Seki E, De Minicis S, Gwak G-Y, Kluwe J, Inokuchi S, Bursill CA, et al. CCR1 and CCR5 promote hepatic fibrosis in mice. J Clin Invest 2009;119:1858-1870. (Reprinted with permission).
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Hepatic fibrosis develops as a response to chronic liver injury and almost exclusively occurs in a proinflammatory environment. However, the role of inflammatory mediators in fibrogenic responses of the liver is only poorly understood. We therefore investigated the role of CC chemokines and their receptors in hepatic fibrogenesis. The CC chemokines MIP-1α, MIP-1β, and RANTES and their receptors CCR1 and CCR5 were strongly upregulated in 2 experimental mouse models of fibrogenesis. Neutralization of CC chemokines by the broadspectrum CC chemokine inhibitor 35k efficiently reduced hepatic fibrosis, and CCR1- and CCR5-deficient mice displayed substantially reduced hepatic fibrosis and macrophage infiltration. Analysis of fibrogenesis in CCR1- and CCR5-chimeric mice revealed that CCR1 mediates its profibrogenic effects in BM-derived cells, whereas CCR5 mediates its profibrogenic effects in resident liver cells. CCR5 promoted hepatic stellate cell (HSC) migration through a redox-sensitive, PI3K-dependent pathway. Both CCR5-deficient HSCs and CCR1- and CCR5-deficient Kupffer cells displayed strong suppression of CC chemokine–induced migration. Finally, we detected marked upregulation of RANTES, CCR1, and CCR5 in patients with hepatic cirrhosis, confirming activation of the CC chemokine system in human fibrogenesis. Our data therefore support a role for the CC chemokine system in hepatic fibrogenesis and suggest distinct roles for CCR1 and CCR5 in Kupffer cells and HSCs.
The liver responds to acute injury with a remarkable capacity to restore normal liver function via a wound healing process involving restitution of liver cell mass and architecture. This is achieved via a coordinated series of events involving repeated division of mature hepatocytes, or when this replicative competence is compromised via expansion and differentiation of liver progenitor cells (LPCs). The process of wound healing and reconstitution of normal liver architecture requires numerous cellular interactions involving a major role for hepatic stellate cells (HSCs) in liver matrix renewal. However, perpetuated wound healing becomes entrenched in chronic forms of liver insult, leading to the deposition of significant extracellular matrix, principally fibrillar collagens, and ultimately to the development of hepatic fibrosis and cirrhosis. Recruitment of immunomodulatory cells including HSCs, LPCs, resident (Kupffer cells) and nonresident macrophages, and lymphocytes to the site of hepatic injury and inflammation is crucial for wound healing and hepatic regeneration to occur. Chemokines and their receptors are pivotal in controlling the flux of cell migration in liver disease and thus are inextricably linked to the processes associated with hepatic fibrogenesis.
