RESUMO
Invariant natural killer T (iNKT) cells are involved in the intrahepatic immune response and in hepatitis. In particular, iNKT lymphocytes are responsible for hepatocyte death in concanavalin A-induced hepatitis in mice. We examined the role of iNKT cells in acute hepatitis induced by a hepatotoxic agent, carbon tetrachloride (CCl(4) ). WT and iNKT cell-deficient (Jα18(-/-) ) mice were challenged with a single dose of 2.4 g/kg CCl(4) and both hepatic physiopathology and immune responses were studied. Plasma alanine and aspartate amino-transferase levels were significantly higher in Jα18(-/-) mice than in WT mice two days after CCl(4) administration. Chemokine CXCL1/keratinocyte-derived chemokine (KC) and MMP-8 were significantly higher in iNKT cell-deficient mice than in control mice. The more severe liver injury in Jα18(-/-) mice was associated with greater leukocyte infiltrate, which was enriched in neutrophils (CD11b(+) CD11c(-) Gr-1(+) cells), in agreement with CXCL1/KC and MMP-8 levels. Complementary experiments with NK-depleted animals indicate a minor role for NK cells in the liver damage found in iNKT-deficient mice. Thus, unlike for ConA-induced hepatitis, we report that iNKT cells protect the liver against acute hepatitis induced by CCl(4) and limit neutrophil infiltration.
Assuntos
Quimiocina CXCL1/metabolismo , Hepatite Animal/imunologia , Fígado/metabolismo , Células T Matadoras Naturais/metabolismo , Neutrófilos/metabolismo , Doença Aguda , Alanina/sangue , Animais , Apoptose/efeitos dos fármacos , Aspartato Aminotransferases/sangue , Tetracloreto de Carbono/administração & dosagem , Tetracloreto de Carbono/toxicidade , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Quimiocina CXCL1/genética , Quimiocina CXCL1/imunologia , Hepatite Animal/sangue , Hepatite Animal/induzido quimicamente , Hepatócitos/patologia , Fígado/efeitos dos fármacos , Fígado/imunologia , Fígado/patologia , Metaloproteinase 8 da Matriz/genética , Metaloproteinase 8 da Matriz/imunologia , Metaloproteinase 8 da Matriz/metabolismo , Camundongos , Camundongos Knockout , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/patologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/patologia , Receptores de Antígenos de Linfócitos T alfa-beta/genéticaRESUMO
Interleukin-33 (IL-33), the most recently identified member of the IL-1 family, induces synthesis of T Helper 2 (Th2)-type cytokines via its heterodimeric ST2/IL-1RAcP receptor. Th2-type cytokines play an important role in fibrosis; thus, we investigated the role of IL-33 in liver fibrosis. IL-33, ST2 and IL-1RAcP gene expression was analysed in mouse and human normal (n= 6) and fibrotic livers (n= 28), and in human hepatocellular carcinoma (HCC; n= 22), using real-time PCR. IL-33 protein was detected in normal and fibrotic liver sections and in isolated liver cells using Western blotting and immunolocalization approaches. Our results showed that IL-33 and ST2 mRNA was overproduced in mouse and human fibrotic livers, but not in human HCC. IL-33 expression correlated with ST2 expression and also with collagen expression in fibrotic livers. The major sources of IL-33 in normal liver from both mice and human beings are the liver sinusoidal endothelial cells and, in fibrotic liver, the activated hepatic stellate cells (HSC). Moreover, IL-33 expression was increased in cultured HSC when stimulated by pro-inflammatory cytokines. In conclusion, IL-33 is strongly associated with fibrosis in chronic liver injury and activated HSC are a source of IL-33.
