Berres ML, Koenen RR, Rueland A, Zaldivar MM, Heinrichs D, Sahin H, et al. Antagonism of the chemokine Ccl5 ameliorates experimental liver fibrosis in mice. J Clin Invest 2010;120:4129-4140. (Reprinted with permission.)
Activation of hepatic stellate cells in response to chronic inflammation represents a crucial step in the development of liver fibrosis. However, the molecules involved in the interaction between immune cells and stellate cells remain obscure. Herein, we identify the chemokine CCL5 (also known as RANTES), which is induced in murine and human liver after injury, as a central mediator of this interaction. First, we showed in patients with liver fibrosis that CCL5 haplotypes and intrahepatic CCL5 mRNA expression were associated with severe liver fibrosis. Consistent with this, we detected Ccl5 mRNA and CCL5 protein in 2 mouse models of liver fibrosis, induced by either injection of carbon tetrachloride (CCl4) or feeding on a methionine and choline–deficient (MCD) diet. In these models, Ccl5−/− mice exhibited decreased hepatic fibrosis, with reduced stellate cell activation and immune cell infiltration. Transplantation of Ccl5-deficient bone marrow into WT recipients attenuated liver fibrosis, identifying infiltrating hematopoietic cells as the main source of Ccl5. We then showed that treatment with the CCL5 receptor antagonist Met-CCL5 inhibited cultured stellate cell migration, proliferation, and chemokine and collagen secretion. Importantly, in vivo administration of Met-CCL5 greatly ameliorated liver fibrosis in mice and was able to accelerate fibrosis regression. Our results define a successful therapeutic approach to reduce experimental liver fibrosis by antagonizing Ccl5 receptors.
Chemokines and their G protein–coupled receptors are increasingly being recognized as crucial mediators in the pathology of chronic disease. Chemokines (chemotactic cytokines) control the movement of immune cells along a concentration gradient to the site of inflammation or tissue injury and are, therefore, intimately associated with the processes involved in wound healing. In chronic liver disease, resident hepatic cells secrete chemokines in response to tissue injury; subsequently, there is additional production by the resulting inflammatory infiltrate, which includes T cells, dendritic cells, and macrophages. Hepatic fibrosis is the result of an ongoing wound-healing response to a persistent hepatic insult. The resulting inflammatory response by the liver to this insult leads to the subsequent activation of hepatic stellate cells, which are responsible for the deposition of fibrillar collagens and the development of hepatic fibrosis and cirrhosis.
A number of different chemokines, including the C-C motif (or CC) chemokines [monocyte chemotaxis protein 1 (MCP-1) or chemokine (C-C motif) ligand 2 (CCL2); macrophage inflammatory protein 1α (MIP-1α) or CCL3; MIP-1β or CCL4; regulated upon activation, normal T cell expressed, and secreted (RANTES) or CCL5; and eotaxin or CCL11] and the C-X-C motif (or CXC) chemokines [monokine induced by interferon-γ or chemokine (C-X-C motif) ligand 9 (CXCL9) and interferon-inducible protein 10 or CXCL10], have been implicated in the pathogenesis of chronic liver disease.1, 2 Likewise, a number of chemokine receptors, including chemokine (C-C motif) receptor 1 (CCR1), CCR2, CCR5, CCR7, and chemokine (C-X-C motif) receptor 3, have been shown to play crucial roles in the development of hepatic fibrosis. There is considerable redundancy within chemokine subfamilies,1 with many receptors being capable of binding more than one chemokine and with the same chemokine eliciting a response from more than one receptor (Fig. 1).
In a recent study, Berres et al.3 examined the role of the CC chemokine RANTES (also called CCL5) in the interaction between immune cells and hepatic stellate cells and thus in the development of hepatic fibrosis. They examined the expression of RANTES in both human chronic liver diseases (hepatitis C virus and nonalcoholic steatohepatitis) and murine models of hepatic fibrosis, and they demonstrated that T cells in the liver are a major source of RANTES. They then evaluated the effects of the genetic inactivation of RANTES on hepatic fibrosis in animal models of liver disease. Finally, they used the RANTES receptor antagonist Met-CCL5 to assess the effects on both hepatic stellate cell activation in vitro and the development (and treatment) of hepatic fibrosis in animal models of liver injury, and they demonstrated the inhibition of stellate cell activation and the accelerated regression of hepatic fibrosis. This study, therefore, describes the potential therapeutic utility of blocking the function of RANTES in the treatment of hepatic fibrosis.
