RESUMEN
The molecular mechanisms that fine-tune Toll-like receptor (TLR)-triggered innate inflammatory responses remain to be fully elucidated. Major histocompatibility complex (MHC) molecules can mediate reverse signaling and have nonclassical functions. Here we found that constitutively expressed membrane MHC class I molecules attenuated TLR-triggered innate inflammatory responses via reverse signaling, which protected mice from sepsis. The intracellular domain of MHC class I molecules was phosphorylated by the kinase Src after TLR activation, then the tyrosine kinase Fps was recruited via its Src homology 2 domain to phosphorylated MHC class I molecules. This led to enhanced Fps activity and recruitment of the phosphatase SHP-2, which interfered with TLR signaling mediated by the signaling molecule TRAF6. Thus, constitutive MHC class I molecules engage in crosstalk with TLR signaling via the Fps-SHP-2 pathway and control TLR-triggered innate inflammatory responses.
Asunto(s)
Antígenos de Histocompatibilidad Clase I/inmunología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/inmunología , Proteínas Proto-Oncogénicas c-fes/inmunología , Receptores Toll-Like/inmunología , Animales , Escherichia coli/inmunología , Inmunidad Innata/inmunología , Immunoblotting , Interferón beta/inmunología , Interleucina-6/inmunología , Listeria monocytogenes/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , Transducción de Señal/inmunología , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
OBJECTIVE: To prepare anti-fps mono-specific serum, and detect the fps antigen in tumors induced by acute transforming avian leukosis/sarcoma virus containing v-fps oncogene. METHODS: Two part of v-fps gene was amplified by RT-PCR using the Fu-J viral RNA as the template. Mono-specific serum was prepared by immuning Kunming white mouse with both two recombinant infusion proteins expressed by the prokaryotic expression system. Indirect immunofluorescent assay was used to detect fps antigen in tumor tissue suspension cells and CEF infected by sarcoma supernatant. Immunohistochemical method was used to detect fps antigen in tumor tissue. RESULTS: The mouse mono-specific serum was specific as it had no cross reaction with classical ALV-J strains. The result reveals that the tumor tissue suspension cells, the CEF infected by sarcoma supernatant, and the slice immunohistochemistry of the sarcoma showed positive results. CONCLUSION: The anti-fps mono-specific serum was prepared, and the detection method was established, which laid the foundation for the study of viral biological characteristics and mechanism of tumourgenesis of acute transforming avian leukosis/sarcoma virus containing v-fps oncogene.
Asunto(s)
Virus de la Leucosis Aviar/inmunología , Virus del Sarcoma Aviar/inmunología , Pollos , Fibrosarcoma/inmunología , Enfermedades de las Aves de Corral/inmunología , Proteínas Proto-Oncogénicas c-fes/inmunología , Animales , Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Leucosis Aviar/inmunología , Leucosis Aviar/virología , Transformación Celular Neoplásica , Fibrosarcoma/virología , Ratones , Enfermedades de las Aves de Corral/virología , Proteínas Proto-Oncogénicas c-fes/genética , ARN Viral/genética , Sarcoma Aviar/inmunología , Sarcoma Aviar/virología , Organismos Libres de Patógenos EspecíficosRESUMEN
Fps/Fes and Fer are members of a distinct subfamily of cytoplasmic protein tyrosine kinases that have recently been implicated in the regulation of innate immunity. Previous studies showed that mice lacking Fps/Fes are hypersensitive to systemic LPS challenge, and Fer-deficient mice displayed enhanced recruitment of leukocytes in response to local LPS challenge. This study identifies physiological, cellular, and molecular defects that contribute to the hyperinflammatory phenotype in Fps/Fes null mice. Plasma TNF-alpha levels were elevated in LPS challenged Fps/Fes null mice as compared with wild-type mice and cultured Fps/Fes null peritoneal macrophages treated with LPS showed increased TNF-alpha production. Cultured Fps/Fes null macrophages also displayed prolonged LPS-induced degradation of IkappaB-alpha, increased phosphorylation of the p65 subunit of NF-kappaB, and defective TLR4 internalization, compared with wild-type macrophages. Together, these observations provide a likely mechanistic basis for elevated proinflammatory cytokine secretion by Fps/Fes null macrophages and the increased sensitivity of Fps/Fes null mice to endotoxin. We posit that Fps/Fes modulates the innate immune response of macrophages to LPS, in part, by regulating internalization and down-regulation of the TLR4 receptor complex.
Asunto(s)
Regulación hacia Abajo/inmunología , Lipopolisacáridos/farmacología , Macrófagos Peritoneales/inmunología , Proteínas Proto-Oncogénicas c-fes/inmunología , Receptor Toll-Like 4/inmunología , Factor de Transcripción ReIA/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Proteínas I-kappa B/inmunología , Proteínas I-kappa B/metabolismo , Inmunidad Innata/efectos de los fármacos , Inflamación/inmunología , Inflamación/metabolismo , Lipopolisacáridos/inmunología , Activación de Macrófagos , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Noqueados , Inhibidor NF-kappaB alfa , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Procesamiento Proteico-Postraduccional/inmunología , Proteínas Proto-Oncogénicas c-fes/deficiencia , Proteínas Proto-Oncogénicas c-fes/metabolismo , Factor de Transcripción ReIA/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
FES and FES-related (FER) comprise a unique subfamily of protein-tyrosine kinases (PTKs) that signal downstream of several classes of receptors involved in regulating hematopoietic cell development, survival, migration, and inflammatory mediator release. Activated alleles of FES are potent inducers of myeloid differentiation, however FES-deficient mice have only subtle differences in hematopoiesis. This may reflect overlapping function of other kinases such as FER. Studies of FES- and FER-deficient mice have revealed more prominent roles in regulating the activation of mature innate immune cells, including macrophages and mast cells. Recently, new insights into regulation of FES/FER kinases has emerged with the characterization of their N-terminal phospholipid-binding and membrane targeting FER/CIP4 homology-Bin/Amphyphysin/Rvs (F-BAR) and F-BAR extension (FX) domains. The F-BAR/FX domains regulate subcellular localization and FES/FER kinase activation. FES kinase activity is also enhanced upon ligand binding to its SH2 domain, which may lead to further phosphorylation of the same ligand, or other ligand-associated proteins. In mast cells, SH2 ligands of FES/FER include KIT receptor PTK, and the high affinity IgE receptor (FceRI) that trigger rapid activation of FES/FER and signaling to regulators of the actin cytoskeleton and membrane trafficking. Recently, FES/FER have also been implicated in growth and survival signaling in leukemias driven by oncogenic KIT and FLT3 receptors. With further definition of their roles in immune cells and their progenitors, FES/FER may emerge as relevant therapeutic targets in inflammatory diseases and leukemias.