Assuntos
Interleucinas/metabolismo , Cirrose Hepática/metabolismo , Animais , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Núcleo Celular/metabolismo , Células Cultivadas , Células Endoteliais/metabolismo , Feminino , Regulação da Expressão Gênica , Células Estreladas do Fígado/metabolismo , Humanos , Inflamação/metabolismo , Inflamação/patologia , Proteína Acessória do Receptor de Interleucina-1/genética , Proteína Acessória do Receptor de Interleucina-1/metabolismo , Interleucina-33 , Interleucinas/genética , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The development of inflammatory granulomas around infected Kupffer cells is necessary for hepatic parasite clearance during visceral leishmaniasis. Invariant NKT (iNKT) cells are predominant T cells in the mouse liver and can synthesize large quantities of IL-4 and IFN-γ, two cytokines involved in granuloma formation. This study analyzed the role of iNKT cells in the hepatic immune response during Leishmania donovani infection, using a murine model of wild-type (WT) and iNKT cell-deficient (Jα18â»/â») C57BL/6 mice sacrificed 15, 30 or 60 days post-infection. We recorded hepatic parasite loads, cytokine expression, and analyzed granulomatous response by immunohistochemistry and hepatic immune cell infiltration by flow cytometry. Whereas WT animals rapidly controlled the infection and developed an inflammatory response associated with a massive influx of iNKT cells observed by flow cytometry, Jα18â»/â» mice had significantly higher parasitic loads on all time points. This lack of control of parasite burden was associated with a delay in granuloma maturation (28.1% of large granulomas at day 60 versus 50.7% in WT). Cytokine transcriptome analysis showed that mRNA of 90/101 genes encoding chemokines, cytokines and their receptors, was underexpressed in Jα18â»/â» mice. Detection of IL-4 and TNF-α by ELISA in liver extracts was also significantly lower in Jα18â»/â» mice. Consistent with flow cytometry analysis, cytokinome profile in WT mice showed a bias of expression towards T cell-chemoattractant chemokines on D15, and displayed a switch towards expression of granulocytes and/or monocytes -chemoattractant chemokines on D60. In Jα18â»/â» mice, the significantly lower expression of CXCL5, MIP-2 and CCL2 mRNA was correlated with a defect in myeloperoxidase positive-cell attraction observed by immunohistochemistry and with a lower granulocyte and monocyte infiltration in the liver, as shown by flow cytometry. These data indicate that iNKT cells play a role in early and sustained pro-inflammatory cytokine response warranting efficient organization of hepatic granulomas and parasite clearance.
Assuntos
Citocinas/metabolismo , Granuloma/patologia , Leishmaniose Visceral/prevenção & controle , Fígado/imunologia , Células T Matadoras Naturais/imunologia , Animais , Citocinas/genética , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/patologia , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , TranscriptomaRESUMO
In cutaneous contact sensitivity there is an early elicited innate cascade of complement, mast cells, and platelets activated via IgM Abs. This response is required to initiate the elicitation of acquired classical contact sensitivity by leading to local recruitment of effector T cells. We recently performed in vivo experiments showing that collaboration is required between innate-like invariant Valpha14+ NKT cells (iNKT) and the innate-like B-1 B cell subset to induce this initiation process. Contact sensitization triggers iNKT cells to produce IL-4 to coactivate the B-1 cells along with specific Ag for production of the initiating IgM Abs. We now describe in vitro collaboration of iNKT and B-1 cells. Normal peritoneal B-1 cells, incubated in vitro with soluble Ag, and with 1-h in vivo immune iNKT cells producing IL-4, are activated to mediate the contact sensitivity-initiation cascade. The three components of this process can be activated by different Ag. Thus, 1-h iNKT cell activation, B-1 cell stimulation, and generation of immune effector T cells can be induced by sensitization with three different Ag to respectively generate IL-4 and Ag-specific IgM Abs, to recruit the Ag-specific effector T cells. These findings have relevance to allergic and autoimmune diseases in which infections can trigger exacerbation of T cell responses to allergens or to autoantigens.
