CCR2 promotes hepatic fibrosis in mice

Authors


  • Potential conflict of interest: Nothing to report.

Abstract

Chemokines and chemokine receptors contribute to the migration of hepatic stellate cells (HSCs) and Kupffer cells, two key cell types in fibrogenesis. Here, we investigate the role of CCR2, the receptor for monocyte chemoattractant protein (MCP)-1, MCP-2, and MCP-3, in hepatic fibrosis. Hepatic CCR2, MCP-1, MCP-2, and MCP-3 messenger RNA expression was increased after bile duct ligation (BDL). Both Kupffer cells and HSCs, but not hepatocytes, expressed CCR2. BDL- and CCl4-induced fibrosis was markedly reduced in CCR2−/− mice as assessed through collagen deposition, α-smooth muscle actin expression, and hepatic hydroxyproline content. We generated CCR2 chimeric mice by the combination of clodronate, irradiation, and bone marrow (BM) transplantation allowing full reconstitution of Kupffer cells, but not HSCs, with BM cells. Chimeric mice containing wild-type BM displayed increased macrophage recruitment, whereas chimeric mice containing CCR2−/− BM showed less macrophage recruitment at 5 days after BDL. Although CCR2 expressed in the BM enhanced macrophage recruitment in early phases of injury, CCR2 expression on resident liver cells including HSCs, but not on the BM, was required for fibrogenic responses in chronic fibrosis models. In vitro experiments demonstrated that HSCs deficient in CCR2−/− or its downstream mediator p47phox−/− did not display extracellular signal-regulated kinase and AKT phosphorylation, chemotaxis, or reactive oxygen species production in response to MCP-1, MCP-2, and MCP-3. Conclusion: Our results indicate that CCR2 promotes HSC chemotaxis and the development of hepatic fibrosis. (HEPATOLOGY 2009.)

Hepatic fibrosis results from many chronic liver diseases, including hepatitis B and C, autoimmune hepatitis, alcoholic liver disease, and nonalcoholic steatohepatitis.1 Hepatic stellate cells (HSCs) are the principal liver cells that promote hepatic fibrosis.2, 3 Upon the activation of HSCs by various stimuli, such as transforming growth factor (TGF)-β and platelet-derived growth factor, HSCs transdifferentiate into myofibroblasts and then produce excessive extracellular matrix proteins (including collagen type I, III, and IV), resulting in hepatic fibrosis. Cirrhosis, the end stage of hepatic fibrosis, may result in hepatic dysfunction, portal hypertension, and hepatocellular carcinoma.1, 4 Kupffer cells, hepatic resident macrophages, induce acute and chronic liver inflammation by producing inflammatory cytokines—including tumor necrosis factor α (TNF-α), IL-6, CC-chemokine ligand 2/monocyte chemoattractant protein (MCP)-1, interleukin (IL)-1, and TGF-β—and by activating HSCs during hepatic fibrosis.3, 5–7

Recruitment of immune cells such as Kupffer cells and HSCs to the site of injury and inflammation is an important event in regeneration, wound healing, and hepatic fibrosis.1 Chemokines and chemokine receptors have a central role in the regulation of cell migration and local inflammation.8 CC-chemokine receptor 2 (CCR2), which is mainly expressed on the surface of monocytes and macrophages, is a functional receptor for MCP-1, MCP-2, and MCP-3 and is involved in the migration of monocytes and macrophages in peritonitis, autoimmune encephalitis, rheumatoid arthritis, tuberculosis, atherosclerosis, and obesity.9–16 Genetic or pharmacological inactivation of CCR2 inhibits pulmonary and renal fibrosis.17–19 The CCR2 ligand MCP-1 is produced by Kupffer cells and HSCs, which promotes hepatic fibrosis by recruitment of macrophages that are associated with HSC activation.7, 20–22

The present study examines the role of CCR2 in two different experimental models of hepatic fibrosis and identifies the CCR2-expressing cell types in the liver. We characterized the distinct roles of CCR2 in Kupffer cells and HSCs between the early phase of liver injury and chronic liver fibrosis. In addition, we assessed the function of CCR2 in HSC activation by stimulating primary cells with the CCR2 ligands MCP-1, MCP-2, and MCP-3 and then investigated the requirement of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in HSC activation and migration.

