Bile Infarcts: New Insights Into the Pathogenesis of Obstructive Cholestasis

SEE ARTICLE ON PAGE 666 Bile infarcts, known as Charcot–Gombault necrosis, were first accurately described in 1876 by Charcot and Gombault in studies in animals after bile duct ligation (BDL). Since that time, the cause of these infarcts has been widely debated. Modern hypotheses first suggested that the accumulation of bile acids in hepatocytes is directly responsible for hepatocyte death because of their cytotoxic detergent properties. Subsequent studies suggested that bile acids cause hepatocyte apoptosis. Currently it is believed that bile acids injure hepatocytes not through their detergent properties but by triggering a cytokine‐mediated inflammatory response. Although considerable evidence now supports this latter hypothesis,1 the sequence of events that lead to bile infarcts and hepatocyte necrosis after BDL remains to be elucidated. In a study described in this issue of Hepatology, Ghallab et al. used an elegant intravital two‐photon‐based imaging system and fluorescent labeled bile acid and other cellular markers to capture, in real time, the live events of bile infarct formation in the liver of a bile‐duct‐ligated mouse.4 During the acute phase (1‐3 days after BDL), hepatic bile acid levels increased, resulting in loss of the cells’ mitochondrial membrane potential. At this point, the apical canalicular membrane in focal areas ruptured, and bile was seen to initially regurgitate into single cells and then into adjacent sinusoids, creating a canalicular bile‐sinusoidal shunt (#4 in Fig. 1). The high concentration of bile acids in these focal areas then injured neighboring cells, resulting in sinusoidal membrane leakage and cell death. Immune cell infiltration followed, resulting in the final formation of the bile infarct. In contrast, bile infarcts were no longer observed in the chronic stages in 21‐day BDL mouse livers, in which the bile acid concentrations in the bile were significantly lower than in the bile from 1‐day BDL mice. Bile infarcts were also not detected in Mdr2‐/‐ mice.Figure 1: Hepatic effects of obstructive cholestasis. #1: cholehepatic shunt. #2: inward blebbing. #3: bile leaks via tight junctions. #4: ruptured apical membrane. #5: cytokine‐mediated inflammation. #6: adaptive response of bile acid synthesis and transport. Abbreviations: BA, bile acids; BDL, bile duct ligation.Together, these series of images revealed a novel sequence of events that lead to hepatocyte necrotic death, resembling the “Charcot–Gombault necrosis” described more than a century ago. By creating a shunt between the bile canaliculus and the blood, bile acid concentrations in the biliary tract diminish, resulting in reductions in bile acid toxicity, while regurgitation of bile into blood results in bile acid clearance into urine. The concentrations of bile acids in bile are high enough to directly kill hepatocytes; thus, these bile infarcts must be initiated by the direct cytotoxicity of bile acids. Because immune cells (leukocytes and neutrophils) migrated to the affected areas after hepatocyte necrosis was first detected, the inflammatory response cannot be the initiating event after BDL but presumably contributes to the formation of the infarct. Because the compromised hepatocytes lose their mitochondrial membrane potential before the apical membrane ruptures, the stressed hepatocytes may simultaneously initiate an inflammatory response. This scenario is very likely because the hepatic expression of proinflammatory cytokines is increased starting 6 hours after BDL in mice, long before histological evidence of cell necrosis is observed.5 These cytokines could then contribute to the loss of the mitochondrial membrane potential and apical membrane rupture. Otherwise, it is difficult to explain why bile infarcts/necrosis are significantly reduced after BDL in mice when the inflammatory response is mitigated because of knockout of proinflammatory genes or drug treatment. Biliary pressure may also play a role.6 Although it was not measured in this study, it is likely highest in the acute phase (1‐3 days) of BDL when bile acid excretion is still fully active and presumably decreases in the chronic phase (21 days) of BDL, when bile secretion is diminished as part of the protective adaptive response to cholestasis (Fig. 1). This would explain why bile infarcts were not found in the livers of 21‐day BDL mice and Mdr2‐/‐ mice in which bile flow is not obstructed. While Ghallab et al.’s impressive images provide a mechanistic explanation for the initiation of bile infarct formation, they do not explain how cholestatic liver injury develops in nonobstructive cholestasis, in which bile regurgitation is very unlikely. The cause of hepatocyte death in these cholestatic livers must occur through alternative mechanisms. Figure 1 illustrates several additional mechanisms that have been described in acute and chronic stages of BDL as well as nonobstructive cholestatic disorders. In obstructive disease, bile acid may also be removed from the expanded and proliferated biliary tree by the apical sodium‐dependent transporter, ABST, and effluxed across the basolateral membrane by OSTα/OSTβ. However, it remains to be determined to what extent this cholehepatic shunt (#1 in Fig. 1) contributes to the overall adaptive response. It has long been known that the apical membrane of the hepatocyte may form blebs in the acute phase of BDL (#2 in Fig. 1). A recent study used intravital laser imaging techniques to demonstrate herniation of vesicles and vacuoles from the bile canalicular membrane into the hepatocyte during the first 1 to 2 hours of BDL, when biliary pressure is at its maximum (#2 in Fig. 1).7 Cytoplasmic blebs (vacuoles) up to 5 µm in diameter were visualized that contain both leaflets of the canalicular membrane from which they bud. These vacuoles then cross the cell to the sinusoidal membrane of the hepatocyte to discharge their contents into the blood. This phenomenon appears to occur in response to the acute increases in biliary pressure and prior to the loss of metabolic integrity of the hepatocyte. Whether “blebbing” continues during the chronic phase of BDL as illustrated is not certain. Alterations in tight junction structure and increases in permeability of the paracellular pathway have also been described in both obstructive and nonobstructive cholestasis (#3 in Fig. 1).8 However, Ghallab et al. did not observe any paracellular leakage of bile. In contrast, the death of hepatocytes in nonobstructive cholestasis is most likely the result of an immune response stimulated by bile acid accumulation in hepatocytes at nondetergent levels. This results in hepatocyte cytokine synthesis and release, which initiates a neutrophil and T‐cell response that then leads to hepatic injury (#5 in Fig. 1). This hypothesis is supported by studies that have shown reductions in bile infarcts/liver necrosis when the response of neutrophils is diminished in mice undergoing BDL, including gene knockout of Icam‐1, Egr‐1, or Ccl2.2 Other studies have demonstrated that pathophysiologic concentrations of bile acids stimulate neutrophil chemotaxis by inducing the expression of proinflammatory genes in mouse and human hepatocytes, but not in nonparenchymal cells or cholangiocytes.2 Finally, in all forms of cholestasis, the hepatocyte undergoes adaptive changes in bile acid synthesis and transport that serve to reduce the extent of liver injury as illustrated (#6 in Fig. 1).10 Although the present study focuses on the role of apical membrane rupture in the formation of bile infarcts, it is likely that all six of these mechanisms are involved in the response to bile duct obstruction. The development of novel therapeutic options for the treatment of cholestasis will need to take these diverse mechanisms into account. Finally, Ghallab and colleagues are to be congratulated for adding yet another mechanism to this picture and elucidating the cause of Charcot–Gombault necrosis, described nearly 150 years ago. Potential conflict of interest Nothing to report.