Glycoprotein Nonmetastatic Melanoma B (Gpnmb)-Positive Macrophages Contribute to the Balance between Fibrosis and Fibrolysis during the Repair of Acute Liver Injury in Mice

<div><p>Background and aims</p><p>Glycoprotein nonmetastatic melanoma B (Gpnmb), a transmembrane glycoprotein that is expressed in macrophages, negatively regulates inflammation. We have reported that Gpnmb is strongly expressed in the livers of rats fed a choline-deficient, L-amino acid-defined (CDAA) diet. However, the role of macrophage-expressed Gpnmb in liver injury is still unknown. This study aimed to clarify the characteristics of infiltrating macrophages that express Gpnmb, and the involvement of Gpnmb in the repair process in response to liver injury.</p><p>Methods</p><p>C57BL/6J, DBA/2J [DBA] and DBA/2J-Gpnmb⁺ [DBA-g+] mice were treated with a single intraperitoneal injection of carbon tetrachloride (CCl₄) at a dose of 1.0 mL/kg body weight. Mice were sacrificed at predetermined time points, followed by measurement of serum alanine aminotransferase (ALT) levels and histological examination. Expression of Gpnmb, pro-/anti-inflammatory cytokines, and profibrotic/antifibrotic factors were examined by quantitative RT-PCR and/or Western blotting. Immunohistochemistry, fluorescent immunostaining and flow cytometry were used to determine the expression of Gpnmb, CD68, CD11b and α-SMA, phagocytic activity, and the presence of apoptotic bodies. We used quantitative RT-PCR and ELISA to examine TGF-β and MMP-13 expression and the concentrations and supernatants of isolated infiltrating hepatic macrophages transfected with siGpnmb.</p><p>Results</p><p>In C57BL/6J mice, serum ALT levels increased at two days after CCl₄ injection and decreased at four days. Gpnmb expression in the liver was stimulated four days after CCl₄ injection. Histological examination and flow cytometry showed that Gpnmb-positive cells were almost positive for CD68-positive macrophages, contained engulfed apoptotic bodies and exhibited enhanced phagocytic activity. Isolated infiltrating hepatic macrophages transfected with siGpnmb showed high MMP-13 secretion. There was no significant difference in the magnitude of CCl₄-induced liver injury between DBA-g+ and DBA mice. However, hepatic MMP-13 expression, as well as α-SMA expression and collagen production, increased significantly in DBA-g+ compared with DBA mice.</p><p>Conclusions</p><p>Gpnmb-positive macrophages infiltrate the liver during the recovery phase of CCl₄–induced acute liver injury and contribute to the balance between fibrosis and fibrolysis in the repair process following acute liver injury.</p></div

Effects of Gpnmb expression on hepatic macrophages.

<p>In infiltrating hepatic macrophages isolated from injured livers, (A) co-culture with apoptotic hepatocytes does not affect mRNA expression of Gpnmb, TGF-β, or MMP-13, but does increase TGF-β and MMP-13 secretion. Values are mean ± SEM (<i>n</i> = 3). * <i>P</i> < 0.05 (Mann-Whitney U test). Moreover, (B) mRNA expression of TGF-β and MMP-13 is not affected by inhibited Gpnmb expression, however secretion of MMP-13, but not TGF-β is significantly decreased by inhibition of Gpnmb expression. Values are mean ± SEM (<i>n</i> = 3). * <i>P</i> < 0.05 vs. control (Tukey’s HSD test).</p

Characteristics of gpnmb-positive cells infiltrating liver tissue.

<p>In LMNCs isolated from injured liver, (A) more than 90% of hepatic macrophages positive for F4/80 are also positive for CD68, whereas CD11b is expressed in approximately 10% of F4/80-positive cells. Gpnmb expression is detected in approximately 50% of CD68-positive cells. (B) Immunofluorescent double staining shows that some CD68-positive cells are also positive for Gpnmb (scale bar: upper, 100 μm; lower, 50 μ m). (C) Some Gpnmb-positive cells show phagocytosis of apoptotic cells (scale bar: left side, 100 μm; right side, 50 μm). (D) Phagocytic activity of CD68-positive cells is significantly higher than that of CD11b-positive cells. Additionally, in CD68-positive cells, phagocytic activity of Gpnmb-positive cells is significantly increased compared with that in Gpnmb-negative cells. Values are mean ± SEM (<i>n</i> = 3). * <i>P</i> < 0.05 (Mann-Whitney U test).</p

Features of DBA-g+ and DBA mice.