Abbreviations

ALT, alanine aminotransferase; BDL, bile duct ligation; BM, bone marrow; BMT, bone marrow transplantation; CCR, CC-chemokine receptor; DPI, diphenilene-iodonium; ERK, extracellular signal-regulated kinase; GFP, green fluorescent protein; 4-HNE, 4-hydroxy-nonenal; HSC, hepatic stellate cells; IL, interleukin; MCP, monocyte chemoattractant protein; MIP-1β, macrophage inflammatory protein 1β; mRNA, messenger RNA; NADPH, nicotinamide adenine dinucleotide phosphate; PCR, polymerase chain reaction; qPCR, quantitative polymerase chain reaction; ROS, reactive oxygen species; α-SMA, α-smooth muscle actin; TGF, transforming growth factor; TIMP-1, tissue inhibitor of metalloproteinase 1; TLR4, Toll-like receptor 4; TNF-α, tumor necrosis factor α; WT, wild-type.

Materials and Methods

Animals and Models of Hepatic Fibrosis.

Specific pathogen-free wild-type (WT) C57BL/6J mice, CCR2−/− mice (Jackson Laboratories, Bar Harbor, ME), and p47phox−/− mice (Taconic, Hudson, NY) were used for this study. For the bile duct ligation (BDL) model, 8- to 12-week-old mice were anesthetized.7, 23 After laparotomy, the common bile duct was ligated twice and closed at the abdomen. The sham operation was performed similarly without BDL. For the CCl4 model, the mice were injected with CCl4diluted 1:3 in corn oil (Sigma-Aldrich, St. Louis, MO) or vehicle (corn oil) intraperitoneally at a dose of 0.5 μL/g body weight twice a week for a total of 12 injections.7 The mice received humane care according to National Institutes of Health recommendations outlined in the Guide for the Care and Use of Laboratory Animals. All animal experiments were approved by the Columbia University and University of California, San Diego, Institutional Animal Care and Use Committees.

Bone Marrow Transplantation.

Bone marrow transplantation (BMT) experiments were performed as described.7, 24 Because only 30% of Kupffer cells are reconstituted by donor-derived BM cells 6 months after BMT, mice received liposomal clodronate injection (200 μL) before irradiation to deplete Kupffer cells and accelerate tissue macrophage turnover in order to obtain fully reconstituted BM-derived cells.25, 26 1 × 107 BM cells from the tibias and femurs of donor mice were injected into the tail vein of lethally irradiated (11 Gy) recipient mice. As we have reported, the mice were transplanted with BM isolated from β-actin promoter-driven green fluorescent protein (GFP)-transgenic mice, and the liver tissue was fixed and stained with macrophage marker (F4/80) or HSC marker (Desmin) confirming the reconstitution of Kupffer cells by GFP-positive cells and GFP-negative HSCs (Supporting Fig. 1A,B).7 We also demonstrated that GFP-positive Kupffer cells in GFP-transgenic BM-transplanted mice was the same as that in GFP-transgenic mice through fluorescence-activated cell sorting (FACSCanto, BD Bioscience) (Supporting Fig. 1C-E). The CCR2-chimeric mice were generated by WT mice receiving CCR2−/− BM and vice versa. They were subjected to BDL or CCl4 treatment 12 weeks after BMT. To demonstrate the success of BMT in CCR2−/− and WT mice, spleen cells were isolated from all CCR2-chimeric mice, and CCR2 messenger RNA (mRNA) expression was measured by means of quantitative polymerase chain reaction (qPCR).

HSC and Kupffer Cell Isolation and Culture.

HSCs were isolated through collagenase-pronase perfusion of livers followed by 8.2% Nycodenz (Accurate Chemical and Scientific Corporation, Westbury, NY) two-layer discontinuous density gradient centrifugation7, 23 resulting in 99% purity of HSCs as confirmed by retinoid autofluorescence. To avoid Kupffer cell contamination in HSCs isolated from BDL mice, contaminated Kupffer cells were depleted by means of magnetic antibody sorting (MACS; Miltenyi Biotec, Auburn, CA) using F4/80 (eBiosience, San Diego, CA) and CD-11b (Miltenyi Biotec) antibodies.27 The method of Kupffer cell isolation has been described.28, 29

Immunohistochemistry and Immunofluorescence.

Liver specimens were fixed in 10% buffered formalin and incubated with monoclonal antibody to α-smooth muscle actin (α-SMA) (DakoCytomation, Carpinteria, CA) using an M.O.M. kit (Vector Laboratories, Burlingame, CA), monoclonal antibody to F4/80 (eBioscience, San Diego, CA), or rabbit anti–4-hydroxy-nonenal (4-HNE) antibody (Alpha Diagnostic, San Antonio, TX).7, 23 For immunofluorescent staining, liver specimens were fixed in 4% paraformaldehyde and subsequently incubated in phosphate-buffered saline containing 30% sucrose and frozen at −80°C. Frozen sections were incubated with antibody for CCR2 (Novus Biologicals, Littleton, CO), desmin (Neomarkers, Fremont, CA), F4/80, and pan-CK (Biolegend, San Diego, CA) and imaged with confocal microscopy. Immunocytofluorescence against CCR2 was performed as described.23

Western Blotting.