Assuntos
Subpopulações de Linfócitos B/imunologia , Dermatite de Contato/imunologia , Haptenos/administração & dosagem , Haptenos/imunologia , Células Matadoras Naturais/imunologia , Cooperação Linfocítica/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Subpopulações de Linfócitos B/metabolismo , Células Cultivadas , Dermatite de Contato/metabolismo , Feminino , Injeções Subcutâneas , Células Matadoras Naturais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Camundongos Knockout , Camundongos Transgênicos , Cloreto de Picrila/administração & dosagem , Cloreto de Picrila/imunologia , Soroalbumina Bovina/administração & dosagem , Soroalbumina Bovina/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
We showed that hepatic Valpha14+ invariant natural killer T (iNKT) cells, via their rapid interleukin (IL)-4 production, activate B-1 cells to initiate contact sensitivity (CS). This innate collaboration was absent in IL-4(-/-) and signal transducer and activator of transcription (STAT)-6(-/-) mice and was inhibited by anti-IL-4 treatment. These mice have defective CS because they fail to locally recruit the sensitized effector T cells of acquired immunity. Their CS is reconstituted by transfer of downstream-acting 1-day immune B-1 cells from wild-type mice. Responses were not reconstituted with B-1 cells from IL-4 receptor-alpha(-/-) or STAT-6(-/-) mice, nor by IL-4 treatment of B cell-deficient mice at immunization. Finally, IL-4 was preferentially and transiently produced by hepatic iNKT cells within 7 min after sensitization to mediate collaboration between innate-like iNKT cells and the B-1 B cells that participate in the recruitment of effector T cells in vivo.
Assuntos
Subpopulações de Linfócitos B/imunologia , Dermatite de Contato/imunologia , Interleucina-4/imunologia , Células Matadoras Naturais/imunologia , Subpopulações de Linfócitos T/imunologia , Transferência Adotiva , Animais , Antígenos de Diferenciação de Linfócitos B/análise , Comunicação Celular/imunologia , Dermatite de Contato/etiologia , Feminino , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe II/análise , Tolerância Imunológica/imunologia , Interleucina-4/biossíntese , Fígado/imunologia , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Cloreto de Picrila , Receptores de Antígenos de Linfócitos T alfa-beta/análise , Receptores de Interleucina-4/imunologia , Fator de Transcrição STAT6/imunologiaRESUMO
LPS stimulated B-1 cell polyclonal in vivo IgM responses depend on IL-4 release by invariant Valpha14+Jalpha18+ NKT (iNKT) cells. The IgM Abs can recruit effector T cells to mediate contact sensitivity. LPS activates the B-1 cell response just 1 day later, and depends on CD1d, iNKT cells, IL-4, TLR4, and MyD88. LPS in vivo and in vitro stimulates rapid preferential production of IL-4 in hepatic iNKT cells within 2 h. TLR4 were demonstrated in iNKT cells by flow cytometry and functional studies. Thus, innate microbial stimulation via TLR can activate iNKT cell and B-1 cell collaboration. The result is polyclonal IgM Ab responses capable of recruiting Ag-specific T cells into tissues. This may be involved in the promotion of autoimmunity by infectious agents.
Assuntos
Interleucina-4/biossíntese , Células Matadoras Naturais/imunologia , Subpopulações de Linfócitos T/imunologia , Receptor 4 Toll-Like/metabolismo , Animais , Antígenos CD1/metabolismo , Antígenos CD1d , Linfócitos B/imunologia , Imunidade Inata , Imunoglobulina M/biossíntese , Técnicas In Vitro , Lipopolissacarídeos/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Cooperação Linfocítica/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/genéticaRESUMO
Allergic asthma is a multifaceted syndrome consisting of eosinophil-rich airway inflammation, bronchospasm, and airway hyper-responsiveness (AHR). Using a mouse model of allergic asthma, we previously reported that invariant NKT (iNKT) cells increase the severity of this disease. Herein, we demonstrate that a single i.v. injection of alpha-galactosylceramide (alpha-GalCer), 1 h before the first airway allergen challenge of OVA-sensitized mice, abrogates elicitation of AHR, airway eosinophilia, IL-4 and IL-5 production in bronchoalveolar lavage fluid, and specific anti-OVA IgE antibodies. Further, alpha-GalCer administered intranasally also strongly inhibited the major symptoms of asthma in sensitized and challenged mice. Alpha-GalCer treatment induces iNKT cell accumulation in the lungs, and shifts their cytokine profile from pro-asthmatic IL-4 to a protective IFN-gamma production. The role of IFN-gamma from iNKT cells in protection was shown by adoptive transfer of sorted iNKT cells from OVA-sensitized and alpha-GalCer-treated mice which protected immunized recipients from manifesting asthma by an IFN-gamma-dependent pathway. Our findings demonstrate for the first time that alpha-GalCer administered locally inhibits asthma symptoms, even in predisposed asthmatic mice, through an iNKT cell- and IFN-gamma-dependent pathway.
