Macrophage secretory products induce an inflammatory phenotype in hepatocytes

Figure 1 Macrophage-conditioned media induces a fibroblast-like morphological change in hepatocytes. Phase contrast microscopy of HepG2 (A and B) and Huh7 cells (C and D) grown in complete media (A and C) or 50% macrophage-conditioned media in complete medium (B and D). The images shown are representative of six experiments of cells after 24 h of culture in the indicated medium.

Figure 2 Macrophage-conditioned media induces a transient change in hepatocytes. Phase contrast microscopy of HepG2 cells grown in complete media (A) for 72 h, macrophage-conditioned media (MφCM) for 72 h (B) or MφCM for 24 h followed by washing and culture in complete medium for a further 48 h (C). The images shown are representative of cells after 72 h of culture in the indicated medium.

Figure 3 Expression of E-cadherin is reduced and vimentin is increased in hepatocytes treated with macrophage-conditioned media. A, D: Following culture in complete media or 50% macrophage-conditioned media (MφCM) for 24 h, E-cadherin (A) and vimentin (D) mRNA levels in HepG2 and Huh7 cells were measured by real-time polymerase chain reaction. Results are expressed as fold of untreated cells (mean ± SEM, n = 8) (P < 0.05 vs untreated); B, C: Immunofluorescence staining for E-cadherin (green) in untreated (B) and MφCM-treated (C) Huh7 cells; E, F: Immunofluorescence staining for vimentin (green) in untreated (E) and MφCM-treated (F) Huh7 cells. 4',6-diamidino-2-phenylindole stained nuclei blue (63 × magnification). Untr: Untreated.

Figure 4 Lipocalin-2 mRNA expression was increased in hepatitis C virus-infected patients with increasing stage of fibrosis (A), grade of inflammation (B) or grade of steatosis (C). D and E: Lipocalin-2 (LCN2) staining by immunohistochemistry was minimal in hepatitis C virus (HCV)-infected patients with stage 1 fibrosis (D; 400 × magnification) compared with stage 4 fibrosis (E; 400 × magnification); F: Increased LCN2 protein expression in HCV-infected patients with stage 3/4 fibrosis. P < 0.05 vs fibrosis stage 0 (A), inflammation grade 1/2 (B) steatosis grade 0 (C) or fibrosis stage 1 (D).

Figure 5 Matrix metalloproteinase-9 mRNA expression is significantly increased in macrophages compared with THP-1 monocytes. A: Results are expressed as fold of monocytes (mean ± SEM, n = 6) (P < 0.05 vs monocytes); B: Western blotting analysis (top) and zymography (bottom) of matrix metalloproteinase (MMP)-9 in MonoCM and macrophage-conditioned media (MφCM) from three independent experiments; C: Generation of MφCM in the presence of MMP-9 inhibitor I (100 μmol) prevented the MφCM-induced morphological change in HepG2 cells; D: Zymography gel confirming MMP-9 inhibitor I reduces MMP-9 activity in MφCM at 100 μmol; E-G: MMP-9 Inhibitor I significantly attenuated downregulation of E-cadherin (E) and upregulation of transforming growth factor-β1 (TGF-β1) (F) but not lipocalin-2 (LCN2) (G) mRNA expression in response to MφCM. Results are expressed as fold of untreated cells (mean ± SEM, n = 5), P < 0.05 vs untreated.

Figure 6 In patients with chronic hepatitis C, increased hepatic mRNA expression of the macrophage marker CD163 (A) as well as matrix metalloproteinase-9 (B) was significantly associated with increasing stage of fibrosis (P < 0. 05 vs F0). C-E: Immunofluorescence in liver biopsies from patients with chronic hepatitis C demonstrated matrix metalloproteinase (MMP)-9 expression in CD163⁺ macrophages.