they suggest that reduced activation or curtate expression of SHIP1 can be compensated by pharmacological inhibition of BTK and vice versa.
Assuntos
Tirosina Quinase da Agamaglobulinemia/metabolismo , Antígenos/metabolismo , Imunoglobulina E/metabolismo , Mastócitos/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/deficiência , Animais , Células da Medula Óssea/citologia , Cálcio/metabolismo , Degranulação Celular , Citocinas/biossíntese , Diglicerídeos/metabolismo , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Mastócitos/fisiologia , Camundongos , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Fosfolipase C gama/metabolismo , Fosforilação , Receptores de IgE/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: Antigen (Ag)/IgE-mediated mast cell (MC) responses play detrimental roles in allergic diseases. MC activation via the high-affinity receptor for IgE (FcεRI) is controlled by the Src family kinase Lyn. Lyn-deficient (-/-) bone marrow-derived MCs (BMMCs) have been shown by various laboratories to exert stronger activation of the PI3K pathway, degranulation, and production of pro-inflammatory cytokines compared to wild-type (wt) cells. This mimics the phenotype of BMMCs deficient for the SH2-containing inositol-5'-phosphatase 1 (SHIP1). In this line, Lyn has been demonstrated to tyrosine-phosphorylate and activate SHIP1, thereby constituting a negative feedback control of PI3K-mediated signals. However, several groups have also reported on Lyn-/- BMMCs degranulating weaker than wt BMMCs. RESULTS: Lyn-/- BMMCs, which show a suppressed degranulation response, were found to exhibit abrogated tyrosine phosphorylation of SHIP1 as well. This indicated that even in the presence of reduced SHIP1 function MC degranulation is dependent on Lyn function. In contrast to the reduced immediate secretory response, pro-inflammatory cytokine production was augmented in Lyn-/- BMMCs. For closer analysis, Lyn/SHIP1-double-deficient (dko) BMMCs were generated. In support of the dominance of Lyn deficiency, dko BMMCs degranulated significantly weaker than SHIP1-/- BMMCs. This coincided with reduced LAT1 and PLC-γ1 phosphorylation as well as Ca(2+) mobilization in those cells. Interestingly, activation of the NFκB pathway followed the same pattern as measured by IκBα phosphorylation/degradation as well as induction of NFκB target genes. This suggested that Ag-triggered NFκB activation involves a Ca(2+)-dependent step. Indeed, IκBα phosphorylation/degradation and NFκB target gene induction were controlled by the Ca(2+)-dependent phosphatase calcineurin. CONCLUSIONS: Lyn deficiency is dominant over SHIP1 deficiency in MCs with respect to Ag-triggered degranulation and preceding signaling events. Moreover, the NFκB pathway and respective targets are activated in a Lyn- and Ca(2+)-dependent manner, reinforcing the importance of Lyn for MC activation.
Assuntos
Mastócitos/metabolismo , Fenótipo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética , Quinases da Família src/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Camundongos , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/deficiência , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Fosfolipase C gama/metabolismoRESUMO
The cAMP-responsive element modulator alpha (CREMα) plays a role in autoimmunity and, in particular, in systemic lupus erythematosus. CREMα negatively regulates IL-2 transcription and activates IL-17 expression by direct transcriptional mechanisms. To understand the role of CREM in autoimmunity, we recently generated a mouse with a transgenic overexpression of CREMα selectively in T cells. This mouse is characterized by enhanced IL-17 and IL-21 expression. We, herein, dissect the transcriptional mechanisms of enhanced IL-21 transcription in these mice. T cells of CREMα transgenic mice display an enhanced binding of CREMα to the CD3ζ chain promoter resulting in decreased CD3ζ chain expression. This is accompanied by a decreased excitation threshold and enhanced Ca2+ influx, which is known to induce IL-21 expression via NFATc2 activation. However, CREMα directly binds to cAMP-response element (CRE) half-site within the Il-21 promoter, which results in enhanced promoter activity shown by promoter reporter assays. CREMα-induced IL-21 transcription is not abrogated in the presence of cyclosporine A but depends on an intact CRE site within the IL-21 promoter, which suggests that CREM largely enhances IL-21 expression by direct transcriptional regulation. IL-21 transcription is critical for IL-17 generation in these mice, since IL-21 receptor blockade downregulates IL-17 transcription to wild-type levels. Finally, this is of functional relevance since CREMα transgenic mice display enhanced disease activity in dextran sodium sulfate-induced colitis accompanied by higher local IL-21 expression. Thus, we describe two novel mechanisms of CREMα-dependent IL-21 transcription. Since T cells of systemic lupus erythematosus patients are characterized by enhanced IL-21 transcription, this might also be of functional relevance in humans.
