Volume 127, Issue 1, Pages 261-274 (July 2004) Regurgitation of bile acids from leaky bile ducts causes sclerosing cholangitis in Mdr2 (Abcb4) knockout mice  Peter Fickert, Andrea Fuchsbichler, Martin Wagner, Gernot Zollner, Arthur Kaser, Herbert Tilg, Robert Krause, Frank Lammert, Cord Langner, Kurt Zatloukal, Hanns-Ulrich Marschall, Helmut Denk, Michael Trauner  Gastroenterology  Volume 127, Issue 1, Pages 261-274 (July 2004) DOI: 10.1053/j.gastro.2004.04.009

Figure 1 Tight junctions are altered severely in Mdr2−/−. Double-immunofluorescence labeling of CK 8/18 (in green) and the tight junction protein ZO-1 (in red) was performed in 2-week-old (A-C) Mdr2+/+ (+/+) and (D-F) Mdr2−/− (−/−). (B, C) ZO-1 staining of bile ducts in Mdr2+/+ shows a sharp staining pattern outlining the cell contacts between the BECs. (E, F) In contrast, Mdr2−/− show strikingly altered tight junction morphology characterized by a tortuous, partially disrupted, ZO-1 staining pattern between BECs. Bar = 20 μm. Gastroenterology 2004 127, 261-274DOI: (10.1053/j.gastro.2004.04.009)

Figure 2 Basement membranes are disrupted frequently in Mdr2−/−. Double-immunofluorescence labeling of CK 8/18 (green) and laminin (red) was performed in 2-week-old (A-C) Mdr2+/+ (+/+) and (D-F) Mdr2−/− (−/−). (C) Double-immunofluorescence of Mdr2+/+ shows a regular continuous pattern of laminin around bile ducts (bd), hepatic arteries (ha), and the portal vein (pv), and outlining the peribiliary plexus (∗). (E, F) In contrast, laminin staining around bile ducts is discontinuous in Mdr2−/−. (F) Note also that the peribiliary plexus is separated from the biliary epithelium in Mdr2−/−. Bar = 20 μm. Gastroenterology 2004 127, 261-274DOI: (10.1053/j.gastro.2004.04.009)

Figure 3 Ultrastructural characteristics of sclerosing cholangitis in Mdr2−/− comprising pathologic cell-to-cell contacts, disrupted basement membranes, intraepithelial lymphocytes, and periductal edema. Electron microscopy was performed in 2-week-old (A, B) Mdr2+/+ (+/+) and (C, D) Mdr2−/− (−/−). (A) Interlobular bile duct (bd) in Mdr2+/+ with regular architecture and normal peribiliary plexus. A hepatic artery (ha) branch is near the bile duct. (B) Higher magnification showing regular cell-to-cell contacts between the BECs and a continuous basement membrane is indicated by the arrows. (C) In contrast, the Mdr2−/− shows severely altered BECs of a bile duct (bd) with widened intercellular spaces (indicated by arrows). Note the appearance of intraepithelial lymphocytes and also the periductal edematous tissue containing leukocytes and activated fibroblasts. (D) Higher magnification showing severely injured BECs with partially intact basement membrane (indicated by arrows) and disruption of the basement membrane (indicated by arrowheads). Note also that collagen bundles (indicated by stars) are most prominently present near the leak in the basement membrane. ∗Pathologic cell-to-cell contacts of BECs in Mdr2−/−. (A, C) Bar = 5 μm, (B, D) bar =1 μm. Gastroenterology 2004 127, 261-274DOI: (10.1053/j.gastro.2004.04.009)

Figure 4 Leakage of fluorescent UDCA into the portal tract in Mdr2−/−. Fluorescent UDCA was visualized in 2-week-old (A) Mdr2+/+ and (B, C) Mdr2−/− as described in the Materials and Methods section. (A) Fluorescent UDCA stains bile canaliculi and the bile duct lumen as well as the BECs in Mdr2+/+ liver. (B) Leakage of fluorescent UDCA (indicated by arrows) into the portal tract in Mdr2−/− liver. The basal cell border of BECs is indicated by the white ellipse. (C) Bile infarct in Mdr2−/− liver impregnated by fluorescent UDCA. These findings suggest that periportal hepatocytes and hepatocytes surrounding bile infarcts face the highest bile acid levels within the liver lobule in Mdr2−/−. bd, bile duct. Bar = 20 μm. Gastroenterology 2004 127, 261-274DOI: (10.1053/j.gastro.2004.04.009)

