RESUMO
BACKGROUND: There is little information about the involvement of galectin-9 (Gal-9) in allergic inflammation. Thus, we investigated the role of Gal-9 in asthma model guinea pigs. METHODS: Airway resistance (R(aw)) was measured using a double-flow plethysmograph system. Gal-9 expression in the lung was assessed by Western blot and immunohistochemistry. Eosinophil chemotactic activity was evaluated in a chamber containing a polyvinylpyrolidone-free membrane. Cell apoptosis was analyzed on a flowcytometry with propidium iodide. RESULTS: In cloning guinea pig Gal-9 we identified three isoforms that differ only in the length of their linker peptides, just as with human Gal-9. Guinea pig Gal-9 was found to be a chemoattractant for eosinophils and to promote induction of apoptosis in sensitized but not non-sensitized T lymphocytes. In allergic airway hypersensitivity model, a low level of Gal-9 expression was observed in the nonsensitized/nonchallenged group, but upregulation was detected at 7 h after challenge and sustained up to 24 h. Such upregulation correlated with elevation of eosinophil peroxidase activity but not with increased R(aw). CONCLUSIONS: The present results provide evidence that Gal-9 is not involved in airway hypersensitivity, but is partly involved in prolonged eosinophil accumulation in the lung.
Assuntos
Resistência das Vias Respiratórias/fisiologia , Eosinófilos/fisiologia , Galectinas/fisiologia , Eosinofilia Pulmonar/fisiopatologia , Hipersensibilidade Respiratória/fisiopatologia , Processamento Alternativo , Sequência de Aminoácidos , Animais , Apoptose , Asma/etiologia , Asma/fisiopatologia , Sequência de Bases , Líquido da Lavagem Broncoalveolar/citologia , Linfócitos T CD4-Positivos/metabolismo , Quimiotaxia de Leucócito , DNA Complementar/genética , Modelos Animais de Doenças , Peroxidase de Eosinófilo/análise , Eosinófilos/citologia , Éxons/genética , Galectinas/biossíntese , Galectinas/química , Galectinas/genética , Galectinas/isolamento & purificação , Cobaias , Humanos , Imunização , Pulmão/química , Pulmão/patologia , Masculino , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Ovalbumina/toxicidade , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Eosinofilia Pulmonar/patologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/fisiologia , Hipersensibilidade Respiratória/etiologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
Galectin-9, a mammalian lectin with affinity for beta-galactosides, is known as an apoptosis inducer of activated T lymphocytes. In the present study, we examined the properties of galectin-9-mediated cell death of Jurkat T cells. Galectin-9NC (wild-type), consisting of two CRDs (N-terminal and C-terminal carbohydrate recognition domains), and derivatives of it, galectins-9-NN and -9-CC, induced Jurkat T-cell apoptosis. However, a single CRD (galectin-9NT or -CT) had no effect, suggesting the stable dimeric structure of two CRDs is required for the activity. The apoptosis was inhibited by pretreatment with an N-glycan synthesis inhibitor, indicating that the expression of N-glycans in the cells is essential for galectin-9-induced apoptosis. We previously showed that the apoptosis of MOLT-4 cell is mediated by galectin-9 via a Ca(2+)-calpain-caspase-1-dependent pathway. In Jurkat cells, the cell death by galectin-9, was insufficiently suppressed by caspase inhibitors, Ca(2+)-chelator or calpain inhibitor. Furthermore, we observed the loss of mitochondrial membrane potential and significant AIF release in galectin-9-treated cells. These findings suggest that caspase-dependent and-independent death pathways exist in Jurkat cells, and the main pathway might vary with the T-cell type.