In this study, Berres et al.3 demonstrated that RANTES was associated with progressive fibrosis in patients with hepatitis C virus, and the distributions of HapMap CCL5 haplotypes were significantly different for patients with mild fibrosis (F0-F1) versus patients with more advanced fibrosis (F2-F4). This difference was principally due to the increased prevalence of the CCL5_H3 haplotype among those with advanced fibrosis (2.6-fold versus those with mild fibrosis). This haplotype is tagged by rs11652536, which is in strong linkage disequilibrium with a functional single-nucleotide polymorphism in the CCL5 promoter that has previously been shown to increase RANTES expression.4 However, this study did not find any significant increases in serum RANTES levels in patients with the minor rs11652536 allele. The involvement of RANTES in progressive fibrosis was also demonstrated in a separate cohort of subjects with nonalcoholic steatohepatitis. The authors suggested that genetically determined serum levels of RANTES may contribute only marginally to increased fibrosis in risk allele carriers.
Berres et al.3 then examined the expression of RANTES [messenger RNA (mRNA) and protein] in two different mouse models of hepatic fibrosis; they used either carbon tetrachloride (CCl4) injections or a methionine and choline–deficient (MCD) diet. Although previous studies have demonstrated elevated expression of RANTES mRNA in animal models of hepatic fibrosis,5 these authors went further by demonstrating that a significant number of RANTES+ cells in the liver were in fact CD3+ T cells. This work also used bone marrow chimeras and Ccl5−/− mice to examine the most likely source of RANTES-expressing cells in CCl4-treated mice. RANTES protein expression was markedly reduced (50%-65%) in mice when the bone marrow was transplanted from Ccl5−/− mice to wild-type (WT) mice (in comparison with both WT→WT mice and WT→Ccl5−/− mice). This experiment showed quite convincingly that hematopoietic cells are likely to be a major source of RANTES associated with hepatic fibrosis, at least in CCl4-induced liver injury.
The Ccl5−/− mice were then used to fully assess the impact of a loss of RANTES expression on the development of hepatic fibrosis in both the CCl4 and MCD models of liver injury. Hepatic fibrosis, which was assessed with Sirius red histochemistry, was markedly suppressed by approximately 65% to 70% in both models of liver injury in comparison with WT mice subjected to these fibrotic stimuli. This was confirmed by the significant decrease in hydroxyproline levels and the suppression of transforming growth factor β1, procollagen α1(I), tissue inhibitor of metalloproteinase 1, interleukin-6, and matrix metalloproteinase 9 mRNA expression. There are two additional observations of note from this study. The first is the statistically significant decrease in serum alanine aminotransferase levels observed in Ccl5−/− mice as early as 24 hours after a single injection of CCl4. This suggests that in the absence of RANTES expression, there is an early reduction in hepatocyte damage; thus, a role for RANTES-induced inflammatory cells (T cells and macrophages) in hepatocyte damage and/or clearance (as evidenced by the release of alanine aminotransferase) is implied. This is an interesting observation worthy of further investigation. The second observation is the loss of α-smooth muscle actin–positive hepatic stellate cells and myofibroblasts in vivo with RANTES gene inactivation; this is perhaps not unexpected, but the fact that this was not replicated in vitro is interesting because hepatic stellate cells isolated from both WT and Ccl5−/− mice and cultured for up to 5 days on plastic showed similar expression levels of α-smooth muscle actin and procollagen α1(I) mRNA. This suggests that hepatic stellate cells require immune cell activation in vivo. In both models, there was a significant reduction in the number of CD3+ T cells and CD68+ macrophages in the livers of Ccl5−/− mice versus WT mice.
