Macrophage-mediated phagocytosis of apoptotic cholangiocytes contributes to reversal of experimental biliary fibrosis

Abstract

Studies have suggested the reversibility of liver fibrosis, but the mechanisms of fibrosis reversal are poorly understood. We investigated the possible functional link between apoptosis, macrophages, and matrix turnover in rat liver during reversal of fibrosis secondary to bile duct ligation (BDL). Biliary fibrosis was induced by BDL for 4 wk. After Roux-en-Y (RY)-bilio-jejunal-anastomosis, resolution of fibrosis was monitored for up to 12 wk by hepatic collagen content, matrix metalloproteinase (MMP) expression and activities, and fibrosis-related gene expression. MMP expression and activities were studied in macrophages after engulfment of apoptotic cholangiocytes in vitro. Hepatic collagen decreased to near normal at 12 wk after RY-anastomosis. During reversal, profibrogenic mRNA declined, whereas expression of several profibrolytic MMPs increased. Fibrotic septa showed fragmentation at week 4 and disappeared at week 12. Peak histological remodeling at week 4 was characterized by massive apoptosis of cytokeratin 19+ cholangiocytes, >90% in colocalization with CD68+ macrophages, and a 2- to 7.5-fold increase in matrix-degrading activities. In vitro, phagocytosis of apoptotic cholangiocytes induced matrix-degrading activities and MMP-3, -8, and -9 in rat peritoneal macrophages. We concluded that reconstruction of bile flow after BDL leads to an orchestrated fibrolytic program that results in near complete reversal of advanced fibrosis. The peak of connective tissue remodeling and fibrolytic activity is associated with massive apoptosis of cholangiocytes and their phagocytic clearance by macrophages in vivo. Macrophages upregulate MMPs and become fibrolytic effector cells upon apoptotic cholangiocyte engulfment in vitro, suggesting that phagocytosis-associated MMP induction in macrophages significantly contributes to biliary fibrosis reversal.

Fig. 1.

Histological features of fibrosis reversal, changes in hepatic collagen content, and collagenase activities after bilio-jejunal anastomosis. A: connective tissue staining (Sirius Red, representative images) of liver sections of rats after 4 wk of bile duct ligation (BDL) (a) and 7 and 14 days and 4 and 12 wk after Roux-en-Y (RY)-anastomosis (b, RY7d; c, RY14d; d, RY4w and e, RY12w, respectively). Original magnification: ×200. B and C: relative (μg/g of liver) and total (per liver) hepatic collagen content as determined by hydroxyproline in livers of rats after 4 wk of BDL (n = 6) and 3, 7, and 14 days and 4 and 12 wk after RY-anastomosis (n = 4 for RY3d and n = 6 for all other time points). D and E: interstitial collagenase and gelatinase activities in liver homogenates as determined by degradation of DQ-collagen type I and DQ-gelatin, respectively. Sham-operated rats served as normal and BDL as fibrotic controls (n = 6). MMP, matrix metalloproteinase. *P < 0.05 compared with the sham-operated group; +P < 0.05 compared with the peak of fibrosis group (BDL). #P < 0.05 compared with the preceding time-point (ANOVA, Tukey posttest).

Fig. 2.

Temporal patterns of profibrogenic gene expression during fibrosis reversal. Hepatic transforming growth factor (TGF)-β1, procollagen-α1(I), tissue inhibitor of MMP (TIMP)-1, TGF-β2, integrin-β6, and plasminogen activator inhibitor (PAI)-1 transcript levels as quantified by quantitative RT-PCR. Note the rapid decline of transcripts that are primarily expressed by activated cholangiocytes (A) and the slow decline of transcripts that are characteristic of activated hepatic stellate cells/myofibroblasts (HSC/MF) (B) after RY-anastomosis. Data are expressed as means ± SE and in arbitrary units relative to β-2 microglobulin (β2MG) mRNA. *P < 0.05 compared with the sham-operated group; +P < 0.05 compared with the peak fibrosis group (BDL).

Fig. 3.

Cholangiocyte apoptosis is a prominent feature of biliary fibrosis reversal. A: double staining for bile duct epithelial (cytokeratin 19, CK19) and apoptotic cells (TdT-mediated dUTP nick end labeling, TUNEL) demonstrates a sharp increase in double-positive cells 3 days (b) and 4 wk (e) after RY-anastomosis (arrows), whereas there are no apoptotic cholangiocytes at peak fibrosis (a). Original magnification: ×200. B: high-power magnification (×600) of a representative section 4 wk after RY-anastomosis when maximal apoptosis occurs, demonstrating that almost all CK19+ cholangiocytes (red staining) forming bile ducts are TUNEL positive (brown nuclear staining), whereas other portal cells and hepatocytes are TUNEL negative (light blue nuclear counterstain). C: quantification of apoptosis: CK19-negative/TUNEL-positive (open bars) and CK19/TUNEL-double-positive cells (shaded bars) were counted in 3–5 animals per group in at least 10 random fields at a magnification of ×200. Numbers are expressed as positive cells/10 high-power fields (HPF), means ± SE. D–F: detection of apoptotic cholangiocytes by immunofluorescent double-labeling for TUNEL and the alternative cholangiocyte marker pan-cytokeratin (pan-CK), confirming the results obtained using TUNEL/CK-19 double immunohistochemistry. D: no detectable cholangiocyte apoptosis is found at the peak of fibrosis (BDL4w). Small TUNEL-positive nuclei are rare and belong to intraepithelial (inflammatory) cells in proliferating bile ducts (inset). E: early apoptotic cholangiocyte at the peak of resolution (RY4w) identified by the large TUNEL+ nucleus, pan-CK+ cytoplasm, typical morphology and its location within a bile duct (inset). F: late apoptotic cholangiocytes within a vanishing bile duct. TUNEL+, p-CK+ material demonstrates colocalization in the merged image (yellow). Original magnification, ×100.