Assuntos
Apoptose , Galectinas/metabolismo , Linfócitos T/citologia , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Caspase 1/metabolismo , Linhagem Celular Tumoral , Inibidores de Cisteína Proteinase/farmacologia , Humanos , Células Jurkat , Proteínas Recombinantes/metabolismoRESUMO
Little is known about the roles of galectins, a family of beta-galactoside-binding lectins, in myeloid cell differentiation. In the present experiments, we used HL-60 cells as a model of myeloid cell differentiation. The HL-60 cells were differentiated into eosinophil-, monocyte-, and neutrophil-like cells by coculture with sodium butyrate under a mild alkaline condition, phorbol 12-myristate 13-acetate, and dimethyl sulfoxide, respectively. Thus, the expression of galectins in HL-60 cells during differentiation into three different lineages was assessed. Reverse transcriptase-polymerase chain reaction analyses revealed that undifferentiated HL-60 cells expressed galectin-1, -3, -8, -9, and -10 (identical to Charcot Leyden crystal) mRNAs, and galectin-2, -4, and -7 were negligible before and after the differentiations. We failed to detect evident changes in the mRNA levels of galectin-1 and -8 during the differentiations. However, during the eosinophilic differentiation, galectin-9 mRNA expression was gradually decreased, whereas galectin-10 mRNA expression was increased. During the course of monocytic differentiation, galectin-9 mRNA expression was down-regulated, whereas galectin-3 mRNA expression was up-regulated. Moreover, only galectin-10 mRNA expression was enhanced in the process of neutrophilic differentiation. These changes in galectin expressions were confirmed by Western blot and flow cytometry analyses. It is thus suggested that changes in the expressions of galectin-3, -9, and -10 are potentially important for myeloid cell differentiation into specific lineages.
Assuntos
Butiratos/farmacologia , Dimetil Sulfóxido/farmacologia , Galectinas/fisiologia , Células Mieloides/citologia , Acetato de Tetradecanoilforbol/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula , Eosinófilos/citologia , Galectina 3/biossíntese , Galectina 3/genética , Galectina 3/fisiologia , Galectinas/biossíntese , Galectinas/classificação , Galectinas/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glicoproteínas/biossíntese , Glicoproteínas/genética , Glicoproteínas/fisiologia , Células HL-60/efeitos dos fármacos , Humanos , Lisofosfolipase , Monócitos/citologia , Células Mieloides/efeitos dos fármacos , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Neutrófilos/citologia , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We investigated the role of galectin-9 (Gal-9) in maturation of dendritic cells (DC). Culture of immature DCs with exogenous Gal-9 markedly increased the surface expression of CD40, CD54, CD80, CD83, CD86, and HLA-DR in a concentration-dependent manner, although Gal-9 had no effect on differentiation of human monocytes into immature DCs. Gal-9-treated DCs secreted IL-12 but not IL-10, and they elicited the production of Th1 cytokines (IFN-gamma and IL-2), but not that of the Th2 cytokines (IL-4 and IL-5) by allogeneic CD4(+) T cells. These effects of Gal-9 on immature DCs were not essentially dependent on its lectin properties, given that they were only slightly inhibited by lactose. We further found that a Gal-9 mutant that lacks beta-galactoside binding activity reproduced the above activities, and that an anti-Gal-9 mAb suppressed them. Gal-9 induced phosphorylation of the p38 MAPK and ERK1/2 in DCs, and an inhibitor of p38 signaling, but not inhibitors of signaling by either ERK1/2 or phosphatidylinositol 3-kinase, blocked Gal-9-induced up-regulation of costimulatory molecule expression and IL-12 production. These findings suggest that Gal-9 plays a role not only in innate immunity but also in acquired immunity by inducing DC maturation and promoting Th1 immune responses.
Assuntos
Células Dendríticas/imunologia , Galectinas/farmacologia , Animais , Antígenos CD/metabolismo , Diferenciação Celular , Células Cultivadas , Citocinas/metabolismo , Galectinas/fisiologia , Humanos , Interleucina-12/metabolismo , Linfócitos T/imunologia , Células Th1/metabolismoRESUMO
Galectin-9 (Gal-9) is a tandem-repeat-type member of the galectin family associated with diverse biological processes, such as apoptosis, cell aggregation, and eosinophil chemoattraction. Although the detailed sugar-binding specificity of Gal-9 has been elucidated, molecular mechanisms that underlie these functions remain to be investigated. During the course of our binding study by affinity chromatography and surface plasmon resonance (SPR) analysis, we found that human Gal-9 interacts with immobilized Gal-9 in the protein-protein interaction mode. Interestingly, this intermolecular interaction strongly depended on the activity of the carbohydrate recognition domain (CRD), because the addition of potent saccharide inhibitors abolished the binding. The presence of multimers was also confirmed by Ferguson plot analysis of result of polyacrylamide gel electrophoresis and matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Moreover, this intermolecular interaction was observed between Gal-9 and other galectin members, such as Gal-3 and Gal-8, but not Gal-1. Because such properties have not been reported yet, they may explain an unidentified mechanism underlying the diverse functions of Gal-9.