<p>(A) Expression of Gpnmb is observed in DBA-g+ but not DBA mice following a single injection of CCl₄. However, (B) sequential changes in serum ALT levels and the degree of liver injury are not affected by the lack of Gpnmb-positive macrophages (original magnification, x100). Conversely, (C) the areas of fibrosis and (D) the number of α-SMA-positive cells are significantly decreased in the liver tissues of DBA mice compared to DBA-g+ mice (original magnification, x200). Additionally, (E) although lack of Gpnmb-positive macrophages does not affect expression of TGF-β or Col1α1, expression of MMP-9, MMP-13, and TIMP-1 are significantly decreased in mice lacking Gpnmb expression at six or eight days after single injection of CCl₄. Values are mean ± SEM (<i>n</i> = 4). * <i>P</i> < 0.05 (Mann-Whitney U test).</p

Impact on liver injury repair process by depletion of infiltrating hepatic macrophages during the recovery phase.

<p>(A) Hepatic expression of IL-1β, IL-10, and CCL2 are significantly decreased (clodronate liposome injection group [<i>n</i> = 3, only survival] vs. PBS injection group [<i>n</i> = 4)]). Values are mean ± SEM. * <i>P</i> < 0.05 (Mann-Whitney U test)). (B) Collagen deposition and α-SMA expression are decreased (original magnification, x400). (C) Hepatic expression of TGF-β, Col1α1, and MMP-13 are significantly decreased (clodronate liposome injection group [<i>n</i> = 3, only survival] vs. PBS injection group [<i>n</i> = 4]). Values are mean ± SEM. * <i>P</i> < 0.05 (Mann-Whitney U test)).</p

Sequential changes in and localization of Gpnmb expression.

<p>Gpnmb expression is enhanced in the recovery phase (A) in whole liver as determined by Western blotting and (B) in isolated hepatic macrophages as determined by quantitative real-time polymerase chain reaction (<i>n</i> = 5 at each day). (C) Moreover, Gpnmb expression is observed immunohistochemically around injured lesions in a pattern similar to that in F4/80-positive macrophages, and Gpnmb-positive cells exhibit partial phagocytosis.</p

Primer lists.

<p>Primer lists.</p

Impact on liver injury by depletion of infiltrating hepatic macrophages during the recovery phase.

<p>After clodronate liposome injection at the 2^nd day after CCl₄ injection (<i>n</i> = 6 vs. PBS injection group (<i>n</i> = 4)), (A) the survival rate is decreased and weight loss is remarkable. (B) Centrilobular necrosis persists (original magnification, x200) and (C) serum ALT levels increase significantly (clodronate liposome injection group (<i>n</i> = 3 (only survival)) vs. PBS injection group (<i>n</i> = 4)). Values are mean ± SEM. * <i>P</i> < 0.05 (Mann-Whitney U test)).</p

Sequential changes of ALT and histological examination in ccl₄-induced acute liver injury.

<p>At four days after CCl₄ injection, (A) Serum ALT decreases and centrilobular necrosis are reduced (original magnification, x100). Values are mean ± SEM (n = 4). * <i>P</i> < 0.05 vs. the 0^th day (Tukey’s HSD test). (B) Hepatic IL-1β, IL-10 and CCL2 expression reveals that at the 4^th day after CCl₄ injection the repair process is in the recovery phase. Values are mean ± SEM (<i>n</i> = 4). * <i>P</i> < 0.05 vs. the 0^th day (Tukey’s HSD test). (C) Hepatic macrophages infiltrating into the liver injury (original magnification, x40 and x200).</p