Chemokines are chemotactic cytokines that control the migration of immune cells along an increasing chemokine concentration gradient to the site of injury in the damaged tissue. Resident cells of the liver secrete chemokines in response to liver injury with subsequent further production by the resultant inflammatory infiltrate. At present, 50 different chemokines and 18 chemokine receptors have been identified. These can be divided into four distinct structurally and functionally related subfamilies on the basis of the position of conserved cysteine residues within a conserved tetracysteine motif at their N-terminus1-3: (1) C-C motif (or CC) chemokines contain two consensus cysteines in juxtaposition; (2) CXC chemokines contain a nonconserved single amino acid between the two N-terminal cysteine residues. These two subfamilies account for 47 of the known chemokines with the remaining three chemokines attributed to either the (3) CX3C subfamily, with three amino acids between the two cysteines; and (4) C chemokines (also annotated as XC), which are devoid of two of the four canonical cysteines.1, 3 Chemokines mediate their chemotactic effects on their respective target cells via seven-transmembrane spanning G protein–coupled receptors. There exists considerable redundancy within each subfamily, with many of the chemokine receptors able to bind more than one chemokine and indeed chemokines able to bind to more than one receptor.2
HSCs are intimately associated with the processes of hepatic wound healing and fibrogenesis. They produce a number of different chemokines which aid in the establishment of an inflammatory infiltrate such as monocyte chemoattractant protein-1 (MCP-1), RANTES (Regulated upon Activation, Normal T-cell Expressed, and Secreted), interleukin-8 (IL-8) (CINC-1 in rats), and macrophage inflammatory protein-2 (MIP-2). Indeed HSCs respond to a variety of chemokines through the elaboration of chemokine receptors such as the chemokine (C-C motif) receptors CCR5 and CCR7, in addition to the chemokine (CXC motif) receptor 3 (CXCR3). Thus, in addition to producing scar tissue, HSCs are crucially involved in regulating cell migration events associated with the inflammation required to initiate fibrogenesis. However, the precise role of HSC chemokine/receptor interaction in the processes associated with wound healing is poorly understood. Two of the most potent HSC chemokines are MCP-1 and RANTES, which along with MIP-1α and MIP-1β, belong to the CC chemokine subfamily. In recent years, there has been significant interest in the contribution of CC chemokines to the development of hepatic fibrosis. Given the inherent redundancy in CC chemokine/receptor interaction, investigations are now focused on the role of both chemokines and their multiple receptors in regulating the processes of wound healing.
In the recent article by Seki et al., the authors examined the potential role of CC chemokine receptors CCR1 and CCR5, and their ligands, RANTES, MIP-1α, and MIP-1β, in promoting HSC activation and hepatic fibrosis.4 They evaluated the effect of CC chemokine neutralization, as well as genetic inactivation of CCR1 and CCR5 on hepatic fibrosis in animal models of both hepatocellular and cholestatic fibrogenesis. In this study, they demonstrated that both CCR1 and CCR5 contribute to the cellular interaction between HSCs and Kupffer cells, but that these CC chemokine receptors influence the processes of fibrogenesis via a difference in temporal expression and via different cell populations. The authors demonstrated a marked hepatic expression of CCR1, CCR5, as well as RANTES, MIP-1α, and MIP-1β messenger RNA (mRNA) in mice subjected to either bile duct ligation (BDL) or carbon tetrachloride (CCl4) to induce hepatic fibrosis. They showed that CCR1 was predominantly expressed by fluorescently activated cell sorting (FACS)-purified Kupffer cells, whereas CCR5 was expressed in both Kupffer cells and HSCs isolated from fibrotic mouse livers. This expression pattern was verified using immunofluorescence staining for CCR1 and CCR5 in mouse liver from both BDL-induced and CCl4-induced hepatic fibrosis models. To assess the contribution of CC chemokines to the fibrotic process, they then used a broad spectrum soluble inhibitor of CC chemokines derived from vaccinia virus, called 35k. Using adenoviral overexpression of 35k (Ad35k) in fibrotic versus wild-type mice, the authors showed an approximate 50% reduction in histological fibrosis. This was accompanied by a 40%-60% inhibition in α-smooth muscle actin protein and hydroxyproline levels, and in genes associated with fibrosis including transforming growth factor-β1, procollagen α1(I) and tissue inhibitor of metalloproteinase-1 (TIMP-1). The incomplete inhibition of hepatic fibrosis using Ad35k can be attributed to one of several possibilities including a potential role for the CXC chemokine system in the development of hepatic fibrosis. In addition, there is clearly an inefficient blockade of CC chemokines, as acknowledged by the authors, due to the fact that 35k does not inhibit murine RANTES.5 It could be suggested that more selective targeting of CC chemokine suppression, perhaps using specific neutralizing antibodies to RANTES, MIP-1α, or MIP-1β, might be a better approach to assess the actual contribution of CC chemokines in fibrogenesis.
