Intrahepatic cholestasis in common chronic liver diseases

Authors


Abstract

Background and objective

Cholestasis represents the consequence of impaired bile formation and decrease in bile flow, generally classified as extra- and intrahepatic. Cholestasis is the pivotal hallmark of the so-called primary cholestatic liver diseases but may also emerge in other forms of chronic liver injury. The aim now was to summarise the current state of knowledge on intrahepatic cholestasis related to chronic liver diseases.

Methods

For this overview on intrahepatic cholestasis in chronic liver disorders other than the ‘classic’ cholestatic liver diseases, selected references were retrieved by literature search in MEDLINE and textbooks were reviewed. All articles were selected that discussed pathophysiological and clinical aspects of intrahepatic cholestasis in the context of alcoholic liver disease, nonalcoholic fatty liver disease, chronic hepatitis B and C virus infections as well as drug-induced and granulomatous liver diseases. Titles referring to primary biliary cirrhosis and sclerosing cholangitis were excluded.

Results and conclusions

Dependent on the aetiology, intrahepatic cholestasis is present at variable frequencies and in different disease stages in chronic liver diseases. Cholestasis secondary to chronic liver injury may denote a severe disease course and development of end-stage liver disease or specific disease variants. These findings indicate that ‘secondary intrahepatic cholestasis’ (SIC) can occur in the natural course of chronic liver diseases other than the primary cholestatic diseases, in particular in the setting of advanced disease progression.

Introduction

Bile formation is maintained by a coordinated network of hepatobiliary membrane transporters [1]. Bile acids, the major solute in bile, predominantly drive bile formation. Any impairment of bile formation and reduction in bile flow result in cholestasis. Clinically, cholestatic patients present with pruritus (itching), fatigue and, in severe forms, jaundice, reflected by elevated serum bilirubin levels. In the early stages of the condition, symptoms might be absent and only increased serum activities of alkaline phosphatase (AP) and/or γ-glutamyl-transpeptidase (γ-GT) indicate a cholestatic condition. Cholestasis may be classified as acute or chronic (i.e. more than 6 months) and as intrahepatic or extrahepatic cholestasis [2], the former developing from obstruction of the intrahepatic biliary tree or hepatocellular defects. The vast majority of reports on cholestasis refer to chronic cholestatic liver diseases such as primary biliary cirrhosis (PBC), primary sclerosing cholangitis, secondary sclerosing cholangitis or hereditary defects in hepatobiliary transporters, such as progressive familial intrahepatic cholestasis. In this review, we focus on intrahepatic cholestasis in chronic liver diseases other than the ‘classic’ cholestatic liver diseases (Table 1, Fig. 1), excluding paediatric liver diseases, and we propose to call this phenomenon ‘secondary intrahepatic cholestasis’ or SIC.

Table 1. Characteristics of secondary intrahepatic cholestasis in chronic liver diseases
Liver diseaseIntrahepatic cholestasis
Alcoholic liver diseaseMild in alcoholic steatosis
Potentially severe in alcoholic hepatitis
Variants of alcoholic liver disease
Nonalcoholic fatty liver diseaseSubgroup of patients
Advanced disease
Variant with cholestatic features
Chronic hepatitis BSubgroup of patients
Hepatitis B reactivation
Liver cirrhosis
Fibrosing cholestatic hepatitis in liver transplant
Chronic hepatitis CSubgroup of patients
Mild nonspecific bile duct lesions
Liver cirrhosis
Cholestatic hepatitis in liver transplant
Granulomatous liver diseasesSarcoidosis-type granulomas in portal spaces
Granulomas with epithelioid and giant cells and centripetal hyaline fibrosis
Drug-induced cholestasisPredominantly hepatocellular cholestasis
Portal inflammation with neutrophils and plasma cells
Figure 1.

Proposed classification of chronic intrahepatic cholestasis. Cholestatic liver diseases can be grouped as primary cholestatic liver diseases of genetic or unknown origin and secondary cholestasis either due to hepatocellular injury (secondary intrahepatic cholestasis, SIC) or secondary sclerosing cholangitis (SSC). SIC can be defined as cholestasis in chronic liver diseases other than the ‘classic’ cholestatic liver diseases. For SSC, which is beyond the scope of this review, we may refer to [124].

