Neutrophil depletion blocks early collagen degradation in repairing cholestatic rat livers

Abstract

Biliary obstruction results in a well-characterized cholestatic inflammatory and fibrogenic process; however, the mechanisms and potential for liver repair remain unclear. We previously demonstrated that Kupffer cell depletion reduces polymorphonuclear cell (neutrophil) (PMN) and matrix metalloproteinase (MMP)8 levels in repairing liver. We therefore hypothesized that PMN-dependent MMP activity is essential for successful repair. Male Sprague-Dawley rats received reversible biliary obstruction for 7 days, and the rat PMN-specific antibody RP3 was administered 2 days before biliary decompression (repair) and continued daily until necropsy, when liver underwent morphometric analysis, immunohistochemistry, quantitative RT-PCR, and in situ zymography. We found that RP3 treatment did not reduce Kupffer cell or monocyte number but significantly reduced PMN number at the time of decompression and 2 days after repair. RP3 treatment also blocked resorption of type I collagen. In addition, biliary obstruction resulted in increased expression of MMP3, MMP8, and tissue inhibitor of metalloproteinase 1. Two days after biliary decompression, both MMP3 and tissue inhibitor of metalloproteinase 1 expression declined toward sham levels, whereas MMP8 expression remained elevated and was identified in bile duct epithelial cells by immunohistochemistry. PMN depletion did not alter the hepatic expression of these genes. Conversely, collagen-based in situ zymography demonstrated markedly diminished collagenase activity following PMN depletion. We conclude that PMNs are essential for collagenase activity and collagen resorption during liver repair, and speculate that PMN-derived MMP8 or PMN-mediated activation of intrinsic hepatic MMPs are responsible for successful liver repair.

Figure 1

Experimental design and indicators of injury and repair. Biliary suspension surgery was performed, and cholestatic injury occurred over the following 7 days. Saline or RP3 (5 ml/rat) was administered i.p. 2 days before bile duct decompression and continued daily until necropsy. The day of decompression is defined here as day 0. Rats were sacrificed at day 0 and at 2 days of repair for each group (A). Measurements of the liver enzymes AST (B) and ALT (C) as well as bilirubin (D) were significantly (^∗P ≤ 0.05) elevated following 7 days of injury and returned to sham levels following decompression. Although RP3 treatment resulted in an apparent attenuation in injury-induced increases in AST, ALT, and bilirubin, these differences were not statistically significant. Difference scores of presurgical weight and necropsy weight (E) show a significant (^∗P ≤ 0.05) weight loss following injury with a significant weight recovery following decompression. No significant difference was noted with RP3 treatment.

Figure 2

Complete blood cell counts (CBCs) with differential were performed on peripheral blood from all groups. Values from lymphocytes, PMNs, and monocytes are represented in stack bar graphs. The absolute number of lymphocytes, PMNs, and monocytes are significantly elevated after 7 days of injury (A); however, unlike lymphocytes and monocytes, PMN cells counts do not decline following decompression in saline treated animals. RP3 treatment (B) does not alter the injury-related increases in lymphocytes and monocytes; however, PMN cell counts are not different from sham controls. Repairing animals following RP3 treatment show sustained increases in lymphocytes and monocytes but not PMNs, which remain at sham levels.

Figure 3

Inflammatory cells of the liver. Esterase (A)-, ED1⁺ (B)-, and ED2⁺ (C)-stained liver sections derived from sham, injured (day 0), and repairing (day 2) animals treated with saline or RP3 were digitally imaged and counted. Representative images of stained liver sections from injured animals accompany each graph and illustrate staining quality (×400). The number of esterase, ED1⁺, and ED2⁺ cells following cholestatic injury are significantly (^∗P ≤ 0.05) elevated when compared with saline sham controls. Declines in ED1⁺ and ED2⁺ cells following decompression are in contrast with PMN cell counts that remain elevated. RP3 treatment significantly depletes PMNs from with liver without depleting ED1⁺ and ED2⁺ cells.

Figure 4

Fibrotic repair. Representative images of Sirius red-stained liver sections (A) derived from injured and repairing animals treated with either saline or RP3 (×40) illustrate the data shown in the bar graph. Collagen (B) is expressed as a percentage of total area section area. Biliary obstruction significantly (*P ≤ 0.05) increased the deposition of collagen both in saline- and RP3-treated groups following 7 days of injury. Collagen content, as measured by Sirius red, was significantly (*P ≤ 0.05) decreased in the saline-treated animals following decompression (day 2). RP3 treatment blocked the decompression related decline in collagen. Measures of collagen content were not different from injured animals and significantly (**P ≤ 0.05) elevated when compared with saline-treated matched controls.

Figure 5

Quantitative real-time RT-PCR. MMP2 (A), MMP3 (B), MMP8 (C), MMP8 (D), MMP13 (E), MMP14 (F), TIMP1 (G), and TIMP2 (H) RNA expression was quantified in liver homogenates derived from sham, injured, and repairing animals following saline or RP3 treatment. Data are relative to 18S expression and are given as fold increases over saline sham. Significant increases over saline sham (^∗P ≤ 0.05) are noted. ▵ represents significant difference in TIMP-1 expression (P < 0.05) from control repairing animals.

Figure 6

MMP 8 Immunohistochemistry. Representative images of liver sections from bile duct obstructed cholestatic-injured animals treated with saline (A and C) or RP3 (B and D). Double immunofluoresent labeling of MMP8 (red) and neutrophils (green) demonstrate MMP8 immunostained bile duct epithelial cells (red) and colocalized PMNs (green) in proximity (A and B). MMP8 immunostaining confirms the bile duct hepatic source of MMP8 protein during injury. Immunofluorescent labeling is contrasted with the nuclear counterstain 4′,6′-diamidino-2-phenylindole in C and D.

Figure 7

Gelatinase/collagenase in situ zymography. A: Representative images of gelatinase activity in sham, injured, and repairing animals are shown at both low (×40) and high magnification (×400). Arrows point to gelatinolytic activity in and around the injured and repairing portal tracts. B: RP3-mediated neutrophil depletion does not alter gelatinolytic activity or localization during injury or repair. C: Collagenolytic activity in saline-treated animals is evident (arrows) in highly restricted regions of the portal tract in sham animals, and this activity is absent during injury. Repairing animals show a substantial increase in collagenase activity (arrows) primarily in apparent bile duct epithelial cells but also in individual cells within the portal tracts. D: RP3 treatment reduces collagenase activity during repair to sham levels. Arrowheads indicate the regions of collagenase activity which is markedly reduced in the absence of neutrophils compared to saline controls in C. Note there is no difference collagenase activity in sham or repaired livers without neutrophils.