Protein extracts were electrophoresed and subsequently blotted.7, 23 Blots were incubated with antibodies for α-SMA (Sigma-Aldrich), phospho–extracellular signal-regulated kinase (ERK), phospho-AKT, AKT (Cell Signaling, Danvers, MA) and ERK2 (Santa Cruz Biotechnology, Santa Cruz, CA), with secondary horseradish peroxidase–conjugated antibody, and visualized using enhanced chemiluminescence (Amersham Biosciences).

Measurement of Hepatic Collagen Content.

Hepatic hydroxyproline content was measured as described.7, 23 Hepatic collagen content was also quantitated by Sirius red staining of paraffin-embedded sections. Sirius red–positive areas were analyzed in six random fields (magnification ×100) on each slide and quantified using NIH imaging software.

Real-Time qPCR and Reverse-Transcription PCR.

RNA was extracted, and real-time qPCR using primer-probe sets (Applied Biosystems, Foster City, CA) was performed as described.7 Reverse-transcription PCR for CCR2 and β-actin was performed using primers 5′-AGAGGTCTCGGTTGGGTTGT-3′ and 5 ′-ATCATAACGTTCTGGGCACC-3′ for 33 cycles and primers 5′-GATGACGATATCGCTGCGCTG-3′ and 5′-GTACGACCAGAGGCATACAGG-3′ for 27 cycles at 95°C for 45 seconds, 60°C for 45 seconds, and 72°C for 60 seconds.

Cell Chemotaxis Assay.

Cell migration assays was performed using a modified Boiden-Chamber as described.7, 30 Briefly, HSCs isolated from WT, CCR2−/−, or p47phox−/− mice were placed onto the upper chamber (4 × 104 cells/well) in Dulbecco's modified Eagle's medium without serum and exposed to vehicle, MCP-1, MCP-2, or MCP-3 (R&D Systems, Minneapolis, MN) placed in the lower chamber. After 24 hours of incubation at 37°C, cells migrated to the lower side of the chamber were counted in eight randomly chosen (magnification ×100) fields. In some experiments, the NADPH oxidase inhibitors diphenilene-iodonium (DPI) (Sigma-Aldrich) was used. Ten micromolar DPI was incubated for 30 minutes before treatment with chemokines.

Measurement of Intracellular Reactive Oxygen Species.

HSCs were preincubated with the redox-sensitive dye DCFDA (8 μM) (Molecularprobe, Eugene, OR) for 20 minutes and then stimulated with MCP-1, MCP-2, or MCP-3 (100 ng/mL).31 DCFDA fluorescence was measured with a multiwell fluorescence scanner (Fluostar Optima, BMG).

Statistical Analysis.

All data are expressed as the mean ± standard error of the mean. Multiple groups were compared using one-way analysis of variance with post hoc Bonferroni's correction (GraphPad Prism 4.02, GraphPad Software). Two groups were compared using an unpaired Student t test (two-tailed). P values less than 0.05 were considered statistically significant.

Results

Expression of CCR2 and its Ligands in Hepatic Fibrosis.

Although the expression of MCP-1 is increased in acute liver injury and hepatic fibrosis,7, 20, 22, 32 its receptor CCR2 and the other CCR2 ligands, MCP-2 and MCP-3, have not been investigated. Expression of hepatic CCR2, MCP-1, MCP-2, and MCP-3 mRNA levels were elevated after BDL (Fig. 1A) and CCl4 treatment (Fig. 1B). Next, we examined what cell types express CCR2 in the liver. We costained CCR2 with desmin (HSC marker) (Fig. 1C), F4/80 (Kupffer cell marker) (Fig. 1D), or pan-CK (hepatocyte and cholangiocyte marker) (Fig. 1E) and found that CCR2 is expressed both in HSCs and Kupffer cells, but not in hepatocytes or biliary epithelial cells, in the BDL liver. We also demonstrated CCR2 expression in mouse primary HSCs (Supporting Fig. 1A-C) and Kupffer cells (Supporting Fig. 1D,E) by means of immunofluorescence and CCR2 mRNA using reverse-transcription PCR (Fig. 1F).

Figure 1.