Alcoholic liver disease

Alcoholic liver disease encompasses a broad spectrum of liver injury, ranging from simple steatosis to alcoholic hepatitis (AH), chronic hepatitis with hepatic fibrosis and cirrhosis. There is often considerable overlap of these subtypes in any given patient, as steatosis might persist in later stages of the disease [3]. Clinical jaundice and histological features of intrahepatic cholestasis may be present in all stages of alcoholic liver disease [4].

Molecular mechanisms of cholestasis in alcoholic liver disease

Alcohol-mediated processes leading to steatosis, inflammation and fibrosis have been studied in detail (reviewed by Gao and Bataller [5]). In contrast, our understanding of the pathophysiology of cholestasis in alcoholic liver disease is not that well established. We have known for some time that compression of intrahepatic biliary radicles [6] and increased permeability of the bile ductules [7] appears to mediate alcohol-induced cholestasis. Subsequently, the potential effects of ethanol on microtubule assembly have been described in detail by Tung and Carithers [8] with the knowledge that the cytoskeleton machinery plays a critical role in bile acid secretion [9]. Under these conditions, acetaldehyde binding may impair microtubule function and contribute to alcohol-induced cholestasis. Data supportive of this model have been observed in polarised hepatic WIF-B cells, where alcohol-induced microtubule acetylation has been shown to correlate with defective hepatic membrane trafficking [10].

Serum bile acid concentrations are elevated in almost all patients with AH, particularly chenodeoxycholic acid [11]. Indeed, serum bile acid levels have been correlated with the histological AH score in patients with biopsy-proven disease but no consistent association between AH and cholestasis scores was observed [11, 12].

Hepatocellular transport of bile acids is mediated by multiple sinusoidal and canalicular membrane transporters that can be modulated by inflammatory processes. Cholestasis typically occurs during sepsis mediated by lipopolysaccharide (LPS) or endotoxin activation of pro-inflammatory cytokines such as tumour necrosis factor-α, interleukin (IL)-1β and IL-6. The activation of pro-inflammatory cytokines inhibits hepatobiliary transport function leading to a reduction in bile acid flow and the accumulation of bile acids and toxins in liver and serum [13]. Inflammation also plays a central role in the progression of alcoholic liver disease, particularly in patients with AH. The ethanol-induced translocation of LPS into the portal venous system and the subsequent induction of the inflammatory cascade contribute to the development of alcoholic steatohepatitis (ASH) [14]. Furthermore, a significant downregulation of critical bile acid transporters (Fig. 2), such as Na+-taurocholate cotransporting polypeptide [NTCP, solute carrier (SLC) 10A1], organic anion-transporting polypeptide 1B1 (OATP1B1, SLCO1B1) and bile salt export pump [BSEP, ATP-binding cassette (ABC) transporter B11], the major determinant of canalicular bile salt secretion, has been documented within liver samples derived from patients with AH and other inflammatory cholestasis [15]. Because BSEP is rate limiting for bile salt secretion, this transporter rather than the uptake systems is likely the most important factor [9].

Figure 2.

Major hepatobiliary transport proteins. The uptake of bile acids from the sinusoidal blood is mediated by the Na+-taurocholate cotransporter (NTCP, SLC10A1) and Na+ - independent via organic anion-transporting polypeptides (OATPs, SCLOs). The canalicular excretion of monovalent bile acids occurs via the bile salt export pump (ABCB11), and divalent bile acids and different organic anions are excreted by the conjugate export pump multidrug resistance-related protein 2 (MRP2, ABCC2). Phosphatidylcholine is excreted at the canalicular membrane by the ‘floppase’ action of multidrug resistance 3 P-glycoprotein (MDR3, ABCB4), and aminophospholipids are transported from the outer to the inner leaflet of the canalicular membrane by the ‘flippase’ P-type ATPase FIC1 (ATP8B1). Cholesterol excretion is driven by the ATP-binding cassette (ABC) hemitransporters ABCG5 and ABCG8. MRP3 (ABCC3), MRP4 (ABCC4) and the heterodimeric organic solute transporter OSTα/β (SLC51) are expressed at the basolateral membrane and mediate bile acid excretion into sinusoidal blood, which may be regarded as a backup system for bile acid export in cholestatic conditions when expression levels of these transporters are induced.

These studies suggest a role for inflammatory mediators in regulating the expression of hepatobiliary transporters in cholestatic patients with AH. Because hepatic progenitor cell markers also correlate with the severity of AH [16], failure to regenerate functional biliary epithelial cells might be an important feature of persistent secondary cholestasis in alcoholic liver disease.