In a series of elegantly designed studies using CCR1 and CCR5 knockout mice, Seki et al. showed that both CCR1 and CCR5 are crucial for hepatic fibrosis to develop, but that both the timing of events regulated by these receptors and the cell populations involved are different.4 Hepatic fibrosis induced by either BDL or CCl4 was inhibited by 50% in CCR1−/− mice (at 21 days), which was accompanied by a significant reduction in F4/80+ macrophage (Kupffer cells) and NK1.1 cell infiltration, both of which play a role in HSC activation and fibrogenesis. Of interest, significant reductions in TGF-β1, procollagen α1(I), and TIMP-1 mRNA were observed 5 days after BDL, i.e., quite early in the process of fibrogenesis. In comparison, in CCR5−/− mice, although there was a significant, but less impressive 25%-40% reduction in hepatic fibrosis, hydroxyproline levels and F4/80+ macrophage infiltration after 21 days, the expression of TGF-β1, procollagen α1(I), and TIMP-1 mRNA was not significantly altered 5 days after BDL. However, these genes were all significantly reduced by 21 days after BDL. These data imply that CCR1 may be involved early in fibrogenesis, perhaps more associated with initiation events, whereas CCR5 is principally involved later and may impact on perpetuating fibrosis; thus, CCR1 and CCR5 may regulate fibrogenesis-related cell migration via distinctly different mechanisms.
Using a combination of γ-irradiation and clodronate-induced Kupffer cell depletion prior to bone marrow transplantation, the authors generated CCR1-chimeric and CCR5-chimeric mice to assess the relative contribution of CCR1-expressing or CCR5-expressing resident liver cells, versus bone marrow–derived cells, to the processes associated with hepatic fibrosis. CCR1-chimeric mice with CCR1-deficient bone marrow showed a similar (∼50%) reduction in hepatic fibrosis and hydroxyproline content as in CCR1−/− mice. However, chimeric mice that were CCR1-deficient in the liver but expressed normal CCR1 in the bone marrow showed almost identical hepatic fibrosis as wild-type BDL or CCl4-treated mice. In contrast to these results, CCR5-chimeric mice expressing CCR5 in bone marrow–derived cells but with CCR5 knockout in resident liver cells displayed a similar reduction in hepatic fibrosis as seen in mice with complete CCR5 knockout. Correspondingly, CCR5-chimeric mice expressing CCR5 in resident hepatic cell populations but with CCR5 knockout in the bone marrow displayed a similar degree of fibrosis as wild-type BDL or CCl4-treated mice. Thus, this study demonstrated that the profibrogenic effects of CCR1 were predominantly mediated by a bone marrow–derived cell population, whereas the profibrogenic effects of CCR5 were principally effected via resident liver cells. To further substantiate the differing origins of CC chemokine receptor–expressing cells in fibrogenesis, CCR5−/− mice were transplanted with CCR1−/− bone marrow resulting in a more significant suppression of hepatic fibrosis compared to CCR5−/− alone, showing the distinct mechanisms governing fibrogenesis in these models.
Finally, Seki et al. isolated Kupffer cells and HSCs from CCR1−/−, CCR5−/−, and wild-type mice and assessed their chemotactic responses to RANTES, MIP-1α, and MIP-1β. They showed that although these CC chemokines resulted in cell migration via a reactive oxygen species–induced signal transduction pathway involving phosphoinositol 3-kinase and Akt phosphorylation, the chemokine-induced migration of Kupffer cells isolated from either CCR5−/− or CCR1−/− mice was markedly reduced. Likewise, cell migration, reactive oxygen species generation, and Akt phosphorylation in HSCs isolated from CCR5−/− mice was significantly inhibited whereas HSC chemotaxis from CCR1−/− mice was only partially blocked. Therefore, CCR5 mediates its profibrogenic effects through HSCs, whereas CCR1 promotes fibrogenesis principally via Kupffer cells. Thus, the authors conclude from their study that under conditions of hepatic insult, CC chemokines promote hepatic fibrosis via two distinct mechanisms: (1) the CCR1-dependent early migration of macrophages to the liver which promotes HSC activation and fibrogenesis; and (2) the CCR5-dependent migration of HSCs to the site of hepatic injury, leading to the recruitment of Kupffer cells and other inflammatory cells which activate HSCs and initiate fibrogenesis (Fig. 1).