Expression of CCR2 and CCR2 ligands are increased in hepatic fibrosis. Livers were analyzed from mice after BDL (n = 6) or 12 CCl4 treatments (n = 6) at the indicated time points. (A, B) Hepatic mRNA levels of CCR2, MCP-1, MCP-2, and MCP-3 were measured after treatment with (A) BDL or (B) CCl4 using qPCR. (C-E) CCR2 expression (in green) was detected by means of costaining with (C) desmin (in red), (D) F4/80 (in red), and (E) pan-cytokeratin (in red), which was visualized using confocal microscopy. Arrows indicate CCR2-positive and (C) desmin-positive or (D) F4/80-positive cells. (E) The dashed lines indicate intrahepatic bile ducts. (F) CCR2 mRNA in Kupffer cells, HSCs, and hepatocytes was measured using reverse-transcription PCR. β-Actin mRNA served as an internal control.

CCR2 Mediates Hepatic Fibrosis After BDL.

Twenty-one days after BDL, WT mice had significant hepatic fibrosis, as demonstrated by Sirius red staining and hydroxyproline content. In contrast, CCR2−/− mice had less fibrosis as demonstrated by reduced collagen deposition and hydroxyproline level (Fig. 2A,B). Expression of α-SMA, a marker of HSC activation, was increased in WT mice, but not in CCR2−/− mice as assessed by immunohistochemistry and immunoblotting (Fig. 2C,D). Early fibrogenic responses were demonstrated 5 days after BDL, as represented by mRNA expression of markers for fibrogenesis including collagen α1(I), α-SMA, TGF-β1, and tissue inhibitor of metalloproteinase 1 (TIMP-1). WT mice showed an increased expression of these genes, whereas CCR2−/− mice had suppression in fibrogenic gene expression (Fig. 2E). Gene expression of proinflammatory mediators, such as TNF-α, MCP-1, RANTES, macrophage inflammatory protein 1β (MIP-1β), IL-1β, and IL-6 were decreased in CCR2−/− mice compared with WT mice (Fig. 3A). Moreover, CCR2−/− mice showed less hepatic macrophage infiltration, as assessed by expression of CD68 mRNA and immunohistochemistry for F4/80 (Fig. 3B,C). The lipid peroxidation product 4-HNE is a marker for the generation of reactive oxygen species (ROS). The level of 4-HNE was increased in WT but not CCR2−/− mice (Fig. 3D). Serum alanine aminotransferase (ALT) levels at 21 days after BDL were suppressed in CCR2−/− mice compared with WT mice, whereas we did not find significant differences in alkaline phosphatase or total bilirubin levels between WT and CCR2−/− mice after BDL (Fig. 3E).

Figure 2.

Reduction of hepatic fibrogenesis in CCR2−/− mice after BDL. WT mice (n = 6) and CCR2−/− mice (n = 6) underwent BDL. (A) Fibrillar collagen deposition was evaluated by means of Sirius red staining and its quantification 21 days after BDL. (B) Hepatic hydroxyproline content was measured. (C, D) Expression of α-SMA was determined by immunohistochemistry (C) and western blotting (D). (E) Hepatic mRNA levels of collagen α1(I), α-SMA, TGF-β1, and TIMP-1 were measured in WT mice (n = 5) and CCR2−/− mice (n = 5) 5 days after BDL by qPCR. *P < 0.05 for bile duct–ligated CCR2−/− mice versus bile duct–ligated WT mice. (Original magnification ×100 [A, C].)

Figure 3.

Reduction of hepatic inflammation and macrophage infiltration in CCR2−/− mice after BDL. Hepatic mRNA levels of (A) TNF-α, MCP-1, RANTES, MIP-1β, IL-1β, and IL-6 and (B) CD68 were measured in WT mice (n = 5) and CCR2−/− mice (n = 5) 5 days after BDL by means of qPCR. (C) Macrophage infiltration was determined by immunohistochemistry for F4/80 21 days after BDL. (D) 4-HNE was detected by immunohistochemistry. (E) Serum ALT, alkaline phosphatase, and total bilirubin levels 5 or 21 days after BDL were measured. *P < 0.05 for bile duct–ligated CCR2−/− mice versus bile duct–ligated WT mice. (Original magnification ×200 [C, D].)

CCR2 Mediates CCl4-Induced Hepatic Fibrosis.

Hepatic fibrosis was induced in WT mice after 12 injections of CCl4, as assessed through Sirius red staining (Fig. 4A,B) and hepatic hydroxyproline content (Fig. 4C). In contrast, CCR2−/− mice showed a marked reduction of hepatic fibrosis (Fig. 4A–C). During CCl4-induced hepatic fibrosis, the expression of α-SMA was significantly increased in WT mice, but not in CCR2−/− mice (Fig. 4D,E). However, serum ALT levels after 12 injections of CCl4 were compatible between WT and CCR2−/− mice (Fig. 4F), suggesting a similar hepatocellular injury occurred by CCl4 in WT and CCR2−/− mice.