Alcoholic steatosis

Fatty liver is the earliest consequence of chronic alcohol abuse. With alcohol abstinence, simple fatty liver is generally reversible, whereas about 35% of individuals with continuous heavy alcohol abuse develop advanced alcoholic liver disease [5]. The typical laboratory abnormalities include elevated γ-GT activity and a moderate increase in aminotransferase levels to less than twice the upper limits of normal. Elevated AP activities are observed in up to 50% of patients, and minor increments in serum bilirubin concentrations are found in 20–30% of patients. Histopathology usually shows macrovesicular steatosis with early changes in perivenular hepatocytes within zone 3. In cases of more severe liver injury, periportal hepatocytes within zones 2 and 1 are also affected.

Of note, intrahepatic cholestasis may accompany fatty changes. Histological features of cholestasis have been described in 19% of patients with alcoholic steatosis [17]. In a long-term follow-up study of 258 noncirrhotic individuals with excessive alcohol consumption, histological signs of cholestasis on initial biopsy were not associated with an increased risk of cirrhosis [18]. If present, cholestasis in patients with alcoholic steatosis usually appears to be mild in most patients. However, Ballard et al. [19] reported five patients with clinically overt jaundice and liver biopsies showing severe steatosis as well as marked cholestasis. Indeed, fulminant hepatic failure and death were reported in two of three patients with alcohol-induced macrovesicular and microvesicular steatosis combined with severe cholestasis in the absence of histological AH [6]. In exceptional cases, patients may present with Zieve syndrome with a combination of jaundice, hyperlipidaemia, haemolytic anaemia and alcoholic steatosis [20]. In a case series, these patients showed features of an acquired pyruvate kinase deficiency predisposing to haemolysis [21], potentially via the circulating haemolysin lysolecithin [20, 22, 23].

Alcoholic hepatitis

Alcoholic hepatitis might develop after several years of chronic and excessive alcohol use but can also occur after an acute episode of heavy alcohol consumption. Most patients with liver disease secondary to excessive alcohol use generally present with superimposed ‘acute’ or more strictly speaking acute-on-chronic deterioration of advanced liver disease. In milder cases, nonspecific symptoms such as nausea and vomiting are present, while in more severe forms of AH, jaundice, fever and right upper quadrant pain are often present. Serum aminotransferase activities are typically five- to eightfold elevated, the aspartate aminotransferase to alanine aminotransferase (ALT) ratio is typically > 2, and serum bilirubin and AP levels are generally elevated. Histopathological findings of ASH, the predominant cause of AH, are characterised by the coexistence of steatosis, centrilobular ballooning of hepatocytes, infiltrate with polymorphonuclear leucocytes, Mallory bodies, a ‘chicken-wire’-like pattern of fibrosis and frequently cirrhosis [4, 24] (Fig. 3a).

Figure 3.

(a) Histopathology of alcoholic hepatitis with ballooned hepatocytes, neutrophilic infiltrates, hepatocyte necrosis, Mallory's hyaline and intrahepatic cholestasis (H&E stain) (provided by Prof. Dr. R. M. Bohle, Institute of Pathology, Saarland University Medical Center). (b) Histopathology of granulomatous hepatitis with moderate fibrosis and absence of bile ducts (H&E stain) (provided by Prof. Dr. J. Lorenzen, Institute of Pathology, Klinikum Dortmund).

The concomitant appearance of cholestasis and AH has been well appreciated for a long time. Sixty years ago, explorative laparotomy was performed in specific cases, where extrahepatic biliary obstruction was suspected to be responsible for prolonged jaundice in patients with decompensated alcoholic liver disease [25, 26]. Several studies have reported a variable prevalence of histologically confirmed cholestasis in AH, depending on the severity of disease (Table 2). For example, Chedid et al. [17] observed cholestasis in a quarter of patients with AH and found that cholestasis was more common in patients with cirrhosis and superimposed AH as compared to non-cirrhotic AH patients. In these studies, moderate-to-severe (grade II) cholestasis in AH, as scored on liver biopsies, was a significant prognostic factor of outcome; histologically, the severity of cholestasis was correlated with histological changes such as parenchymal necrosis, portal inflammation and fibrosis stage [12]. In a large trial evaluating the efficacy of corticosteroid treatment in AH, histological features of cholestasis were found in up to 50% of patients, who all had cirrhosis [27]. Similarly, severe intraparenchymal cholestasis was observed in another series of 39% of 163 patients with AH who were hospitalised due to clinical decompensation [28]. In jaundiced patients with AH, liver biopsy does not consistently reveal cholestasis. For example, in the study by Chedid et al. [17], 55% of patients presented with jaundice, while cholestasis was found in only 25% of liver biopsies.