Assuntos
Galectinas/química , Cromatografia de Afinidade , Galectinas/isolamento & purificação , Humanos , Imuno-Histoquímica , Oligossacarídeos/química , Oligossacarídeos/isolamento & purificação , Proteínas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , TermodinâmicaRESUMO
Maturation of dendritic cells (DCs) is critical for initiation of immune responses and is regulated by various stimulatory signals. We assessed the role of galectin (Gal)-9 in DC maturation. Culture of immature DCs with exogenous Gal-9 markedly increased the surface expression of CD40, CD54, CD80, CD83, CD86, and HLA-DR in a dose-dependent manner, although Gal-9 had no or little effect on differentiation of human monocytes into immature DCs. Gal-9-treated DCs secreted IL-12 but not IL-10, and they elicited the production of Th1 cytokines (IFN-gamma and IL-2) but not that of the Th2 cytokines (IL-4 and IL-5) by allogeneic CD4+ T cells. These effects of Gal-9 on immature DCs were not essentially dependent on its lectin properties, given that they were inhibited only slightly by lactose. We further found that a Gal-9 mutant that lacks beta-galactoside binding activity reproduced the above activities and that an anti-Gal-9 mAb suppressed them. Gal-9 induced phosphorylation of the MAPK p38 and ERK1/2 in DCs, and an inhibitor of p38 signaling, but not inhibitors of signaling by either ERK1/2 or PI3K, blocked Gal-9-induced up-regulation of costimulatory molecule expression and IL-12 production. These findings suggest that Gal-9 plays a role not only in innate immunity but also in acquired immunity by inducing DC maturation and promoting Th1 immune responses.
Assuntos
Células Dendríticas/fisiologia , Galectinas/fisiologia , Monócitos/citologia , Apresentação de Antígeno , Diferenciação Celular , Células Dendríticas/efeitos dos fármacos , Humanos , Interleucina-4/farmacologia , Lactose/farmacologia , Lipopolissacarídeos/farmacologia , Teste de Cultura Mista de Linfócitos , Fosforilação , Células Th1/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Ecalectin/galectin-9 was recently described as a novel eosinophil chemoattractant highly expressed in immune tissues. We investigated the regulation of galectin-9 expression and release in Jurkat (a T cell line) cells. We demonstrated that medium and long-sized galectin-9 isoforms were constitutively expressed, and phorbol 12-myriastate 13-acetate (PMA) upregulated the level of galectin-9 mRNA in Jurkat cells. Western blotting and flow cytometry analyses revealed that PMA stimulation resulted in the upregulation of both intracellular and surface galectin-9 protein. The stimulated Jurkat cells simultaneously released evident eosinophil chemoattractant activity (ECA). Main ECA was adsorbed by both lactose and anti-galectin-9 antibody affinity column, suggesting that the ECA was ascribed to galectin-9. When Jurkat cells were stimulated with PMA in the presence of a BB94, a matrix metalloproteinase (MMP) inhibitor, but not tissue inhibitor of metalloproteinase-1 (TIMP-1), the release of galectin-9 was suppressed in a dose-dependent manner. We further found that calphostin c, a protein kinase c (PKC) inhibitor, weakly but significantly suppressed the release of galectin-9. The present data suggested that galectin-9 production in Jurkat cells is provoked by the stimulation with PMA and that some MMP and PKC is, at least, partly involved in the release of galectin-9 from Jurkat cells.
Assuntos
Galectinas , Células Jurkat/metabolismo , Lectinas/genética , Fenilalanina/análogos & derivados , Animais , Formação de Anticorpos , Antineoplásicos/farmacologia , Western Blotting , Carcinógenos/farmacologia , Fatores Quimiotáticos de Eosinófilos/metabolismo , Primers do DNA/química , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Lectinas/metabolismo , Metaloendopeptidases/antagonistas & inibidores , Naftalenos/farmacologia , Fenilalanina/farmacologia , Proteína Quinase C/antagonistas & inibidores , RNA Mensageiro/metabolismo , Coelhos , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Acetato de Tetradecanoilforbol/farmacologia , Tiofenos/farmacologia , Inibidor Tecidual de Metaloproteinase-1/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
We first cloned galectin-9 (Gal-9)/ecalectin as a T cell-derived eosinophil chemoattractant. Gal-9 plays a role in not only accumulation but also activation of eosinophils in experimental allergic models and human allergic patients, because Gal-9 induces eosinophil chemoattraction in vitro and in vivo and activates eosinophils in many aspects. Gal-9 requires divalent galactoside-binding activity but not the linker peptide of Gal-9 to exhibit its biological functions, and an unidentified matrix metalloproteinase is involved in the release of Gal-9. Our recent studies also showed that Gal-9 has other functions, such as cell differentiation, aggregation, adhesion, and death. Now, we and other groups are on the way of investigating the regulation and function of Gal-9 in a variety of physiological and pathological conditions. In this article, we will show the possible role of Gal-9 in physiological and pathological conditions by using our recent findings.