Figure 4.

CCR2 deficiency inhibits hepatic fibrosis after CCl4 treatment. WT mice (n = 6) and CCR2−/− mice (n = 6) were treated with 12 injections of CCl4 (0.5 μL/g). (A, B) Fibrillar collagen deposition was evaluated by (A) Sirius red staining and (B) quantitation after 12 injections of CCl4. (C) Hepatic hydroxyproline content was measured. (D, E) Expression of α-SMA was determined by (D) immunohistochemistry and (E) western blotting. (F) Serum ALT levels after 12 injections of CCl4 were measured. *P < 0.05 for CCl4-treated CCR2−/− mice versus CCl4-treated WT mice. (Original magnification ×100 [A, D].)

CCR2 on Resident Cells, but not BM-Derived Cells, Are Critical for Hepatic Fibrosis.

Because CCR2 is important in two models of hepatic fibrosis (Figs. 2–4) and is expressed both in Kupffer cells and HSCs (Fig. 1C–F, Supporting Fig. 2A-E), we investigated which cell types are critical in CCR2-mediated hepatic fibrosis. We generated CCR2-chimeric mice by using a combination of Kupffer cell depletion, irradiation, and BMT.7 Because hepatocytes do not express CCR2 (Fig. 1E,F), this protocol reconstitutes Kupffer cells, but not HSCs, with BM-derived cells (Supporting Fig. 1A-E). Thus, we generated two types of CCR2-chimeric mice: (1) WT mice with transplanted CCR2−/− BM, which contained CCR2−/− Kupffer cells and WT HSCs; and (2) CCR2−/− mice with transplanted WT BM, which contained WT Kupffer cells and CCR2−/− HSCs. We confirmed successful BMT by measuring CCR2 mRNA expression in spleen cells from both types of CCR2-chimeric mice (Fig. 5A). At 5 days after BDL, chimeric mice containing WT BM cells showed increased inflammatory gene expressions (TNF-α, MCP-1, RANTES, and MIP-1β) (Fig. 5B) with macrophage accumulation (Fig. 5C,D). In contrast, the mice with CCR2−/− BM cells had reduced inflammatory gene expression (Fig. 5B) and Kupffer cell recruitment (Fig. 5C,D), suggesting that inflammation and macrophage recruitment requires CCR2 expressed on BM cells in the early phase of liver injury. There are no significant differences in serum ALT levels among all types of chimeric mice at 5 days after BDL (Fig. 5E).

Figure 5.

CCR2 on BM-derived cells is required for the early phase of macrophage infiltration after BDL. Chimeric mice were generated by transplanting WT or CCR2−/− BM into irradiated and clodronate-treated WT mice or CCR2−/− mice (n = 4-5 each group). Chimeric mice were then subjected to BDL for 5 days. (A) Successful BMT was confirmed by low levels of CCR2 mRNA in splenocytes from WT mice, but normal CCR2 mRNA in CCR2−/− mice with transplanted WT BM. (B) Hepatic mRNA levels of proinflammatory cytokines (TNF-α, MCP-1, RANTES, and MIP-1β) in chimeric mice were measured with qPCR. (C, D) Macrophage infiltration was determined by (C) immunohistochemistry for F4/80 and (D) qPCR for CD68. (E) Serum ALT levels in chimeric mice 5 days after BDL were measured. (Original magnification ×200 [C].) *P < 0.05. **P < 0.01.

