RESUMO
Mast cells and their mediators are implicated in the pathogenesis of many different diseases. One possible therapeutic intervention in mast cell-associated diseases can be to reduce the number of tissue mast cells by inducing mast cell apoptosis. In this study, we demonstrate that mast cells exhibit a high sensitivity to ABT-737, a BH3-only mimetic molecule that induces apoptosis through high-affinity binding to the prosurvival proteins, Bcl-2, Bcl-XL, and Bcl-w. Primary mast cells as well as mast cell lines tested succumbed to apoptosis in response to the inhibitor at varying but seemingly low concentrations compared with other leukocytes investigated. I.p. injections of ABT-737 in mice resulted in a total abolishment of mast cells in the peritoneum. Confocal microscopy analysis of peritoneal cells revealed apoptotic bodies of mast cells being phagocytosed by macrophages. In addition, ex vivo treatment of human skin biopsies with ABT-737 demonstrated increased mast cell apoptosis. The data we present in this article show exceptional mast cell sensitivity to ABT-737, a selective inhibitor of antiapoptotic proteins, rendering a possible application for BH3-only mimetic compounds like ABT-737 in mast cell-associated diseases, such as mastocytosis, allergy, asthma, and other chronic inflammations.
Assuntos
Apoptose/efeitos dos fármacos , Compostos de Bifenilo/farmacologia , Mastócitos/efeitos dos fármacos , Nitrofenóis/farmacologia , Sulfonamidas/farmacologia , Animais , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Mastócitos/citologia , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Pele/citologia , Pele/efeitos dos fármacos , Pele/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/deficiência , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/deficiência , Proteína X Associada a bcl-2/genéticaRESUMO
FcepsilonRI-activation-induced survival of mast cells is dependent on the expression and function of the prosurvival protein A1. The expression of A1 in lymphocytes and monocytes has previously been described to be transcriptionally regulated by NF-kappaB. Here we demonstrate that the expression of A1 in mast cells is not dependent on NF-kappaB but that NFAT plays a crucial role. FcepsilonRI-induced A1 expression was not affected in mast cells overexpressing an IkappaB-alpha super-repressor or cells lacking NF-kappaB subunits RelA, c-Rel, or c-Rel plus NF-kappaB1 p50. In contrast, inhibition of calcineurin and NFAT by cyclosporin A abrogated the expression of A1 in mast cells on FcepsilonRI-activation but had no effect on lipopolysaccharide-induced expression of A1 in J774A.1 monocytic cells. Cyclosporin A also inhibited luciferase expression in an A1 promoter reporter assay. A putative NFAT binding site in the A1 promoter showed inducible protein binding after FcepsilonRI crosslinking or treatment with ionomycin as detected in a band shift assay or chromatin immunoprecipitation. The binding protein was identified as NFAT1. Finally, mast cells expressing constitutively active NFAT1 exhibit increased expression of A1 after FcepsilonRI-stimulation. These results indicate that, in FcepsilonRI stimulated mast cells, A1 is transcriptionally regulated by NFAT1 but not by NF-kappaB.
Assuntos
Mastócitos/imunologia , Mastócitos/metabolismo , Fatores de Transcrição NFATC/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores de IgE/imunologia , Transcrição Gênica/genética , Animais , Sobrevivência Celular , Células Cultivadas , Reações Cruzadas/imunologia , Ciclosporina/farmacologia , Ionomicina/farmacologia , Macrófagos/efeitos dos fármacos , Mastócitos/citologia , Mastócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Antígenos de Histocompatibilidade Menor , NF-kappa B/deficiência , NF-kappa B/genética , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/genética , Regiões Promotoras Genéticas/genéticaRESUMO
Activation of mast cells through either FcepsilonRI or FcgammaRI leads to release of mediators contributing to the inflammatory response. One of the biologic characteristics of mast cells in allergic pathology is that these cells have the capacity to recover and regranulate after aggregation of FcepsilonRI. We have previously demonstrated that the pro-survival protein A1/Bfl-1 is required for mast cells to survive IgE-mediated activation. In the present study, we have investigated whether human mast cells show similar induction of bfl-1 and activation-induced survival after aggregation of FcgammaRI. Human cord blood-derived mast cells were activated by aggregation of either FcepsilonRI or FcgammaRI, and activation-induced survival and induction of bfl-1 was measured. We found that aggregation of FcgammaRI-induced expression of Bfl-1 and caused a comparable activation-induced mast cell survival as FcepsilonRI does. These data suggests that activation through Fc-receptors contribute to mast cell survival during antibody-dependent mast cell mediated inflammatory responses.
Assuntos
Mastócitos/imunologia , Mastócitos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Receptores de IgG/fisiologia , Regulação para Cima/imunologia , Degranulação Celular/imunologia , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Células Cultivadas , Sangue Fetal/citologia , Humanos , Interferon gama/fisiologia , Antígenos de Histocompatibilidade Menor , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Agregação de Receptores/imunologia , Receptores de IgE/fisiologia , Receptores de IgG/biossíntese , Receptores de IgG/metabolismo , Regulação para Cima/genéticaRESUMO
OBJECTIVE: The aim of this study was to determine the importance of the prosurvival factors Bcl-2 and Bcl-XL for mast cell development and survival. METHODS: bcl-x(-/-) and bcl-2(-/-) mouse embryonic stem cells were maintained in medium supplemented with either interleukin (IL)-3 or IL-3 in combination with stem cell factor (SCF) to favor mast cell development. The development of Bcl-2 family deficient embryonic stem cell-derived mast cells (ESMCs) was monitored and Bcl-2 family gene expression and cell numbers were analyzed. RESULTS: Deficiency in either bcl-x or bcl-2 totally inhibited the development of ESMCs when IL-3 alone was used as a mast cell growth factor. Intriguingly, when IL-3 was used in combination with SCF, the ESMCs developed normally the first 2 weeks but thereafter the cell numbers dropped drastically. The remaining ESMCs express mouse mast cell protease 1, suggesting a mucosal-like phenotype. ESMCs lacking bcl-x or bcl-2 exhibited strong expression of A1, another prosurvival Bcl-2 family member. CONCLUSION: For the first time we provide direct evidence that both bcl-x and bcl-2 are indispensable for mast cell survival during the late phase of their development.