CCR2 promotes hepatic fibrosis in mice - PubMed (original) (raw)
CCR2 promotes hepatic fibrosis in mice
Ekihiro Seki et al. Hepatology. 2009 Jul.
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 CCl(4)-induced fibrosis was markedly reduced in CCR2(-/-) mice as assessed through collagen deposition, alpha-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.
Figures
Figure 1. Expression of CCR2 and CCR2 ligands are increased in hepatic fibrosis
Livers were analyzed from mice after BDL (n=6) or 12 times repeated CCl4 treatment (n=6) at indicated time points. (A, B) Hepatic mRNA levels of CCR2, MCP-1, MCP-2 and MCP-3 were measured after BDL (A) or CCl4 treatment (B) by qPCR. (C-E) CCR2 expression (in green) was detected by co-stained with desmin (in red, C), F4/80 (in red, D) and pan-Cytokeratin (in red, E), which was visualized by confocal microscopy. Arrows indicate CCR2-positive and desmin- (C) or F4/80-positive cells (D). The dashed lines indicate intrahepatic bile ducts (E). (F) CCR2 mRNA in Kupffer cells, HSCs and hepatocytes was measured by RT-PCR. β-actin mRNA served as an internal control.
Figure 2. Reduction of hepatic fibrogenesis in CCR2-/- mice after BDL
WT (n=6) and CCR2-/- (n=6) mice underwent BDL. (A) Fibrillar collagen deposition was evaluated by 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, TGFβ1 and TIMP-1 were measured in WT (n=5) and CCR2-/- (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 is ×100 (A, C).
Figure 3. Reduction of hepatic inflammation and macrophage infiltration in CCR2-/- mice after BDL
(A, B) Hepatic mRNA levels of TNFα, MCP-1, RANTES, MIP-1β, IL-1β and IL-6 (A) and CD68 (B) were measured in WT (n=5) and CCR2-/- (n=5) 5 days after BDL by qPCR. (C) Macrophage infiltration was determined by immunohistochemistry for F4/80 21 days after BDL. (D) 4-hydoxy-nonenal was detected by immunohistochemistry. (E) Serum ALT, ALP and T-Bil 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 is ×200(C, D).
Figure 4. CCR2 deficiency inhibits hepatic fibrosis after CCl4 treatment
WT (n=6) and CCR2-/- (n=6) mice were treated with 12 injections of CCl4 (0.5μl/g). (A, B) Fibrillar collagen deposition was evaluated by Sirius red staining (A) and quantitation (B) after 12 injection of CCl4. (C) Hepatic hydroxyproline content was measured. (D, E) Expression of α-SMA was determined by immunohistochemistry (D) and western blotting (E). (F) Serum ALT levels after 12 injection of CCl4 were measured. *: p<0.05 for CCl4-treated CCR2-/- mice versus CCl4-treated WT mice. Original magnification is ×100 (A, D).
Figure 5. CCR2 on bone marrow-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). Then chimeric mice were 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 transplanted with WT BM. (B) Hepatic mRNA levels of proinflammatory cytokines (TNF-α, MCP-1, RANTES and MIP-1β) in chimeric mice were measured by qPCR. (C, D) Macrophage infiltration was determined by immunohistochemistry for F4/80 (C) and qPCR for CD68 (D). (E) Serum ALT levels in chimeric mice 5 days after BDL were measured. Original magnification is ×200 (C). * p<0.05, ** p<0.01.
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 is ×100 (A, C) and ×200 (D, E). * p<0.05, ** p<0.01.
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 is ×100 (A, C) and ×200 (D, E). * p<0.05, ** p<0.01.
Figure 7. CCR2 on recipient-originated cells is crucial for CCl4-induced hepatic fibrosis
(A-F) 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 injection of CCl4. (A, B) Hepatic fibrosis in chimeric mice was assessed by Sirius red staining (A), quantitation (B), and measurement of hydroxyproline content (C). (D, E) α-SMA expression was determined by immunohistochemistry (D) and western blotting (E). (F) Serum ALT levels in chimeric mice after 12 injection of CCl4 after BDL were measured. Original magnification is ×100 (A, D). * p<0.05, ** p<0.01.
Figure 8. CCR2-dependent NADPH oxidase-dependent ROS production and chemotaxis in HSCs in response to MCP-1, MCP-2 and MCP-3
(A-F) 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 (50ng/ml) was 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), αSMA, TIMP-1, IL-6 and PCNA 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 (8μM) for 20 minutes and incubated with MCP-1, MCP-2 and MCP-3 (100ng/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 (50ng/ml) was placed in the lower chamber. HSCs isolated from WT or p47phox-/- mice were placed in the upper chamber, with or without pretreatment of DPI (10μM) for 30 minutes. Migration of HSCs into the lower chamber was counted 24 hours after stimulation. *: p<0.05.
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