Assuntos
Fenômenos Fisiológicos Celulares , Galectinas/metabolismo , Neoplasias/metabolismo , Apoptose , Eosinófilos/metabolismo , Galectinas/biossíntese , Galectinas/química , Humanos , Neoplasias/diagnóstico , Neoplasias/patologiaRESUMO
Galectin-9 (Gal-9) induced the apoptosis of not only T cell lines but also of other types of cell lines in a dose- and time-dependent manner. The apoptosis was suppressed by lactose, but not by sucrose, indicating that beta-galactoside binding is essential for Gal-9-induced apoptosis. Moreover, Gal-9 required at least 60 min of Gal-9 binding and possibly de novo protein synthesis to mediate the apoptosis. We also assessed the apoptosis of peripheral blood T cells by Gal-9. Apoptosis was induced in both activated CD4(+) and CD8(+) T cells, but the former were more susceptible than the latter. A pan-caspase inhibitor (Z-VAD-FMK) inhibited Gal-9-induced apoptosis. Furthermore, a caspase-1 inhibitor (Z-YVAD-FMK), but not others such as Z-IETD-FMK (caspase-8 inhibitor), Z-LEHD-FMK (caspase-9 inhibitor), and Z-AEVD-FMK (caspase-10 inhibitor), inhibited Gal-9-induced apoptosis. We also found that a calpain inhibitor (Z-LLY-FMK) suppresses Gal-9-induced apoptosis, that Gal-9 induces calcium (Ca(2+)) influx, and that either the intracellular Ca(2+) chelator BAPTA-AM or an inositol trisphosphate inhibitor 2-aminoethoxydiphenyl borate inhibits Gal-9-induced apoptosis. These results suggest that Gal-9 induces apoptosis via the Ca(2+)-calpain-caspase-1 pathway, and that Gal-9 plays a role in immunomodulation of T cell-mediated immune responses.
Assuntos
Apoptose/fisiologia , Sinalização do Cálcio , Calpaína/fisiologia , Caspase 1/fisiologia , Galectinas/fisiologia , Adjuvantes Imunológicos/fisiologia , Clorometilcetonas de Aminoácidos/farmacologia , Linfócitos B/citologia , Linfócitos B/enzimologia , Linfócitos B/fisiologia , Cálcio/metabolismo , Cálcio/fisiologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/imunologia , Inibidores de Caspase , Inibidores de Cisteína Proteinase/farmacologia , Relação Dose-Resposta Imunológica , Células HL-60 , Humanos , Células Jurkat , Monócitos/citologia , Monócitos/enzimologia , Monócitos/fisiologia , Células Mieloides/citologia , Células Mieloides/enzimologia , Células Mieloides/fisiologia , Oligopeptídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Linfócitos T/citologia , Linfócitos T/enzimologia , Linfócitos T/fisiologia , Células Tumorais CultivadasRESUMO
BACKGROUND: There is no information whether galectin-9 (a novel eosinophil chemoattractant) was associated with pathogenesis of eosinophilic disorders. METHODS: We assessed the expression of galectin-9 with imunostaining and in situ hybridization both in the lesion of angiolymphoid hyperplasia with eosinophilia, and peripheral blood eosinophils of eosinophilic patients (E-Eos) in comparison with those of normal volunteers (N-Eos). Regulation of expression of galectin-9 on eosinophils and the effect of galectin-9 on apoptosis of eosinophil were also evaluated. RESULTS: Many eosinophils infiltrating the site were positive for galectin-9. Surface and intracellular immunoreactive galectin-9 was more evident in E-Eos than N-Eos. When eosinophils were cultured with IL-5 in vitro, the surface galectin-9 expression of E-Eos was significantly downregulated, although that of N-Eos was not affected. Treatment of eosinophils with dexamethasone or anti-Fas antibody significantly upregulated the surface galectin-9 expression of E-Eos. In contrast, dexamethasone partially downregulated the surface galectin-9 of N-Eos, although anti-Fas antibody failed to affect on the surface galectin-9 expression. We also found that recombinant galectin-9 significantly suppressed apoptosis of E-Eos (p = 0.0431), whereas it apparently enhanced apoptosis of N-Eos (p = 0.0173). Furthermore, dexamethasone-induced apoptosis of N-Eos was significantly suppressed by galectin-9 (p = 0.0431), whereas galectin-9 failed to induce significant change in dexamethasone-induced apoptosis of E-Eos. In contrast, apoptosis induced by anti-Fas antibody in both N-Eos (p = 0.0431) and E-Eos (p = 0.0431) was enhanced by galectin-9. CONCLUSIONS: These findings suggested that galectin-9 was produced by eosinophils, and galectin-9 showed heterogeneous effects and kinetics to eosinophils, and this factor might be one of crucial factors in eosinophilic inflammation.
