RESUMO
Although several groups have investigated the role of SHIP in macrophage (M) development and function, SHIP's contribution to the generation, maturation, and innate immune activation of dendritic cells (DCs) is poorly understood. We show herein that SHIP negatively regulates the generation of DCs from bone marrow precursors in vitro and in vivo, as illustrated by the enhanced expansion of DCs from SHIP(-/-) GM-CSF cultures, as well as increased numbers of DCs in the spleens of SHIP-deficient mice. Interestingly, however, these SHIP(-/-) DCs display a relatively immature phenotype and secrete substantially lower levels of IL-12 after TLR ligand stimulation than wild type DCs. This, in turn, leads to a dramatically reduced stimulation of Ag-specific T cell proliferation and Th1 cell responses in vitro and in vivo. This immature phenotype of SHIP(-/-) DCs could be reversed with the PI3K inhibitors LY294002 and wortmannin, suggesting that SHIP promotes DC maturation by reducing the levels of the PI3K second messenger phosphatidylinositol-3,4,5-trisphosphate. These results are consistent with SHIP being a negative regulator of GM-CSF-derived DC generation but a positive regulator of GM-CSF-derived DC maturation and function.
Assuntos
Diferenciação Celular/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Monoéster Fosfórico Hidrolases/fisiologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Diferenciação Celular/genética , Proliferação de Células , Células Cultivadas , Células Dendríticas/citologia , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/fisiologia , Inibidores do Crescimento/deficiência , Inibidores do Crescimento/genética , Inibidores do Crescimento/fisiologia , Imunidade Inata/genética , Inositol Polifosfato 5-Fosfatases , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Monoéster Fosfórico Hidrolases/deficiência , Monoéster Fosfórico Hidrolases/genética , Células-Tronco/citologia , Células-Tronco/imunologia , Células-Tronco/metabolismo , Receptores Toll-Like/fisiologia , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
We demonstrate herein that SHIP negatively regulates the proliferation, differentiation, and survival of FL-DCs from BM precursors, as shown by a more rapid appearance and higher numbers of CD11c(+) DCs from SHIP-/- cultures as well as increased survival of mature FL-DCs in the absence of Flt3L. This increased survival, which is lost with low levels of the PI3K inhibitor, LY, correlates with an enhanced constitutive activation of the Akt pathway. Interestingly, however, these SHIP-/- FL-DCs display a less-mature phenotype after TLR ligand stimulation, as far as MHCII, CD40, and CD86 are concerned. Unexpectedly, SHIP-/- FL-DCs activated with TLR ligands, which use MyD88-independent pathways, are markedly impaired in their ability to stimulate Ag-specific T cell proliferation, and SHIP-/- FL-DCs activated by TLRs, which exclusively use the MyD88-dependent pathway, are as capable as WT FL-DCs. There is also a more pronounced T(H)1 skewing by the SHIP-/- FL-DCs than by WT FL-DCs, which is consistent with our finding that SHIP-/- FL-DCs secrete higher levels of IL-12 and TNF-α in response to LPS or dsRNA than their WT counterparts. These results suggest that SHIP negatively regulates FL-DC generation but positively regulates the maturation and function of FL-DCs induced by TLRs, which operate via MyD88-independent pathways.
Assuntos
Células Dendríticas/fisiologia , Monoéster Fosfórico Hidrolases/genética , Animais , Western Blotting , Células Cultivadas , Cruzamentos Genéticos , Primers do DNA , Células Dendríticas/citologia , Células Dendríticas/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Inositol Polifosfato 5-Fosfatases , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fator 88 de Diferenciação Mieloide/genética , Monoéster Fosfórico Hidrolases/deficiência , RNA/genética , RNA/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/imunologiaRESUMO
OBJECTIVE: The activity of the SH2-containing-phosphatidylinositol-5'-phosphatase (SHIP, also known as SHIP1), a critical hematopoietic-restricted negative regulator of the PI3 kinase (PI3K) pathway, is regulated in large part via its protein levels. We sought to determine the mechanism(s) involved in its downregulation by BCR-ABL and by interleukin (IL)-4. MATERIALS AND METHODS: We used Ba/F3(p210-tetOFF) cells to study the downregulation of SHIP by BCR-ABL and bone marrow-derived macrophages to study SHIP's downregulation by IL-4. RESULTS: We show herein that BCR-ABL downregulates SHIP, but not SHIP2 or PTEN, and this can be blocked with the Src kinase inhibitor PP2, which inhibits the tyrosine phosphorylation of SHIP, or with the proteasomal inhibitor MG-132. We also show, using anti-SHIP immunoprecipitates, that c-Cbl and Cbl-b are associated with SHIP and that BCR-ABL induces SHIP's polyubiquitination. This ubiquitination can be blocked with PP2, consistent with the tyrosine phosphorylation of SHIP acting as a signal for its ubiquitination. In bone marrow-derived macrophages, IL-4 also leads to the proteasomal degradation of SHIP but, unlike in Ba/F3(p210-tetOFF) cells, SHIP2 is also proteasomally degraded and the degradation of both inositol phosphatases can be prevented with PP2 or MG-132. CONCLUSION: Our results suggest that SHIP protein levels can be reduced via BCR-ABL and/or Src family member-induced tyrosine phosphorylation of SHIP because this triggers its polyubiquitination and degradation within the proteasome.