At 21 days after BDL, WT mice containing CCR2−/− BM had increased collagen deposition as assessed by Sirius red staining and hydroxyproline content (Fig. 6A,B). Meanwhile, CCR2−/− mice containing WT BM had reduced fibrosis similar to CCR2−/− mice transplanted with CCR2−/− BM (Fig. 6A,B). In agreement with the results on collagen deposition, WT mice with CCR2−/− BM showed an increased expression of α-SMA comparable to WT mice with transplanted WT BM, whereas CCR2−/− mice containing WT BM decreased α-SMA expression comparable to CCR2−/− mice with transplanted CCR2−/− BM (Fig. 6C). Increased macrophage recruitment was observed in WT mice with transplanted WT or CCR2−/− BM and reduced macrophage infiltration was seen in CCR2−/− mice with transplanted WT or CCR2−/− BM, indicating that macrophage recruitment does not require CCR2 in BM-originated cells in the chronic phase of hepatic fibrosis (Fig. 6D). Moreover, WT mice with transplanted WT or CCR2−/− BM showed increased accumulation of 4-HNE compared with CCR2−/− mice with transplanted WT or CCR2−/− BM (Fig. 6E). Serum ALT levels were increased in WT mice with transplanted WT or CCR2−/− BM and reduced in CCR2−/− mice with transplanted WT or CCR2−/− BM (Fig. 6F). To test the importance of CCR2 expressed on the recipient-originated cells, including HSCs in a second model of experimental hepatic fibrosis, hepatic fibrosis was induced by means of chronic injection of CCl4 on CCR2-chimeric mice. Similarly with the results from BDL, CCR2−/− mice containing either WT or CCR2−/− BM cells showed decreased collagen deposition and α-SMA expression (Fig. 7A –E), whereas the WT mice containing either WT or CCR2−/− BM cells had increased collagen deposition and α-SMA expression (Fig. 7A–E). Serum ALT levels were increased in CCR2−/− mice with transplanted WT or CCR2−/− BM and reduced in WT mice with transplanted WT or CCR2−/− BM (Fig. 7F). Taken together, these results suggest that CCR2 expressed on BM cells including Kupffer cells is important in the early phase of hepatic inflammation. However, CCR2 expression on recipient-originated cells including HSCs is required for the late phase of hepatic fibrosis.

Figure 6.

Recipient-originated cells, but not bone marrow-derived cells, mediate CCR2-dependent fibrogenic effects after BDL. (A-F) Chimeric mice were generated by transplanting CCR2−/− BM into irradiated and clodronate-treated WT mice (n=6) or CCR2−/− mice (n=6) and vice versa (n=6 each group). 3 months after BMT, the mice were subjected to BDL and hepatic fibrosis was judged at 21 days after BDL. (A-B) Hepatic fibrosis in chimeric mice was assessed by Sirius red staining (A, left), quantitation (A, right), and hydroxyproline measurement (B). (C) α-SMA expression was shown by immunohistochemistry (C, upper) and western blotting (C, lower). (D) Macrophage infiltration was determined by immunohistochemistry for F4/80. (E) The accumulation of 4-hydoxy-nonenal was measured by immunohistochemistry. (F) Serum ALT levels in chimeric mice 21 days after BDL were measured. (Original magnification ×100 [A, C], ×200 [D, E]). *P < 0.05. **P < 0.01.

Figure 7.

CCR2 on recipient-originated cells is crucial for CCl4-induced hepatic fibrosis. Chimeric mice were generated by transplanting CCR2−/− BM into irradiated and clodronate-treated WT mice (n = 5) or CCR2−/− mice (n = 5) and vice versa (n = 5 each group). Hepatic fibrosis was induced by 12 injections of CCl4. (A, B) Hepatic fibrosis in chimeric mice was assessed by (A) Sirius red staining, (B) quantitation, and (C) measurement of hydroxyproline content. α-SMA expression was determined by (D) immunohistochemistry and (E) western blotting. (F) Serum ALT levels in chimeric mice after 12 injections of CCl4 after BDL were measured. (Original magnification ×100 [A, D].) *P <0.05. **P<0.01.

CCR2 Mediates ROS Production and Migration in HSCs.

On the basis of the above results, the HSC appears to be the primary cell type for CCR2-mediated hepatic fibrosis. We examined intracellular signaling in HSCs by the phosphorylation of AKT and ERK in response to CCR2 ligands (Fig. 8A). MCP-1, MCP-2, and MCP-3 induce the phosphorylation of AKT and ERK in WT HSCs, and CCR2−/− HSCs showed reduced AKT and ERK phosphorylation by CCR2 ligands. Next, we investigated whether HSC migration depends on CCR2 (Fig. 8B). MCP-1, MCP-2, and MCP-3 significantly induced the HSC chemotaxis. In contrast, these chemotactic activities were abolished in CCR2−/− HSCs. We additionally measured fibrogenic genes [collagen α1(I), α-SMA, TIMP-1], an inflammatory cytokine (IL-6), and a proliferative marker (proliferation cell nuclear antigen) in response to CCR2 ligands in HSCs (Fig. 8C). Fibrogenic genes and proliferation cell nuclear antigen were not increased in WT and CCR2−/− HSCs after the stimulation of all CCR2 ligands. IL-6 mRNA levels were elevated in response to MCP-1 and MCP-3 in WT HSCs, and MCP-1 still increased IL-6 mRNA levels in CCR2−/− HSCs. CCR2 ligands do not induce fibrogenic and proliferative responses directly on HSCs and may have inflammatory responses that depend on CCR2 by MCP-3 and independently of CCR2 in response to MCP-1 (Fig. 8C). Next, we investigated ROS production in CCR2 signals. ROS was produced by CCR2 ligands in WT HSCs. In contrast, HSCs deficient in CCR2 or p47phox, which is a critical component of NADPH oxidase, produce little ROS after stimulation with MCP-1, MCP-2, or MCP-3 (Fig. 8D). In p47phox−/− HSCs, the phosphorylation of AKT and ERK were suppressed slightly and significantly, respectively, after stimulation with CCR2 ligands (Fig. 8E). Finally, we tested whether NADPH oxidase is involved in HSC migration. We inactivated NADPH oxidase by using either the inhibitor DPI or p47phox−/− HSCs. DPI treatment and p47phox deficiency dramatically inhibited the migration induced by CCR2 ligands (Fig. 8F). These data indicate that CCR2 ligands MCP-1, MCP-2, and MCP-3 stimulate AKT and ERK activation, ROS production, and HSC migration through CCR2 and p47phox.