Assuntos
Asma/prevenção & controle , Galactosilceramidas/administração & dosagem , Hipersensibilidade/prevenção & controle , Interferon-alfa/imunologia , Células Matadoras Naturais/efeitos dos fármacos , Administração Intranasal , Transferência Adotiva , Animais , Asma/imunologia , Asma/fisiopatologia , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/prevenção & controle , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Citocinas/análise , Citocinas/efeitos dos fármacos , Citocinas/imunologia , Modelos Animais de Doenças , Citometria de Fluxo , Hipersensibilidade/imunologia , Injeções Intravenosas , Células Matadoras Naturais/imunologia , Masculino , Camundongos , Ovalbumina/imunologia , Ovalbumina/farmacologiaRESUMO
We have previously characterized immature hemopoietic cells of the basophil lineage as a lin(-)c-kit(-) population, which responds to IL-3 by enhancing its histamine synthesis through histidine decarboxylase activation. Herein, we show both in vitro and in vivo that exposure to the pro-Th1 cytokines IL-12 and IL-18 promotes Fas-dependent apoptosis of these cells in the spleen. This conclusion was supported by the following findings: 1) A 24-h treatment with IL-12 plus IL-18 enhanced Fas expression and annexin staining among basophil precursor-enriched lin(-)c-kit(-) splenocytes. 2) Fas or Fas ligand deficiency in mutant mice abolished the inhibitory effect of IL-12 plus IL-18 on IL-3-induced histamine production. 3) The large spectrum inhibitor of the caspase cascade, benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone, significantly reduced the effect of IL-12 plus IL-18. The inhibition of histamine production was mediated through NK cells, since it failed to occur upon stimulation of spleen cells from NK cell-deficient mice or after NK cell depletion. IL-12 plus IL-18 rendered NK cells cytotoxic against Fas-transfected target cells and promoted their production of IFN-gamma and TNF-alpha, which are both essential for sensitizing histamine-producing cells to the Fas death pathway. This is the first evidence that pro-Th1 cytokines can promote apoptosis of immature peripheral histamine-producing cells, thus limiting Th2 immune responses. Comparable in vivo data as well as increased histamine production in the spleen of aged Fas-deficient lpr mice support its physiological relevance.
Assuntos
Apoptose/imunologia , Basófilos/citologia , Basófilos/imunologia , Citocinas/fisiologia , Proteínas , Células Th1/imunologia , Células Th1/metabolismo , Receptor fas/fisiologia , Animais , Antígenos/biossíntese , Antígenos de Superfície , Morte Celular/imunologia , Diferenciação Celular/imunologia , Citocinas/biossíntese , Testes Imunológicos de Citotoxicidade , Regulação para Baixo/imunologia , Combinação de Medicamentos , Feminino , Histamina/biossíntese , Antagonistas dos Receptores Histamínicos/administração & dosagem , Antagonistas dos Receptores Histamínicos/farmacologia , Liberação de Histamina/imunologia , Injeções Intravenosas , Interferon gama/biossíntese , Interferon gama/fisiologia , Interleucina-12/administração & dosagem , Interleucina-12/farmacologia , Interleucina-18/administração & dosagem , Interleucina-18/farmacologia , Interleucina-3/antagonistas & inibidores , Interleucina-3/farmacologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Lectinas Tipo C , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos MRL lpr , Camundongos Knockout , Subfamília B de Receptores Semelhantes a Lectina de Células NK , Biossíntese de Proteínas , Baço/citologia , Baço/imunologia , Baço/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/fisiologia , Receptor fas/genéticaRESUMO
Natural killer (NK) T cells are prominent for their prompt IL-4 and IFN-gamma production upon TCR ligation that enables them to influence acquired immune responses. In the present study we provide evidence that the regulatory functions of this particular T cell subset extend to the myeloid compartment of bone marrow and spleen through its production of hematopoietic growth factors. Bone marrow and spleen NKT cells responded to a single injection of their specific ligand alpha-galactosylceramide (alpha-GalCer) by producing both IL-3 and granulocyte-macrophage colony stimulating factor (GM-CSF), whose colony-stimulating activity became detectable in the serum as early as 1 h post treatment. These cytokines were not produced in mice lacking NKT cells (CD1d-/-), whose exclusive involvement in this biological activity was further confirmed by intracellular immuno-staining. Growth factor production was accompanied by significant changes in the myeloid compartment of treated mice, namely mobilization of myeloid progenitors (colony-forming unit cells, CFU-C) and neutrophils from the bone marrow to the periphery. Taken together, our data support the notion that activated NKT cells influence innate immune responses by recruiting myeloid progenitors and granulocytes to the periphery through their production of hematopoietic growth factors.