Table 2. Prevalence of jaundice and histological cholestasis in patients with alcoholic hepatitis in selected studies
Author and year [reference] N DesignJaundice (%)Cholestasisa (%)Moderate–severe cholestasis (%)Cirrhosis (%)Comments
  1. a

    Cholestasis is defined as histological evidence of bile thrombi and is graded on a scale using none, mild, moderate or severe histological cholestasis.

Beckett et al. (1961) [117]7Case series10083NR67Liver histology not available in one patient
Harinasuta and Zimmerman (1971) [118]257Retrospective4732NR37No difference in mortality in patients with or without Mallory bodies
Galambos (1972) [119]76RetrospectiveNR21NR20 
Birschbach et al. (1974) [120]100Prospective38451256 
Chedid et al. (1991) [17]217Prospective5525NR51Cholestasis seen in 32% of cirrhotic, but only in 18% of noncirrhotic patients
Mathurin et al. (1996) [27]122ProspectiveNRNR4395Maddrey's score > 32 or encephalopathy in all patients
Spahr et al. (2011) [28]163ProspectiveNRNR3997Cholestasis in early liver biopsies independent predictive factor of 3-months survival; serum bilirubin levels, age and Maddrey's score correlate to severity of intraparenchymal cholestasis

Rare forms of cholestasis in alcoholic liver disease

In a series of patients with alcoholic liver disease, Glover et al. [29] described three patients who initially presented after excessive alcohol intake with jaundice and histological evidence of severe centrilobular cholestasis but without hepatitis. The authors suggested the term ‘acute alcoholic cholestasis’ for this rare presentation of alcoholic liver disease, where all patients recovered after alcohol cessation. In another study of 23 jaundiced alcoholic patients, Afshani et al. [30] described four patients with distinct polymorphonuclear leucocytes in multiple bile ducts (‘microscopic cholangitis’). They concluded that microscopic cholangitis may be a feature of severe cholestasis that accompanies alcoholic liver injury.

Notwithstanding these rare conditions, the studies above taken together illustrate that common alcoholic liver injury causes cholestasis by multiple mechanical, toxic and inflammatory mechanisms. Whereas intrahepatic cholestasis in alcoholic fatty liver is generally mild, it represents a prognostic marker in AH, indicating that impaired bile formation could determine outcome of critically ill patients.

Nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) exhibits histopathological features similar to alcoholic liver disease but occurs in patients with no or little alcohol consumption [31]. NAFLD is regarded as the hepatic manifestation of the metabolic syndrome that is frequently accompanied by obesity, type 2 diabetes and hyperlipidaemia [32]. NAFLD comprises a histopathological disease spectrum from bland steatosis to nonalcoholic steatohepatitis (NASH) with significant inflammation and fibrosis, progressing to cirrhosis in approximately 20% of patients with NASH [33]. Patients with NAFLD are mostly asymptomatic, and while hepatomegaly is common, jaundice is rarely seen. Laboratory work-up may show modest elevation of serum aminotransferases and γ-GT, normal or mildly increased AP and, rarely, hyperbilirubinaemia. NASH histopathology resembles ASH and cannot reliably differentiate between the two conditions, with the caveat that there are some histological features including cholestasis, which have not typically been described in NAFLD [34]. Minor ductal reaction is frequently present, but may be more prominent in advanced disease [35]. More severe histopathological cholestatic features such as bile duct damage and loss are uncommon in NAFLD; hence, these findings suggest the coexistence of other liver diseases.

A cholestatic variant of NAFLD has been described in 20 NAFLD patients with elevated aminotransferase levels, AP > 500 U/L, γ-GT > 250 U/L and negative serological markers for other liver diseases [36]. The cholestatic patients demonstrated histological evidence of cholangitis, swelling, variable bile duct loss and bile stasis. In comparison with an age-, sex- and body mass index–matched NAFLD group, the cholestatic patients displayed more severe liver disease with advanced bridging fibrosis or cirrhosis. In a second matched group of NAFLD patients with identical histological staging and grading but without biochemical cholestasis, significant bile duct damage was absent in liver biopsies. These findings suggest that cholestasis might promote disease progression in individuals susceptible for the development of NAFLD and bile duct damage. In a large international collaborative trial investigating the long-term morbidity and mortality of patients with NAFLD with advanced fibrosis and cirrhosis, higher serum bilirubin levels were associated with liver-related mortality [37]. Histopathological data were not presented in detail; thus, a correlation of bilirubin levels to a histological cholestasis was not feasible; however, hyperbilirubinaemia may reflect advanced liver dysfunction rather than cholestasis.