RESUMO
Mast cells (MCs) are tissue-resident sentinels of hematopoietic origin that play a prominent role in allergic diseases. They express the high-affinity receptor for IgE (FcεRI), which when cross-linked by multivalent antigens triggers the release of preformed mediators, generation of arachidonic acid metabolites, and the synthesis of cytokines and chemokines. Stimulation of the FcεRI with increasing antigen concentrations follows a characteristic bell-shaped dose-responses curve. At high antigen concentrations, the so-called supra-optimal conditions, repression of FcεRI-induced responses is facilitated by activation and incorporation of negative signaling regulators. In this context, the SH2-containing inositol-5'-phosphatase, SHIP1, has been demonstrated to be of particular importance. SHIP1 with its catalytic and multiple protein interaction sites provides several layers of control for FcεRI signaling. Regulation of SHIP1 function occurs on various levels, e.g., protein expression, receptor and membrane recruitment, competition for protein-protein interaction sites, and activating modifications enhancing the phosphatase function. Apart from FcεRI-mediated signaling, SHIP1 can be activated by diverse unrelated receptor systems indicating its involvement in the regulation of antigen-dependent cellular responses by autocrine feedback mechanisms or tissue-specific and/or (patho-) physiologically determined factors. Thus, pharmacologic engagement of SHIP1 may represent a beneficial strategy for patients suffering from acute or chronic inflammation or allergies.
Assuntos
Monoéster Fosfórico Hidrolases/fisiologia , Receptores de IgE/fisiologia , Transdução de Sinais/fisiologia , Animais , Humanos , Inositol Polifosfato 5-Fosfatases , Mastócitos/fisiologia , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Receptores de IgE/químicaRESUMO
In this study, we report that cytoplasmic granules from in vivo and in vitro derived mouse mast cells (MCs) contain active granzyme B (gzmB) and caspase-3, which is consistent with recent findings. Studying WT and gzmB-deficient mice, we observed that BM-derived MCs (BMMCs) from both strains contain cytosolic pro-caspase-3, but only WT BMMCs expressed active caspase-3 limited to their secretory lysosomes. Confocal microscopy revealed colocalization of active caspase-3 and gzmB in these cytoplasmic granules. The combined data demonstrate that the generation and storage of active caspase-3 is gzmB-dependent. The finding that BMMCs secrete caspase-3 and gzmB after Ag stimulation suggests that both proteases contribute to extracellular MC-mediated proteolytic events. Although the extracellular function of MC-derived caspase-3 remains unclear, we show that BMMC-secreted caspase-3 cleaves IL-33, a cytokine that contributes to the development of asthma and arthritis. We also show that an in vitro propagated cytolytic T-lymphocyte line constitutively expresses gzmB together with active caspase-3, suggesting a novel interaction of these proteases in the execution of multiple innate and adaptive immune responses.
Assuntos
Células da Medula Óssea/imunologia , Caspase 3/imunologia , Exocitose/imunologia , Granzimas/imunologia , Lisossomos/imunologia , Mastócitos/imunologia , Imunidade Adaptativa/fisiologia , Animais , Antígenos/imunologia , Células da Medula Óssea/citologia , Células da Medula Óssea/enzimologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/enzimologia , Linfócitos T CD8-Positivos/imunologia , Caspase 3/biossíntese , Caspase 3/genética , Linhagem Celular , Exocitose/genética , Regulação Enzimológica da Expressão Gênica/genética , Regulação Enzimológica da Expressão Gênica/imunologia , Granzimas/biossíntese , Granzimas/genética , Imunidade Inata/fisiologia , Interleucina-33 , Interleucinas/genética , Interleucinas/imunologia , Interleucinas/metabolismo , Lisossomos/enzimologia , Lisossomos/genética , Mastócitos/citologia , Mastócitos/enzimologia , Camundongos , Camundongos Knockout , ProteóliseRESUMO
Recognition of bacterial constituents by mast cells (MCs) is dependent on the presence of pattern recognition receptors, such as Toll-like receptors (TLRs). The final cellular response, however, depends on the influence of multiple environmental factors. In the current study we tested the hypothesis that the PI3K-activating ligands insulin-like growth factor-1 (IGF-1), insulin, antigen, and Steel Factor (SF) are able to modulate the TLR4-mediated production of proinflammatory cytokines in murine MCs. Costimulation with any of these ligands caused increased LPS-triggered secretion of IL-6 and TNF-α, but attenuated the production of IL-1ß, though all three cytokines were produced in an NFκB-dependent manner. The pan-specific PI3K-inhibitor Wortmannin reverted the altered production of these cytokines. In agreement, MCs deficient for SHIP1, a negative regulator of the PI3K pathway, showed augmented secretion of IL-6/TNF-α and reduced production of IL-1ß in response to LPS alone. The differential effects of IGF-1 on TLR4-mediated cytokine production were also observed in the context of TLR2 and IL-33 receptor-mediated MC activation. Importantly, these effects were seen in both bone marrow-derived and peritoneal MCs, suggesting general relevance for MCs. Using pharmacological and genetic tools, we could show that the p110δ isoform of PI3K is strongly implicated in SF-triggered suppression of LPS-induced IL-1ß production. Costimulation with antigen was affected to a lesser extent. In conclusion, NFκB-dependent production of proinflammatory cytokines in MCs is differentially controlled by PI3K-activating ligand/receptor systems.