Assuntos
Galactosilceramidas/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Interleucina-3/biossíntese , Células Matadoras Naturais/imunologia , Células Mieloides/imunologia , Linfócitos T/imunologia , Animais , Galactosilceramidas/administração & dosagem , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Granulócitos/citologia , Granulócitos/imunologia , Hematopoese Extramedular , Injeções , Interferon gama/genética , Interleucina-3/genética , Interleucina-4/genética , Células Matadoras Naturais/efeitos dos fármacos , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/citologia , Linfócitos T/efeitos dos fármacosRESUMO
The present study was designed to clarify the cytotoxic capacities of invariant V alpha 14 natural killer T (iNKT) cells activated in vivo. We found that as early as 2 h after a single injection of alpha-galactosylceramide (alpha-GalCer), sorted iNKT splenocytes from treated mice kill Fas-transfected target cells. The implication of the Fas pathway in this lysis was strengthened by both the blockage of cytotoxicity in the presence of anti-Fas ligand (FasL) monoclonal antibody (mAb) and the up-regulation of FasL expression on iNKT cells. Sorted NK cells did not participate in the lytic activity at this time point. Yet, they became cytotoxic later on, 24 h post-treatment, when target cell lysis was mainly independent of the Fas pathway. This type of cell killing was predominant at this later time point, even though iNKT cells conserved a slight Fas-dependent cytotoxicity. NK cells failed to acquire the ability to kill target cells when IFN-gamma production in alpha-GalCer-injected mice was blocked by anti-IFN-gamma mAb, underscoring the major role of this cytokine. In conclusion, our findings provide the first direct evidence that iNKT cells can exert Fas-dependent cytotoxicity very shortly after in vivo alpha-GalCer activation and later, through IFN-gamma secretion, enable NK cells to kill target cells in a Fas-independent pathway.
Assuntos
Adjuvantes Imunológicos/farmacologia , Galactosilceramidas/farmacologia , Células Matadoras Naturais/metabolismo , Neuropeptídeos/metabolismo , Receptores do Fator de Necrose Tumoral , Linfócitos T/metabolismo , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Proteína Ligante Fas , Interferon gama/imunologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Camundongos , Neuropeptídeos/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Regulação para Cima , Receptor fasRESUMO
Airway hyperreactivity (AHR), eosinophilic inflammation with a Th2-type cytokine profile, and specific Th2-mediated IgE production characterize allergic asthma. In this paper, we show that OVA-immunized Jalpha18(-/-) mice, which are exclusively deficient in the invariant Valpha14(+) (iValpha14), CD1d-restricted NKT cells, exhibit impaired AHR and airway eosinophilia, decreased IL-4 and IL-5 production in bronchoalveolar lavage fluid, and reduced OVA-specific IgE compared with wild-type (WT) littermates. Adoptive transfer of WT iValpha14 NKT cells fully reconstitutes the capacity of Jalpha18(-/-) mice to develop allergic asthma. Also, specific tetramer staining shows that OVA-immunized WT mice have activated (CD69(+)) iValpha14 NKT cells. Importantly, anti-CD1d mAb treatment blocked the ability of iValpha14 T cells to amplify eosinophil recruitment to airways, and both Th2 cytokine and IgE production following OVA challenge. In conclusion, these findings clearly demonstrate that iValpha14 NKT cells are required to participate in allergen-induced Th2 airway inflammation through a CD1d-dependent mechanism.