Figure 8.

CCR2-dependent NADPH oxidase-dependent ROS production and chemotaxis in HSCs in response to MCP-1, MCP-2 and MCP-3. HSCs isolated from WT, CCR2−/−, and p47phox−/− mice were stimulated with MCP-1, MCP-2, and MCP-3. (A) phospho-AKT (on Ser473), total AKT, phospho-ERK, and total ERK were determined in WT and CCR2−/− HSCs by immunoblotting. (B) Serum-free media containing MCP-1, MCP-2, or MCP-3 (50 ng/mL) were placed in the lower chamber. HSCs isolated from WT or CCR2−/− mice were placed in the upper chamber. Migration of HSCs into the lower chamber was counted 24 hours after stimulation. (C) mRNA expression of collagen α1(I), α-SMA, TIMP-1, IL-6, and proliferation cell nuclear antigen were measured in WT or CCR2−/− HSCs 24 hours after stimulation by qPCR. (D) Mouse HSCs from WT, CCR2−/−, and p47phox−/− mice were loaded with DCFDA (8 μM) for 20 minutes and incubated with MCP-1, MCP-2, and MCP-3 (100 ng/mL) and measured fluorescence intensity for 30 minutes. (E) phospho-AKT (on Ser473), total AKT, phospho-ERK, and ERK2 were determined in WT and p47phox−/− HSCs by immunoblotting. (F) Serum-free media containing MCP-1, MCP-2, or MCP-3 (50 ng/mL) were placed in the lower chamber. HSCs isolated from WT or p47phox−/− mice were placed in the upper chamber, with or without pretreatment of DPI (10 μM) for 30 minutes. Migration of HSCs into the lower chamber was counted 24 hours after stimulation. *P < 0.05.

Discussion

Chronic inflammation leads to continuous hepatocyte damage and subsequently hepatic fibrosis.1–3 The recruitment and migration of Kupffer cells and HSCs are critical events for developing liver inflammation and fibrosis. In human liver diseases, increased MCP-1 is associated with macrophage recruitment and severity of hepatic fibrosis and primary biliary cirrhosis.22, 32 In animal models, the inactivation of MCP-1 attenuates CCl4-induced liver injury and fibrosis by inhibiting macrophage recruitment.20, 21 Our current study demonstrates that CCR2 deficiency lessens liver inflammation and fibrosis (Fig. 2–4) and that CCR2 on Kupffer cells is required for the early phase of Kupffer cell infiltration and inflammation, but is dispensable for the chronic phase of Kupffer cell accumulation, HSC activation, and collagen deposition (Figs. 5 and 6). Intriguingly, CCR2 on recipient-originated cells including HSCs is more important than CCR2 on Kupffer cells in HSC activation and fibrosis in two different models of experimental fibrogenesis (Figs. 6 and 7). BDL increases biliary pressure and stimulates the proliferation of biliary epithelial cells, which is accompanied by inflammation and necrosis in the surrounding portal area, resulting in HSC activation and fibrosis33 (Figs. 2 and 3). In contrast, intoxication with CCl4, which is converted into hepatotoxic metabolites by p450 CYP2E1, directly damages hepatocytes, inducing severe necrosis around the central vein.33 Subsequently, necrotic hepatocytes stimulate immune cells and HSCs, resulting in fibrosis bridging between central veins (Fig. 4). CCl4 induces hepatocyte damage independent of CCR2-mediated inflammatory responses, as demonstrated by similar ALT levels between WT and CCR2−/− mice (Fig. 4F). However, 3 weeks after BDL, ALT levels are suppressed in CCR2−/− mice, suggesting that cholestasis-mediated liver damage requires a CCR2-dependent inflammatory response (Fig. 3E).