Figure 5 Characterization of the inflammatory infiltrate in Mdr2−/−. Double-immunofluorescence labeling of (A) CD11b-, (B) CD8-, (C) CD4-positive cells (red) and CK 8/18 (green) was performed in Mdr2+/+ (+/+) and Mdr2−/− (−/−) at 2, 4, and 8 weeks of age. (A) Comparison between Mdr2+/+ and Mdr2−/− showed an increased number of CD11b-positive cells in Mdr2−/− that decreased from week 4 to week 8 of life. (B, C) In contrast, there was an increasing number of (B) CD8- and (C) CD4-positive cells in Mdr2−/− from week 2 to 8, whereas Mdr2+/+ livers stained almost negative for CD8 and CD4 markers. Bar = 20 μm. Gastroenterology 2004 127, 261-274DOI: (10.1053/j.gastro.2004.04.009)

Figure 6 Increased number of KCs in Mdr2−/−. Immunohistochemistry for detection of KCs was performed in (A-C) Mdr2+/+ (+/+) and (D-F) Mdr2−/− (−/−). (A, D) Higher number of KCs in Mdr2−/− compared with Mdr2+/+, especially in hepatic acinar zone 1 at 2 weeks of age. (B, C, E, F) No differences in KC number at later age between the genotypes. Original magnification, 40×. bd, bile duct. Gastroenterology 2004 127, 261-274DOI: (10.1053/j.gastro.2004.04.009)

Figure 7 Hepatic cytokine levels in Mdr2−/−. Real-time PCR for quantification of relative expression levels of (A) TNF-α, (B) IL-1β, (C) IL-6, (D) interferon-γ, and (E) TGF-1β was performed in 2-, 4-, and 8-week-old Mdr2+/+ and Mdr2−/−. Data are expressed as fold change of the respective age-matched Mdr2+/+ (n = 5 in each group; mean ± SEM; ∗Mdr2+/+) vs. age-matched Mdr2−/− (P ≤ 0.05). ■, Mdr2+/+; □, Mdr2−/−. Gastroenterology 2004 127, 261-274DOI: (10.1053/j.gastro.2004.04.009)

Figure 8 Periductal fibrosis leads to separation of the peribilary plexus from the biliary epithelium in Mdr2−/−. Immunohistochemistry for (A, C) α-SMA-positive cells and (B, D) Sirius red staining for collagen was performed in 4-week-old Mdr2+/+ (+/+) and Mdr2−/− (−/−) (both in red). (A, C) Sirius red staining is enhanced significantly in Mdr2−/−, marking a pronounced periductal fibrotic ring. Note also partial desquamation of the BECs (arrowheads). (B) In Mdr2+/+, only myoepithelial cells of the hepatic artery (ha) and of the peribiliary plexus (arrows) stain positive for α-SMA. (D) In contrast, there are numerous α-SMA-positive myofibroblasts in Mdr2−/− surrounding the bile duct. Note also that the peribiliary plexus (arrows) is separated from the biliary epithelium in Mdr2−/−. pv, portal vein; ha, hepatic artery, bd, bile duct. Original magnification, 40×. Gastroenterology 2004 127, 261-274DOI: (10.1053/j.gastro.2004.04.009)

Figure 9 Suggested pathogenetic sequence of events for sclerosing cholangitis in Mdr2−/− mice. The lack of biliary phospholipid secretion in Mdr2−/− leads to increased biliary concentrations of nonmicellar-bound, free bile acids. As a result, there is damage of tight junctions (in red) and basement membranes (in green) of bile ducts, leading to leakage of potentially toxic bile acids into the periductal area, inducing inflammation with activation of neutrophil granulocytes as well as CD4- and CD8-positive lymphocytes. Infiltrating leukocytes and autocrine cytokines result in activated periductal myofibroblasts. Activated periductal myofibroblasts form a periductal fibrous ring. The fibrotic ring separates the peribiliary plexus from the BECs, leading to atrophy and, finally, death of BECs, which itself leads to fibroobliteration of the bile duct. Gastroenterology 2004 127, 261-274DOI: (10.1053/j.gastro.2004.04.009)