This study clearly demonstrated a requirement for infiltrating immune cells in the development of hepatic fibrosis. This conclusion was confirmed through the use of bone marrow–chimeric mice: CCl5−/− bone marrow was transplanted into WT recipients (Ccl5−/−→WT mice) and vice versa (WT→Ccl5−/− mice) after lethal irradiation, with WT→WT mice serving as controls; all mice were subjected to CCl4 injections for 6 weeks. Histological fibrosis, which was assessed with Sirius red histochemistry, was reduced by approximately 75% in the Ccl5−/−→WT mice, whereas in the WT→Ccl5−/− mice, there was a nonsignificant decrease (10%-15%) in hepatic fibrosis versus the WT→WT controls.
The final set of experiments in this study used the RANTES receptor antagonist Met-CCL5 in a series of elegantly designed in vitro and in vivo investigations to determine its effect on the activation of hepatic stellate cells (which are known to respond to RANTES) and hepatic fibrosis. Met-CCL5 is a recombinant RANTES analogue that acts as a potent antagonist of the murine RANTES receptors CCR1 and CCR5.6, 7 Interestingly, Met-CCL5 has no effect on CCR3, a third RANTES receptor7 (Fig. 1). This study showed that Met-CCL5 significantly inhibited the RANTES-induced chemotaxis of hepatic stellate cells and the RANTES-induced secretion of MCP-1 in vitro. Because immune cells were shown in this study to be the principal source of RANTES, the authors incubated hepatic stellate cells with conditioned media from splenocytes isolated from either WT or Ccl5−/− mice. They showed a dramatic reduction (∼35%-45%) in stellate cell chemotaxis, proliferation, and collagen production with Ccl5−/− splenocytes. This reduction in fibrogenic activity was even greater when stellate cells were pretreated with Met-CCL5 before the treatment with WT splenocyte–conditioned media (∼75%-80%).
In the in vivo studies, Met-CCL5 (administered concomitantly with either CCl4 or the MCD diet) significantly inhibited hepatic fibrosis progression (∼20%-40%) and the expression of hepatic genes associated with fibrogenesis. In both animal models of hepatic fibrosis, CD8+ T cells and CD68+ macrophages were significantly reduced by the in vivo Met-CCL5 treatment, whereas the numbers of natural killer and natural killer T cells, B220+ B cells, and CD11c+ dendritic cells were unchanged. When daily Met-CCL5 treatments were administered after the establishment of fibrosis by an 8-week CCl4 injection regimen (3 days after the final CCl4 injection), they augmented the regression of hepatic fibrosis (∼50%) after 7 days. These histological changes in fibrosis were preceded by the reduced expression of both procollagen α1(I) and tissue inhibitor of metalloproteinase 1 mRNA levels in the liver. These data are particularly interesting because they suggest the potential for the treatment of established fibrosis via the accelerated regression of fibrotic tissue, although further investigations are warranted to evaluate the mechanisms involved in this process.
In a previous study, Ruddell et al.8 identified CD45+ immune cells as a source of RANTES in another murine model of hepatic fibrosis. They used the choline-deficient, ethionine-supplemented dietary model of hepatic injury, liver progenitor cell expansion, and portal fibrosis to demonstrate a role for the tumor necrosis factor family member lymphotoxin β (LTβ) in the process of wound healing and hepatic fibrosis.8 They proposed a novel mechanism for RANTES expression by hepatic stellate cells via direct cell contact between liver progenitor and hepatic stellate cells that is induced by the interaction of cell surface–bound LTβ on liver progenitor cells with the LTβ receptor expressed on hepatic stellate cells. In the same study, significant numbers of CD45+ T cells were also demonstrated to express RANTES in choline-deficient, ethionine-supplemented mouse livers and were observed in close spatial association with liver progenitor cells. Neither Ruddell et al. nor Berres et al.3 examined the relative contributions of either T cells or hepatic stellate cells to RANTES expression in these models of hepatic fibrosis. Although it appears that immune cells are the major source of RANTES at least in the CCl4 and MCD models, the contributions of other resident and nonresident hepatic cells require further investigation.