Macrophages and Kupffer cells are a principal source of inflammatory cytokines and chemokines, including CCR2 ligands. In addition to Kupffer cells, HSCs, biliary epithelial cells, and hepatocytes produce chemokines including CCR2 ligands such as MCP-1.7, 34–36 Our previous study has shown that HSCs promote Toll-like receptor 4 (TLR4)-mediated liver injury and fibrosis.7 Liver injury increases the intestinal permeability by means of the release of cytokines that alter portal circulation and intestinal epithelial tight junctions, allowing intestinal microflora-derived lipopolysaccharide to enter the portal circulation and the portal lipopolysaccharide, then activates HSCs by means of TLR4.7, 37 TLR4 signaling simultaneously down-regulates the TGF-β pseudoreceptor Bambi (BMP and activin membrane-bound inhibitor), which enhances TGF-β signaling in HSCs.7 TLR4 also induces the production of chemokines such as MCP-1, MCP-2, MCP-3, MIP-1α, and MIP-1β (data not shown).7

CCR2 is mainly expressed on macrophages/Kupffer cells and functions to recruit these cells to the site of inflammation.21, 38, 39 Intriguingly, CCR2 expressed on BM mediates Kupffer cell recruitment in the acute phase (Fig. 5), but Kupffer cell recruitment, HSC activation, and fibrogenic responses in chronic injury require CCR2 expression on resident liver cells (Figs. 6–8), with HSCs being the prime candidate as suggested by CCR2 expression and functional studies (Figs. 1 and 8). We speculate that there is a biphasic inflammatory response after BDL and that chronic phase of inflammation contributes to fibrosis. The monocyte/macrophage lineage is highly heterogeneous and consists of a CCR2hi population and CX3CR1hi population.40 A recent report demonstrated that CCR2 is not required for monocyte migration into tissue.41 Thus, macrophage recruitment might occur through other chemokine receptors, such as CX3CR1 in the chronic phase of hepatic fibrosis.

Previous studies have reported that CCR2 is not expressed in human HSCs and rat portal fibroblasts, which are cells primarily activated in cholestasis-mediated hepatic fibrosis.22, 35, 42 In contrast, our study demonstrates that both Kupffer cells and HSCs, but not hepatocytes and biliary epithelial cells, express CCR2 in normal and fibrotic liver (Fig. 1C–F, Supporting Fig. 2A-E). These differences might be explained by the different roles of HSCs or portal fibroblasts among humans, rats, and mice. Previous studies have suggested that MCP-1 activates human HSCs, rat portal fibroblasts, and hepatocytes by an unknown receptor, instead of by CCR2.35, 42, 43 Our results also support a limited role for a non-CCR2 receptor, because MCP-1 increases IL-6 mRNA in CCR2−/− HSCs. Additionally, we demonstrated that CCR2 leads to ERK and AKT phosphorylation, ROS production, and migration in response to MCP-1, MCP-2, and MCP-3 in murine HSCs. We did not find that CCR2 ligands induce proliferation or profibrogenic features in murine HSCs (Fig. 8 and data not shown).35, 42 These results might be explained by less proliferative and profibrogenic characteristics of murine HSCs compared with human and rat HSCs.

NADPH oxidase is a critical component of HSC activation and chemotaxis in hepatic fibrosis.31 We have previously shown that RANTES induces ROS production, proliferation, and migration by means of NADPH oxidase.30 MCP-1 generates ROS production and migration in vascular smooth muscle cells and monocytes, and DPI abolishes migration induced by MCP-1.44, 45 Here, we show that CCR2 ligand-induced ROS production, ERK and AKT phosphorylation, and chemotaxis were attenuated by the inactivation of NADPH oxidase in p47phox−/−- or DPI-treated HSCs (Fig. 8), indicating that NADPH oxidase is an essential component for CCR2-mediated HSC activation.

In conclusion, our study demonstrates that CCR2 has distinct roles in Kupffer cells and HSCs in different phases of liver injury. In the early phase of liver injury, Kupffer cell migration is mediated by CCR2 expressed in BM, whereas CCR2 expression in resident liver cells promotes macrophage recruitment and hepatic fibrosis in chronic liver injury. In conjunction with our in vitro studies, CCR2 expression on HSCs appears to be responsible for its fibrogenic effects. Thus, inhibition of CCR2 in chronic liver disease might represent a potential strategy for the treatment of hepatic fibrosis with minimal suppression of Kupffer cell–mediated immune response.

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