RESUMO
Inside-out signaling regulation of the beta2-integrin leukocyte function-associated antigen-1 (LFA-1) by different cytoplasmic proteins, including 14-3-3 proteins, is essential for adhesion and migration of immune cells. Here, we identify a new pathway for the regulation of LFA-1 activity by Cbl-b, an adapter molecule and ubiquitin ligase that modulates several signaling pathways. Cbl-b-/- mice displayed increased macrophage recruitment in thioglycollate-induced peritonitis, which was attributed to Cbl-b deficiency in macrophages, as assessed by bone marrow chimera experiments. In vitro, Cbl-b-/- bone marrow-derived mononuclear phagocytes (BMDMs) displayed increased adhesion to endothelial cells. Activation of LFA-1 in Cbl-b-deficient cells was responsible for their increased endothelial adhesion in vitro and peritoneal recruitment in vivo, as the phenotype of Cbl-b deficiency was reversed in Cbl-b-/-LFA-1-/- mice. Consistently, LFA-1-mediated adhesion of BMDM to ICAM-1 but not VLA-4-mediated adhesion to VCAM-1 was enhanced by Cbl-b deficiency. Cbl-b deficiency resulted in increased phosphorylation of T758 in the beta2-chain of LFA-1 and thereby in enhanced association of 14-3-3beta protein with the beta2-chain, leading to activation of LFA-1. Consistently, disruption of the 14-3-3/beta2-integrin interaction abrogated the enhanced ICAM-1 adhesion of Cbl-b-/- BMDMs. In conclusion, Cbl-b deficiency activates LFA-1 and LFA-1-mediated inflammatory cell recruitment by stimulating the interaction between the LFA-1 beta-chain and 14-3-3 proteins.
Assuntos
Proteínas 14-3-3/imunologia , Movimento Celular/imunologia , Antígeno-1 Associado à Função Linfocitária/imunologia , Macrófagos Peritoneais/imunologia , Proteína Oncogênica v-cbl/imunologia , Transdução de Sinais/imunologia , Proteínas 14-3-3/genética , Animais , Antígenos CD18/genética , Antígenos CD18/imunologia , Adesão Celular/genética , Adesão Celular/imunologia , Linhagem Celular Tumoral , Movimento Celular/genética , Endotélio Vascular/imunologia , Humanos , Inflamação/genética , Inflamação/imunologia , Integrina alfa4beta1/genética , Integrina alfa4beta1/imunologia , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/imunologia , Antígeno-1 Associado à Função Linfocitária/genética , Camundongos , Camundongos Knockout , Proteína Oncogênica v-cbl/genética , Transdução de Sinais/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/imunologiaRESUMO
In this study we investigated the mechanisms mediating T-cell hyporesponsiveness in chronically immune-activated individuals. We analyzed in healthy and persistently helminth-infected individuals the relationship between immune activation and general T-cell hyporesponsiveness, Th3/regulatory T-cell expression, transforming growth factor-beta (TGF-beta) secretion, CTL-associated antigen 4 (CTLA-4) levels, Casitas B-cell lymphoma-b (Cbl-b) (a negative regulator of T-cell activation) levels and phosphorylation of mitogen-activated protein kinases/extracellular signal-regulated kinase (ERK)-1 and -2. We found a very significant increase in plasma levels of TGF-beta and intracellular pools of CTLA-4 and Cbl-b in association with immune activation, which correlates with decreased T-cell responses to anti-CD3 stimulation. We demonstrate that the impaired activity of ERK of peripheral T cells in highly immune-activated individuals is associated with increased levels of CTLA-4 and Cbl-b. Interestingly, in some, but not in all, of these immune-activated individuals, induction of Cbl-b intracellular pools occurs by TGF-beta or CTLA-4 stimulation. We suggest that the higher levels of CTLA-4 and TGF-beta, both involved in the induction of Cbl-b, point at potential mechanisms underlying general and antigen-specific immune hyporesponsiveness in chronically infected individuals.
Assuntos
Antígenos de Diferenciação/biossíntese , Helmintíase/imunologia , Proteína Oncogênica v-cbl/biossíntese , Fator de Crescimento Transformador beta/biossíntese , Adolescente , Adulto , Idoso , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Antígenos CD , Antígenos de Diferenciação/sangue , Antígenos de Diferenciação/imunologia , Western Blotting , Complexo CD3/imunologia , Antígeno CTLA-4 , Doença Crônica , Ativação Enzimática , Etiópia/etnologia , Feminino , Helmintíase/sangue , Helmintíase/parasitologia , Humanos , Tolerância Imunológica , Israel , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Oncogênica v-cbl/sangue , Proteína Oncogênica v-cbl/imunologia , Linfócitos T/enzimologia , Linfócitos T/imunologia , Fator de Crescimento Transformador beta/sangue , Fator de Crescimento Transformador beta/imunologiaRESUMO
The transcription factor Ikaros, a key regulator of hematopoiesis, has an essential role in lymphocyte development. In mice, fetal lymphoid differentiation is blocked in the absence of Ikaros, and whereas T cells develop postnatally, B cells are totally absent. The significance of Ikaros in the B cell development is evident, but how Ikaros regulates B cell function has neither been established nor previously been studied with B cells that lack Ikaros expression. Here we show that disruption of Ikaros in the chicken B cell line DT40 induces a B cell receptor (BCR) signaling defect with reduced phospholipase Cgamma2 phosphorylation and impaired intracellular calcium mobilization, which is restored by Ikaros reintroduction. Furthermore, we show that lack of Ikaros induces hyperphosphorylation of Casitas B lymphoma protein subsequent to BCR activation. These results indicate that the absolute need of Ikaros for development, cell fate decisions and maintenance of B cells is due to the enhancement of BCR signaling.
