RESUMEN
BACKGROUND AND AIMS: The chemokines CCL19 and CCL21 bind CCR7, which is involved in the organization of secondary lymphoid tissue and is expressed during chronic tissue inflammation. We investigated the expression of CCL21 and CCR7 in chronic hepatitis C. The effects of CCL21 on hepatic stellate cells (HSCs) were also studied. METHODS: Expression of CCL21 was assessed by in situ hybridization and immunohistochemistry. CCR7 on T cells was analyzed by flow cytometry. Cultured human HSCs were studied in their activated phenotype. RESULTS: In patients with chronic hepatitis C, expression of CCL21 and CCR7 was up-regulated. CCL21 was detected in the portal tracts and around inflammatory lymphoid follicles, in proximity to T lymphocytes and dendritic cells, which contributed to expression of this chemokine. Expression of CCR7 was also increased in patients with primary biliary cirrhosis. Intrahepatic CD8(+) T lymphocytes isolated from patients with chronic hepatitis C had a significantly higher percentage of positivity for CCR7 than those from healthy controls, and the expression of CCR7 was associated with that of CXCR3. Cultured HSCs expressed functional CCR7, the activation of which stimulated cell migration and accelerated wound healing in an in vitro model. Exposure of HSCs to CCL21 triggered several signaling pathways, including extracellular signal-regulated kinase, Akt, and nuclear factor kappaB, resulting in induction of proinflammatory genes. CONCLUSIONS: Expression of CCL21 during chronic hepatitis C is implicated in the recruitment of T lymphocytes and the organization of inflammatory lymphoid tissue and may promote fibrogenesis in the inflamed areas via activation of CCR7 on HSCs.
Asunto(s)
Movimiento Celular/inmunología , Quimiocinas CC/metabolismo , Hepatitis C Crónica/inmunología , Cirrosis Hepática/inmunología , Quinasa 1 de Quinasa de Quinasa MAP , Linfocitos T/patología , Movimiento Celular/efectos de los fármacos , Núcleo Celular/inmunología , Núcleo Celular/metabolismo , Células Cultivadas , Quimiocina CCL21 , Quimiocinas CC/genética , Quimiocinas CC/farmacología , Expresión Génica/inmunología , Hepatitis C Crónica/patología , Hepatitis C Crónica/fisiopatología , Humanos , Hígado/citología , Cirrosis Hepática/patología , Cirrosis Hepática/virología , Tejido Linfoide/inmunología , Tejido Linfoide/patología , MAP Quinasa Quinasa 2 , Sistema de Señalización de MAP Quinasas/inmunología , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptores CCR7 , Receptores de Quimiocina/genética , Receptores de Quimiocina/metabolismo , Proteínas Recombinantes/farmacología , Linfocitos T/fisiologíaRESUMEN
The biologic effects of growth factors are dependent on cell adhesion, and a cross talk occurs between growth factors and adhesion complexes. The aim of the present study was to evaluate the influence of cell adhesion on the major intracellular signaling pathways elicited by platelet-derived growth factor (PDGF) in hepatic stellate cells (HSC). PDGF signaling was investigated in an experimental condition characterized by lack of cell adhesion for different intervals of time. Basal and PDGF-induced focal adhesion kinase (FAK) tyrosine phosphorylation was maintained in a condition of cell suspension for 2, 4, and 6 hours, whereas it was completely lost after 12 and 24 hours. We examined MAP kinase activity at 2 and 24 hours, corresponding to the higher and lower levels of FAK phosphorylation. In these experiments, MAP kinase activity correlated with FAK phosphorylation. Stimulation with PDGF was able to cause Ras-GTP loading only in adherent cells. The ability of PDGF to induce phosphatidylinositol 3-kinase (PI 3-K) activity was abrogated in cells maintained in suspension. The Ser473 phosphorylation of Akt was only marginally affected by the lack of cell adhesion. We then evaluated the association of FAK with c-Src. This association was found to be cell adhesion dependent, and it did not appear to be dependent from phosphorylated FAK. These changes in PDGF-induced intracellular signaling were associated with a remarkable reduction of PDGF-proliferative potential in nonadherent cells, although no marked differences in the apoptotic rate were observed. In conclusion, these results suggest that cell adhesion differentially regulates major signaling pathways activated by PDGF in HSC.
Asunto(s)
Hepatocitos/enzimología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Serina-Treonina Quinasas , Proteína Tirosina Quinasa CSK , Calcio/metabolismo , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , División Celular/efectos de los fármacos , División Celular/fisiología , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Humanos , Isoenzimas/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfolipasa C gamma , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Fosfolipasas de Tipo C/metabolismo , Tirosina/metabolismo , Proteínas ras/metabolismo , Familia-src QuinasasRESUMEN
BACKGROUND/AIMS: Little is known about the role of fractalkine (CX3CL1) in the liver. The aim of this study was to investigate the expression patterns of fractalkine and its receptor CX3CR1 in normal human liver and in conditions of injury. METHODS: Distribution and expression of fractalkine and its receptor were investigated using immunohistochemistry, in situ hybridization, flow cytometry and reverse transcriptase-polymerase chain reaction. In vitro experiments were conducted in HepG2 cells. RESULTS: Both fractalkine and CX3CR1 were up-regulated during chronic injury, in areas of portal and lobular inflammation. In severe acute hepatitis, fractalkine and CX3CR1 were expressed at high levels not only in areas of inflammation but also in regenerating epithelial cells within bile duct-like structures, which showed co-expression of fractalkine and cytokeratin-7 or CX3CR1. The human hepatocarcinoma cell line HepG2 expressed fractalkine at the gene and protein level, and HepG2-conditioned medium was chemotactic for cells overexpressing CX3CR1. Transcripts for CX3CR1 were detected in HepG2, and exposure of these cells to recombinant fractalkine induced cell migration. CONCLUSIONS: This study shows that the fractalkine system is up-regulated during liver damage, and suggests that fractalkine may play a role in the recruitment and adhesion of inflammatory cells and in the biology of liver epithelial cells.