In a pilot study, a moderate increase in bile acids was found in liver tissue of patients with NASH as compared to healthy controls [38]. It is possible that elevation of more hydrophobic bile acids, such as deoxycholic and chenodeoxycholic acids, may contribute to hepatic injury in patients with steatohepatitis. A recent study in obese patients with NAFLD [39] suggests that increased serum levels of free fatty (and bile) acids interfere with the suppression of hepatic bile acid synthesis and uptake by the nuclear receptor small heterodimer partner (SHP; NR0B2), which is induced by the central bile acid sensor, the farnesoid X receptor (FXR). In line with this observation in humans, FXR deficiency caused NASH in low-density lipoprotein (LDL) receptor-knockout mice [40]. It was hypothesised that FXR-SHP dysfunction leads to increased bile acid levels, driving the inflammatory cascade and facilitating the progression from NAFLD to NASH. These observations have led to the suggestion that modulation of FXR might represent a new therapeutic approach in selected patients with NASH and disturbed bile acid metabolism.

Taken together, intrahepatic cholestasis occurs in a subgroup of patients with NAFLD and provides a worse prognosis for advanced disease. In general, cholestatic features in patients with NAFLD should prompt physicians to rule out the presence of additional (cholestatic) liver diseases.

Chronic viral hepatitis

Cholestasis is not considered to be a common feature of chronic viral hepatitis, although intrahepatic cholestasis might be an indicator of disease progression. A number of studies have been carried out to investigate clinical, laboratory and histological characteristics of cholestasis in chronic hepatitis B and C virus infections.

Hepatitis B

More than 350 million people worldwide are chronically infected with hepatitis B virus (HBV). The disease spectrum ranges from the ‘inactive’ carrier state to progressive chronic hepatitis, cirrhosis and hepatocellular carcinoma. The complex natural history of chronic HBV infection is diverse and variable. Three phases have been described: (i) immune-tolerant phase that mostly occurs after perinatal transmission with high levels of HBV replication, normal or minimally elevated aminotransferase levels and minimal histological evidence for disease, (ii) immune-active phase with high HBV-DNA levels, elevated or fluctuating aminotransferases, significant necroinflammation and progression of liver fibrosis and (iii) inactive HBV carrier state with HBV-DNA levels <2000 IU/mL, normal aminotransferases and minimal inflammation and fibrosis with a very low risk of progression to cirrhosis and development of hepatocellular carcinoma [41].

Most patients with mild-to-moderate chronic HBV infection are asymptomatic or have nonspecific symptoms such as fatigue. Pruritus occurs in 8% of patients in the inactive HBV carrier state, with laboratory and histological parameters being comparable to those without pruritus [42]. In general, the onset of jaundice can be caused either by a severe progressive disease with the development of decompensated cirrhosis or a flare due to immune reactivation (Table 3). Various studies have reported jaundice in 8–48% of patients with HBV-related cirrhosis [43], with elevated levels of bilirubin, AP and γGT.

Table 3. Prevalence of jaundice and histological cholestasis in patients with chronic viral hepatitis in selected studies
Author and year [reference]Aetiology N DesignJaundice (%)Cholestasisa (%)Moderate–severe cholestasis (%)Cirrhosis (%)Comments
  1. HBV, hepatitis B virus; HCV, hepatitis C virus.

  2. a

    Cholestasis is defined as histological evidence of bile thrombi and is graded on a scale using none, mild, moderate or severe histological cholestasis.