The same authors have previously demonstrated a role for CCR2 in murine hepatic fibrogenesis showing that CCR2 has distinct roles in both Kupffer cells and HSCs during early and late stages of hepatic injury.6 In the early stages of injury, they propose that Kupffer cell infiltration of the liver is mediated by CCR2 expressed in bone marrow, whereas CCR2 expression in resident liver cells (i.e., Kupffer cells and HSCs) promotes macrophage recruitment and hepatic fibrosis in chronic liver injury.6 Other groups have shown increased expression of the CCR2 ligand MCP-1, in chronic human liver disease,7, 8 with a pivotal role for MCP-1 in HSC chemotaxis demonstrated in vitro.8, 9 The expression of MCP-1 appears to be a very early event in fibrogenesis with studies conducted in BDL rats showing a significant six-fold increased hepatic expression of MCP-1 mRNA as early as 3 days after BDL, prior to the elevated expression of both α-smooth muscle actin and procollagen α1(I) mRNA after 14 days.8 This was accompanied by significantly elevated serum MCP-1 levels observed before histological evidence of fibrosis, i.e., METAVIR fibrosis stage 0.8 In this study, MCP-1 was expressed in both bile duct epithelial cells and hepatocytes in BDL rats, a finding replicated in children with either cystic fibrosis liver disease or biliary atresia. MCP-1 secreted by hepatocytes isolated from BDL rats induced a five-fold elevation in HSC chemotaxis (compared to hepatocytes from sham-operated rats) which was inhibited by 80% using neutralizing antibodies to MCP-1. The study proposed that hepatocytes at the scar margin produced MCP-1, which recruited activated HSCs to the growing fibrotic margin and aided in wound healing in acute injury. However, in chronic cholestatic liver disease, this recruitment would exacerbate fibrosis and result in cirrhosis development (Fig. 1). In this study, MCP-1 expression could be induced in normal hepatocytes by the bile acid taurocholate. Previous studies have not been able to reconcile how MCP-1 affects HSC chemotaxis because CCR2 could not be detected on either human HSCs or portal fibroblasts.9, 10 However, a recent study has clearly shown that both murine Kupffer cells and HSCs express CCR2.6
While Seki et al. demonstrated elevated hepatic expression of CCR1, CCR5, and RANTES mRNA, in patients with hepatic cirrhosis, their study did not assess the source of RANTES, MIP-1α, and MIP-1β.4 This is an important piece of the puzzle which would allow a better understanding of the early triggers leading to the recruitment of CCR1-expressing monocytes/macrophages from bone marrow to the liver to commence the fibrogenic process. Activated HSCs are likely to be a source of RANTES,11 although the precise triggers for RANTES expression are unclear. Various studies have shown that bile acids and ethanol can induce RANTES in hepatocytes12 and Kupffer cells.13 A recent study has suggested a novel mechanism for RANTES expression by HSCs during hepatic regeneration via cell-cell contact between LPCs and HSCs, triggered by the interaction of cell surface–bound lymphotoxin-beta (LT-β) on LPCs with the LT-β receptor expressed on HSCs.14 This aids in the chemotaxis of CCR5+ LPCs and inflammatory cells during fibrogenesis and wound healing. Similar to the data of Seki et al.,4 the LT-β–induced expression of RANTES by HSCs and the recruitment of CCR5+ resident hepatic cells may occur as a perpetuating event in fibrogenesis following the earlier extrahepatic recruitment of CCR1+ inflammatory cells. Future examination of the spatial and temporal expression of CC chemokines and their receptors will further assist our understanding of these complex interactive mechanisms. This may aid in the development of more targeted therapeutic approaches to control fibrogenesis and thus manage the processes governing hepatic wound healing and liver regeneration more effectively.