Assuntos
Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Mastócitos/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Receptores Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Inositol Polifosfato 5-Fosfatases , Fator de Crescimento Insulin-Like I/farmacologia , Proteína 1 Semelhante a Receptor de Interleucina-1 , Lipopolissacarídeos/toxicidade , Mastócitos/efeitos dos fármacos , Mastócitos/enzimologia , Camundongos , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Receptores de Interleucina/metabolismo , Transdução de Sinais , Receptor 2 Toll-Like/metabolismoRESUMO
Ag-triggered mast cell (MC) activation follows a bell-shaped dose-response curve. Reduced activation in response to supraoptimal Ag concentrations is thought to be due to preferential engagement of inhibitory-acting proteins like SHIP1, Lyn, and protein kinase C (PKC)-delta. We show in this study that short-term prestimulation with Steel factor (SF) prevents supraoptimal Ag inhibition, resulting in synergistic MC degranulation and IL-6 secretion. These events are preceded by synergistic phosphorylation/activation of numerous signaling proteins, e.g., Erk, p38, and LAT. However, these effects of prestimulation with SF appear not to be due to reduced engagement of the attenuator SHIP1. Pharmacological analyses suggest that the activation of conventional PKCs is important for this synergy. Specifically, although we found that the conventional PKC inhibitor, Gö6976, likely has some PKC-independent targets in MCs, it led us to further studies that established SF plus Ag-induced IL-6 secretion was severely impaired in PKC-beta(-/-) MCs, but not PKC-alpha(-/-) MCs. Thus, PKC-beta joins PI3K and Btk as important players in this synergistic MC activation.
Assuntos
Antígenos/imunologia , Interleucina-6/biossíntese , Interleucina-6/imunologia , Mastócitos/enzimologia , Mastócitos/imunologia , Proteína Quinase C/metabolismo , Fator de Células-Tronco/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Medula Óssea/imunologia , Cálcio/metabolismo , Diferenciação Celular/imunologia , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Interleucina-6/metabolismo , Isoenzimas/deficiência , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Mastócitos/citologia , Mastócitos/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Fosfoproteínas/metabolismo , Fosfotirosina/metabolismo , Proteína Quinase C/deficiência , Proteína Quinase C/genética , Proteína Quinase C beta , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Bruton's tyrosine kinase (Btk) represents an important signaling element downstream of ITAM-containing receptors, e.g. FcepsilonR1 and BCR. Btk is part of the calcium signalosome and thus, critically involved in intracellular calcium mobilization. Loss of Btk or expression of mutant forms results in severe disease phenotypes, X-linked agammaglobulinemia (XLA) and Xid in humans and mice, respectively. Previously, roles for Btk in TLR-mediated signal transduction have been found in monocytes/macrophages. In the present study we show that Btk deficiency moderately enhances or has no influence on the LPS- or lipopeptide-induced secretion of IL-6 and TNF-alpha from murine bone marrow-derived mast cells (BMMCs). Furthermore, activation of p38 kinase, which is required for cytokine production, is comparable in WT and Btk-/- BMMCs. Moreover, stability of the adaptor protein Mal as well as LPS-induced H(2)O(2) production does not vary between WT and Btk-/- cells. Interestingly, PKC-beta deficiency, which results in a Xid-like phenotype as well, has also no negative effect on LPS-induced cytokine secretion, suggesting that proteins of the calcium signalosome are not involved in TLR-mediated BMMC activation. In conclusion, the study reveals that Btk is dispensable for TLR signaling and function in murine BMMCs.
Assuntos
Mastócitos/imunologia , Proteínas Tirosina Quinases/metabolismo , Receptores Toll-Like/metabolismo , Tirosina Quinase da Agamaglobulinemia , Amidas/farmacologia , Animais , Sequência de Bases , Cálcio/metabolismo , Interleucina-6/análise , Interleucina-6/metabolismo , Lipopeptídeos/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/fisiologia , Mastócitos/enzimologia , Camundongos , Monócitos/fisiologia , Nitrilas/farmacologia , Fosforilação , Proteínas Tirosina Quinases/antagonistas & inibidores , Transdução de Sinais , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Changes in cytoplasmic Ca2+ levels regulate a variety of fundamental cellular functions in virtually all cells. In nonexcitable cells, a major pathway of Ca2+ entry involves receptor-mediated depletion of intracellular Ca2+ stores followed by the activation of store-operated calcium channels in the plasma membrane. We have established a mouse line expressing an activating EF hand motif mutant of stromal interaction molecule 1 (Stim1), an ER receptor recently identified as the Ca2+ sensor responsible for activation of Ca2+ release-activated (CRAC) channels in T cells, whose function in mammalian physiology is not well understood. Mice expressing mutant Stim1 had macrothrombocytopenia and an associated bleeding disorder. Basal intracellular Ca2+ levels were increased in platelets, which resulted in a preactivation state, a selective unresponsiveness to immunoreceptor tyrosine activation motif-coupled agonists, and increased platelet consumption. In contrast, basal Ca2+ levels, but not receptor-mediated responses, were affected in mutant T cells. These findings identify Stim1 as a central regulator of platelet function and suggest a cell type-specific activation or composition of the CRAC complex.