Assuntos
Hiperplasia Angiolinfoide com Eosinofilia/imunologia , Apoptose/imunologia , Eosinófilos/imunologia , Galectinas , Lectinas/imunologia , Hiperplasia Angiolinfoide com Eosinofilia/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Murinos , Biópsia , Dexametasona/imunologia , Dexametasona/farmacologia , Eosinófilos/citologia , Citometria de Fluxo , Glucocorticoides/imunologia , Glucocorticoides/farmacologia , Humanos , Imuno-Histoquímica , Hibridização In Situ , Interleucina-5/imunologia , Interleucina-5/farmacologia , Lectinas/genética , Lectinas/metabolismoRESUMO
Galectin-9 expression was examined in 6 human melanoma cell lines. Among them, MM-BP proliferated with colony formation, but MM-RU failed. RT-PCR analysis revealed evident expression of galectin-9 mRNA in MM-BP but not in MM-RU. MM-BP expressed galectin-9 protein both on the surface and in the cytoplasm, whereas MM-RU expressed it only weakly in the cytoplasm. Exogenous galectin-9 induced in vitro both cell aggregation and apoptosis of MM-RU proliferating without colony formation. Association of galectin-9 expression in melanoma cells with prognosis of the patients bearing melanocytic tumors was further examined. Galectin-9 protein was strongly and homogeneously expressed in melanocytic nevi, but down-regulated in melanoma cells especially in metastatic lesions. High galectin-9 expression was inversely correlated with the progression of this disease, suggesting that high galectin-9 expression in primary melanoma lesions links to a better prognosis.
Assuntos
Apoptose/efeitos dos fármacos , Agregação Celular/efeitos dos fármacos , Galectinas , Lectinas/farmacologia , Melanoma/patologia , Neoplasias Cutâneas/patologia , Células Tumorais Cultivadas/efeitos dos fármacos , Anexina A5/metabolismo , Western Blotting , Primers do DNA/química , Intervalo Livre de Doença , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Lectinas/genética , Melanoma/metabolismo , Nevo Pigmentado/metabolismo , Nevo Pigmentado/patologia , Prognóstico , RNA Mensageiro/biossíntese , RNA Neoplásico/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Cutâneas/metabolismo , Taxa de Sobrevida , Células Tumorais Cultivadas/metabolismoRESUMO
Among galectin family members, galectin-9 was first described as a potent eosinophil chemoattractant derived from Ag-stimulated T cells. In the present study a role of galectin-9 in the interaction between eosinophils and fibroblasts was investigated using a human lung fibroblast cell line, HFL-1. RT-PCR, real-time PCR, and Western blot analyses revealed that both galectin-9 mRNA and protein in HFL-1 cells were up-regulated by IFN-gamma stimulation. On the one hand, IL-4, known as a Th2 cytokine, did not affect the galectin-9 expression in HFL-1 cells. We further confirmed that IFN-gamma up-regulated the expression of galectin-9 in primary human dermal fibroblasts. Flow cytometric analysis revealed that IFN-gamma up-regulated surface galectin-9 expression on HFL-1 cells. Stimulation of HFL-1 cells with IFN-gamma up-regulated adhesion of eosinophils, but not neutrophils, to HFL-1 cells. This adherence of eosinophils to HFL-1 cells was inhibited by both lactose and anti-galectin-9 Ab. These findings demonstrate that IFN-gamma-induced galectin-9 expression in fibroblasts mediates eosinophil adhesion to the cells, suggesting a crucial role of galectin-9 in IFN-gamma-stimulated fibroblasts as a physiological modulator at the inflammatory sites.