Fig. 4.

Scar-associated macrophages increase at maximal fibrolytic remodeling and colocalize with apoptotic cholangiocytes. A: double immunohistochemistry staining for the macrophage marker CD68 (red, cytoplasmic) and apoptosis (TUNEL: brown, nuclear) on liver sections from rats 14 days (a and d), 4 wk (b and e, maximal fibrolysis), and 12 wk (c and f) after RY-anastomosis; representative portal areas (top, original magnification, ×200; bottom, high-power magnification, ×400). Apoptotic cells in colocalization with macrophages are highlighted by arrows. A marked increase of colocalized cells is observed at week 4 of reversal. B: double immunofluorescence staining for the macrophage marker CD68 and apoptosis (TUNEL) on liver sections from rats 4 wk after RY-anastomosis, i.e., at maximal fibrolysis; representative portal area (original magnification, ×100). C: quantification of TUNEL-positive apoptotic cells colocalized with CD68-positive macrophages as assessed immunohistochemically and counted at a magnification of ×200 (see materials and methods). Data are expressed as the number of cells per 10 HPF; means ± SE. *P < 0.05 compared with the RY14d group.

Fig. 5.

Activation of collagen-degrading enzymes in the liver during fibrosis reversal. A: gelatin gel zymography of representative liver homogenates demonstrates that MMP-9 is the most prominent gelatinase, reaching highest levels at the peak of fibrolysis (week 4 of reversal), whereas MMP-2 declines. Densitometry of the MMP-9 band was performed from zymographies of all livers (n = 4–6 per bar) and is expressed as arbitrary units relative to the sham-operated controls (means ± SE). B: corresponding changes in MMP-9 protein as detected by Western blotting in representative liver homogenates. C: expression of MMPs and components of the plasmin proteolytic system during fibrosis reversal. Transcript levels of MMPs, urokinase plasminogen activator (uPA) and uPA receptor (UPAR)-1 were quantified by quantitative RT-PCR. Data are expressed as means ± SE and in arbitrary units relative to β2MG mRNA. *P < 0.05 compared with the sham-operated group; +P < 0.05 compared with the peak fibrosis group (BDL).

Fig. 6.

Engulfment of apoptotic cholangiocytes induces MMP expression and matrix-degrading activities in macrophages. A: high intrinsic gelatinase and collagenase activity in macrophage vs. nonmacrophage cell cultures. Freshly isolated rat peritoneal macrophages (pMΦ), the murine cholangiocyte cell line (603B), the rat hepatic stellate cell line CFSC-2G, and human hepatoma cells (Huh-7) were cultured at a density of 30 × 10⁴ cells/well in the presence of self-quenched DQ-gelatin or DQ-collagen substrates for 16 h to measure live cell gelatinase and collagenase activities as described in materials and methods. B: dose-dependent induction of MMP expression in macrophages upon engulfment of apoptotic cholangiocytes. Apoptotic 603B cholangiocytes (apo603B) were prepared as described in materials and methods, and 1–4 × 10⁴ cells were added to freshly isolated rat peritoneal macrophages (3 × 10⁴ cells/well). Relative mRNA levels of MMP-3, -8, and -9 and TIMP-1 were determined by quantitative RT-PCR 12 h after addition of apoptotic cells. C: corresponding increase in secreted MMP-9 protein levels upon phagocytosis as detected by Western blotting of conditioned macrophage supernatants; no MMP-9 detected in the corresponding cell lysate. D: phagocytosis-associated MMP induction results in a net increase in macrophage matrix-degrading activity. Collagenolytic and gelatinolytic activities were determined as outlined above over 16 h after addition of apoptotic 603B cholangiocytes at increasing concentrations. Data are expressed as means ± SE, with each bar representing 3 (B) or 6–8 (A and D) wells per experimental condition. All in vitro experiments were repeated at least 3 times. *P < 0.05 compared with the control group.

Fig. 7.

Proposed pathophysiology of extracellular matrix remodeling during biliary fibrosis progression and reversal. Cholestasis (BDL) triggers cholangiocyte activation and proliferation (1), which upregulate profibrogenic αvβ6 integrin and soluble factors, e.g., TGF-β, PAI-1 and connective tissue growth factor, leading to paracrine myofibroblast activation (2) and enhanced collagen synthesis. Aberrant ductular proliferation (3) further amplifies HSC/MF recruitment and fibrous matrix deposition, leading to fibrosis and loss of normal liver architecture. Upon restoration of bile flow by RY-anastomosis (RY), activated cholangiocytes undergo rapid deactivation (4) and apoptosis (5), which removes the profibrogenic stimuli acting on myofibroblasts. Cholangiocyte apoptosis triggers recruitment of CD68+ macrophages into scarred portal tracts to remove apoptotic cholangiocytes via phagocytosis (6) and to upregulate MMP-3, -8, and-9 to remodel the scar, leading to dissolution of fibrous septa and restoration of normal liver architecture. a, activated; b, proliferating cholangiocyte; c, apoptotic cholangiocyte; d, myofibroblast; e, macrophage; f, fibrotic matrix.