Although a number of studies have examined the role of the RANTES receptors CCR1 and CCR5 or RANTES itself in the various processes associated with hepatic fibrosis, Berres et al.3 took a systematic approach in this very comprehensive study to evaluate the role of RANTES; they assessed both the genetic inactivation of the ligand and the antagonistic blockade of the receptors. A similar type of approach was used by Seki et al.,5 who used the genetic inactivation of either CCR1 or CCR5 to examine the effect on hepatic fibrosis in murine models. They demonstrated that the knockout of either of the RANTES receptors had marked inhibitory effects on histological fibrosis. They showed that the profibrogenic effects of CCR1 appeared to be involved in early fibrosis, whereas CCR5 seemed to be principally involved in perpetuating fibrosis. The effects of CCR1 were predominantly mediated by a bone marrow–derived cell population, whereas the profibrogenic effects of CCR5 principally occurred through resident liver cells such as hepatic stellate cells.2 However, as discussed earlier, these chemokine receptors can have multiple additional activation signals from a variety of different ligands, with both MIP-1α and RANTES acting as ligands of both CCR1 and CCR5 (Fig. 1). The inhibitory effects might be attributed to MIP-1α (via CCR1), or MIP-1α and/or MIP-1β (via CCR5), just as they were attributed to RANTES by Seki et al. Berres et al. assessed the involvement of RANTES in hepatic fibrosis by using both Ccl5−/− mice, and by examining the effects of RANTES receptor antagonism (i.e., via CCR1 and CCR5) with Met-CCL5 and showed very similar effects on the suppression of fibrosis. There are, however, two caveats. Using Ccl5−/− mice leaves other CCR1 and CCR5 agonists (Fig. 1) free to activate these receptors and cause infiltration of profibrogenic cells; this may account for the fact that fibrosis inhibition never reached 100% in this study. In addition, Met-CCL5 does not bind CCR3,7 the third RANTES receptor (Fig. 1), and although a few studies have examined its role in hepatic fibrosis, the potential exists for RANTES (or even eotaxin), that has been produced as a result of hepatic injury in CCl4- or MCD-treated mice, to exert its profibrogenic effects via this alternate receptor in these models of hepatic fibrosis.
Numerous different CCR antagonists that target one of the five different CCRs (CCR1-CCR5) are currently being tested in clinical trials at various stages for the treatment of conditions such as rheumatoid arthritis, asthma, endometriosis, psoriasis, multiple sclerosis, atherosclerosis, chronic obstructive pulmonary disease, cystic fibrosis, and human immunodeficiency virus.9 Previous approaches to the development of chemokine antagonists used neutralizing antibodies for chemokines or their receptors or modified chemokine proteins. Some of these molecules were also found to have limited agonistic properties, which compromised the conclusions drawn in various studies.9 Most compounds in clinical trials are small molecule receptor antagonists; however, neutralizing antibodies remain among those compounds currently being tested, with small peptide–based receptor inhibitors and ribonuclease-resistant RNA aptamers still in preclinical development.9 Chemokine receptor antagonists that block CCR5 have been approved for therapy in patients with human immunodeficiency virus infections.
The RANTES receptor antagonist Met-CCL5 has previously been used in numerous in vitro and animal model studies designed to evaluate the role of RANTES in tissue injury and to potentially treat tissue inflammation occurring as a result of cardiac disease, arthritis, bone disease, and lung disease, among other conditions. Some reports have suggested that Met-CCL5 is a functional antagonist of CCR5 with partial agonistic activity; this has been evidenced by tyrosine kinase phosphorylation, a small but measurable calcium flux, and a slow internalization of CCR5 in T cells or Chinese hamster ovary K1 cells in vitro.10, 11 Others have shown that although Met-CCL5 reduces diet-induced atherosclerosis in animal models,12 RANTES antagonism may not be therapeutically feasible13 because a direct RANTES blockade (as shown in Ccl5−/− mice) may compromise systemic immune responses, impede macrophage-mediated clearance of viral infections,14 and impair routine T cell functions.15 Few studies to date have assessed the therapeutic potential of RANTES receptor antagonism on liver disease progression. One such study demonstrated a decrease in liver disease severity in a concanavalin A–induced hepatitis model of T cell–mediated hepatitis in Ccr5−/− mice and confirmed the role of CCR1+ natural killer cells in the disease process.16 It is apparent that further extensive investigations are required to identify appropriate antagonistic strategies for controlling inflammation and tissue remodeling in clearly defined liver disease contexts. The availability of specific antagonists such as Met-CCL5 will greatly aid us in this endeavor.