Phagocytosis of Apoptotic Bodies by Hepatic Stellate Cells Occurs in Vivo and Is an Important Mechanism in Liver Fibrogenesis

Abstract

Hepatic stellate cells (HSC) are able to phagocytose apoptotic bodies (AB) in vitro, and this results in a fibrogenic response with up-regulation of TGF-β1 and collagen I gene expression. We have previously shown that phagocytosis induces activation of the NADPH oxidase, with generation of superoxide; however, the exact effect of this on fibrogenic signaling is not known. Therefore, our aims were to (1) demonstrate the role of NADPH oxidase in up-regulation of TGF-β1 and procollagen α 1 (I) gene expression, and (2) visualize phagocytic HSC in vivo, in livers undergoing fibrogenesis. Methods: LX-1 cells were exposed to ABs in the presence or absence of NADPH oxidase inhibitor, diphenylene iodonium (DPI, 20 μM). Total RNA was extracted after 48 hours, and quantitative real-time PCR was performed to evaluate TGF-β1 and procollagen α 1 (I) mRNA expression. The copy numbers were normalized to the expression of GAPDH. For the in vivo studies, we used livers from bile duct ligated (BDL) rats, micropigs diets containing 40% of calories as ethanol for 14 weeks, and human liver biopsy samples with recurrent post-transplant hepatitis C. All liver sections were processed for electron microscopy (EM). Results: Our PCR studies demonstrated that expression of procollagen α 1 (I) significantly increased (2.7-fold) in cells phagocytosing ABs, while adding DPI almost entirely prevented this increase. The expression of TGF-β1 also significantly increased (1.94-fold) in cells exposed to ABs, and this was only partially inhibited by DPI. The EM studies in the 3 models of fibrogenesis clearly revealed the presence of myofibroblast-type cells with intracellular ABs, and abundant microfilaments in the same cells. Conclusions: (1) Phagocytosis of ABs by HSC induced up-regulation of procollagen α 1 (I), and this was prevented by inhibition of NADPH oxidase. Phagocytosis also induced up-regulation of TGF-β1, however inhibition of NADPH oxidase did not prevent this effect, suggesting that different signaling pathways linking phagocytosis of ABs to fibrogenesis. (2) Our in vivo, EM studies in fibrogenesis of different etiologies (cholestasis, ethanol, hepatitis C), and in different species, clearly demonstrated that HSC are phagocytosing ABs in vivo. These results strongly suggest that phagocytosis of ABs by HSC may represent an important mechanism contributing to HSC activation during fibrogenesis.