Davies et al. (1991) [54]HBV27Cross-sectional2222229Six of 27 patients with chronic HBV infection developed fibrosing cholestatic hepatitis after liver transplantation
Mason et al. (1993) [53]HBV14Cross-sectionalNR43NR≥ 7Six of the 14 patients with HBV re-infection after liver transplantation developed fibrosing cholestatic hepatitis
Wong et al. (2011) [51]HBV36ProspectiveNR (bilirubin range 23–754 μM)NRNR14Despite initiation of entecavir treatment in patients with spontaneous severe acute exacerbation of chronic HBV, short-term mortality was 19%
Mori et al. (2012) [50]HBV37Prospective100NRNR19Patients with acute HBV exacerbation were followed, bilirubin levels > 5 mg/dL were determinants of hepatic failure
Tong et al. (1995) [58]HCV131Prospective1·5NRNR51Both of the two patients with jaundice had already developed cirrhosis
Chia et al. (1998) [69]HCV8Retrospective02512·563Eight of 151 screened HCV patients presented with severe pruritus and moderate-to-severe fibrosis or cirrhosis; in liver histology, features of chronic cholestasis were subtle compared with primary biliary cirrhosis patients
Kumar et al. (2001) [62]HCV6Retrospective033NR33Of 620 screened patients with chronic HCV, only six presented with cholestatic features, mostly in advanced disease

Intrahepatic cholestasis might also indicate severe HBV reactivation. Reactivation is characterised by a sudden increase in HBV replication in a patient with inactive or mildly active HBV infection [44], accompanied by a substantial elevation of aminotransferase activities. In more severe forms of HBV reactivation, bilirubin, AP and γGT levels may become elevated. In chronic HBe antigen-positive hepatitis B, elevated aminotransferases and cholestasis may denote hepatitis flares in the context of immunological reactivation, often followed by HBe antigen seroconversion to anti-HBe and accompanied by a decrease in HBV-DNA levels. Spontaneous reactivation may occur in patients with HBe antigen-negative hepatitis B, with typical flares preceded by an increase in HBV-DNA, followed by a rise of ALT levels. In severe forms, these flares can be icteric (resembling acute hepatitis), and it might often be difficult to differentiate a flare from acute hepatitis B [45]. In most instances, in HBV carriers, reactivation is triggered by exposure to chemotherapy or immunosuppressive agents or spontaneously in women during pregnancy or after delivery [46]. On rare occasions, immunosuppression may lead to reactivation in HBs antigen-negative individuals who still harbour replicative intermediates of HBV [47]. Reactivation may also occur after withdrawal of antiviral therapy or the development of resistance to antiviral therapy, especially with lamivudine [48]. In a study involving 100 Chinese patients, about half of those with HBV reactivation developed jaundice [49]. Indeed, in a recent prospective study [50], serum bilirubin levels > 5 mg/dL (and prothrombin activity < 45%) were determinants of hepatic failure and liver-related death in jaundiced patients with acute exacerbation of chronic HBV infection. Despite the initiation of antiviral treatment with more potent nucleos(t)ide analogues, such as entecavir and tenofovir, severe acute exacerbations can be fatal [51]. To avoid HBV reactivation, guidelines recommend pre-emptive therapy before immunosuppressive therapy or chemotherapy in HBV carriers and close monitoring in HBs antigen-negative patients with positive anti-HBc antibodies and undetectable HBV-DNA [52].

In the era before institution of universal hepatitis B immune globulin and antiviral medication for prophylaxis, re-infection of the graft after liver transplantation was almost universal resulting in significant reduction in graft survival and patient survival [53]. About 20–40% of patients developed fibrosing cholestatic hepatitis, characterised by rapid development of severe jaundice, graft failure and death within a few months after development of recurrent HBV infection in the allograft [53, 54]. Cases of fibrosing cholestatic hepatitis B have also been reported in other transplant settings such as kidney, heart or bone marrow transplantation and after chemotherapy for leukaemia, lymphoma or small-cell lung cancer [55]. Typical laboratory findings include increased serum bilirubin levels, mild-to-moderate elevation of aminotransaminases and prolonged prothrombin time. Severe periportal fibrosis, intrahepatic cholestasis with marked ductular reaction, widespread hepatocellular ballooning yet mild infiltration of inflammatory cells are the characteristic histopathological findings [54]. In the absence of inflammation, the direct cytopathic effect of uncontrolled HBV replication may cause fibrosing cholestatic hepatitis in organ recipients without immune reconstitution [53], and while antiviral therapy can improve liver function, mortality is still high [56].

In summary, the occurrence of clinical or laboratory signs of intrahepatic cholestasis in patients with chronic hepatitis B mostly indicates severe progressive liver disease or an acute exacerbation of HBV infection.

