Original Article Activated hepatic stellate cells mediate the differentiation of macrophages Jonathan Chang, Tadakazu Hisamatsu, Katsuyoshi Shimamura, Kazuaki Yoneno, Masayuki Adachi, Hiroshi Naruse, Toru Igarashi, Hajime Higuchi, Katsuyoshi Matsuoka, Mina T. Kitazume, Setsu Ando, Nobuhiko Kamada, Takanori Kanai and Toshifumi Hibi Division of Gastroenterology and Hepatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan Aim: Liver macrophages play integral roles in both the pro- gression and resolution of hepatic inflammation and fibrosis, comprising opposing functions that largely coincide with the activation state of nearby hepatic stellate cells (HSC). While cross-talk between HSC and macrophages may be essential at various stages of inflammation and fibrogenesis, many facets of this interaction have yet to be thoroughly explored. Here, we examine the potential roles of HSC-derived signaling mol- ecules as mediators of liver macrophage differentiation. Methods: Human peripheral blood mononuclear cells (PBMC) were differentiated to macrophages in the presence or absence of cultured HSC-derived conditioned media. The phenotype of resulting macrophages was characterized by examination of cell surface marker expression, antigen- presenting capabilities and cytokine secretion. Results: Conditioned media from activated human HSC pro- moted the differentiation of a unique set of macrophages that differed in morphology and function from both classical (M1) and alternative (M2) macrophages, expressing increased levels of CD14 and CD16, as well as a distinct interleukin (IL)-6 high /IL-10 low /transforming growth factor (TGF)-b high expres- sion profile. These macrophages expressed high levels of CD206, CD209, CD80 and human leukocyte antigen DR, though no significant increases in antigen presentation were apparent. HSC-derived macrophages exhibited specific acti- vation of p38 mitogen-activated protein kinase, and inhibition of this activation by p38 inhibitors during differentiation effec- tively reversed increases in IL-6 and TGF-b. Conclusion: The present results suggest that HSC-derived signaling molecules promote differentiation of liver macroph- ages with both pro-inflammatory and profibrotic functions. Furthermore, these effects appear to be mediated, at least partially, in a p38-dependent manner. Key words: fibrosis, hepatic stellate cell, interleukin-6, macrophage, p38 mitogen-activated protein kinase, transforming growth factor-b INTRODUCTION T HE PIVOTAL ROLE of hepatic stellate cells (HSC) in the progression of chronic liver disease has been well established in recent years. 1 Though normally found in a quiescent state, HSC undergo a process of differentiation in response to liver injury whereby they assume a more contractile, myofibroblast-like pheno- type. This latter state is characterized by excessive production of a-smooth muscle actin and various extra- cellular matrix (ECM) proteins including collagen types I and III. 2–4 While numerous resident fibroblast popula- tions are also known to produce ECM components, acti- vated HSC are likely to be the principal contributor of such fibrotic components in most forms of fibrosis. 5–11 Consequently, HSC are widely regarded as critical mediators of hepatic fibrogenesis. Not surprisingly, elucidation of the conditions whereby HSC activation may be triggered, controlled or reversed has become an area of particular interest. Because HSC are located in the space of Disse, and consequently have close proximities to both hepatocytes and immune cells of the hepatic sinusoid, there are numerous potential avenues of cellular cross-talk to be explored. Indeed, research to date has revealed a complex network of cellular activities and interactions, Correspondence: Dr Toshifumi Hibi and Dr Tadakazu Hisamatsu, Division of Gastroenterology and Hepatology, Department of Internal Medicine, School of Medicine, Keio University, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan. Email: thibi@sc.itc.keio.ac.jp; hisamachi@a7.keio.jp Received 14 April 2012; revision 23 August 2012; accepted 24 September 2012. Hepatology Research 2013; 43: 658–669 doi: 10.1111/j.1872-034X.2012.01111.x © 2012 The Japan Society of Hepatology 658