Hepatitis C

Hepatitis C virus (HCV) infection is a leading cause of chronic liver disease affecting approximately 180 million people worldwide. The natural history of chronic HCV infection is variable, with 15–30% of patients progressing to liver cirrhosis within three decades [57]. Jaundice is rarely seen in chronic HCV infection and seldom in acute HCV infection now that blood transfusions are no longer a source of disease. In a study of 131 HCV patients [58], only two presented with jaundice, both of whom had already developed cirrhosis.

Characteristic but nonspecific findings in liver biopsies from patients with chronic HCV infection are portal lymphoid aggregates, mild steatosis and some inflammatory bile duct damage [59, 60]. Bile duct lesions are usually mild, with small- or medium-sized bile ducts affected, and bile duct loss is rarely seen [61]. The high variability of bile duct injury in liver biopsies reported in previous studies, ranging from 8% to 91%, might be attributable to either the various comorbidities associated with high-risk individuals or differences in histopathological assessment and reporting bias of studies with smaller cohorts [62]. In the study of Kaji et al. [61], advanced disease and a higher degree of necroinflammatory infiltrates, particularly in portal tracts, were associated with the presence of bile duct lesions. Cytopathic effects of HCV and immune-mediated mechanisms have been suggested to be causative for bile duct lesions, although the exact mechanisms have yet to be defined [62]. The diagnostic value of bile duct lesions has been questioned by some authors, because biliary destruction is reportedly absent in liver biopsies from a large cohort of patients with chronic HCV infection [63].

Clinical or laboratory findings of cholestasis are uncommon in chronic HCV infection (Table 3). In a data set of 620 patients with hepatitis C, those with either AP > 400 U/L or AP > 250 U/L and pruritus were defined as presenting with cholestasis [62]. Of the 32 patients identified meeting these criteria, 24 had to be excluded because of confounding factors such as heavy alcohol abuse and/or lack of liver histology. Four of the remaining six patients presented with pruritus, bile duct loss was observed in four patients, and most patients exhibited an advanced fibrosis stage in liver biopsy.

Giannini et al. [64] assessed whether the presence of bile duct lesions in liver biopsies from patients with chronic HCV infection is reflected by serum parameters. An association with γ-GT levels was found, but virological parameters such as HCV-RNA and HCV genotype were not implicated [64]. In an earlier study, the same group found bile duct lesions more often in older patients with advanced disease and biochemical evidence of cholestasis [65]. In contrast, γ-GT serum levels were not correlated with the presence of bile duct damage in 201 patients with chronic HCV infection [66], but increased γ-GT levels were associated with more advanced liver disease. Similarly, a significant association with increased γ-GT activities and hepatic fibrosis was reported from another study but no association with serum bile acid concentrations or with histological cholestasis scores was found [67]. These studies suggest that increased γ-GT levels may reflect more advanced liver disease rather than intrahepatic cholestasis exclusively.

In a chart review of patients with chronic HCV infection, the typical symptom of cholestasis, pruritus, was observed in 5% of subjects [68]. Serum bile acids were the only laboratory parameters that were significantly increased in pruritic patients compared with controls. Most pruritic patients had advanced liver disease accompanied by bile duct abnormalities. Similarly, in a series of eight patients with severe pruritus, moderate-to-severe fibrosis was present in all patients; liver histology showed subtle features of cholestasis, whereas biochemical markers of cholestasis were not strikingly elevated [69]. In a prospective study, 20% of 119 patients with chronic HCV infection reported pruritus, but laboratory and histological parameters were similar to those without [42].

The number of HCV patients with cholestatic features described in the different studies is too small to draw general conclusions concerning the virological response to antiviral therapy. On the whole, the reduced reported rates were in the range expected for patients with advanced fibrosis. In part, it is possible that the lower response rates might be related to increased serum bile acid levels, which have been proposed as a predictive marker for failure to achieve sustained virological response to antiviral therapy [70]. In vitro studies have demonstrated that bile acids may enhance replication of HCV genotype 1 via a pathway involving the transcription factor FXR [71], upregulate HCV replicon expression [72] and reduce the antiviral effect of interferon [73]. Therefore, it has been suggested that FXR antagonists or bile acid sequestrants may be useful adjuncts to antiviral therapy in patients with increased serum bile acid levels [74].

The potential effect of bile acids on response rates to antiviral therapy is also indicated by studies involving genetic variants of hepatic bile acid transporters. A common variant (p.A444V) of the hepatocanalicular BSEP (ABCB11) is associated with significant reduction in response rates for patients infected with HCV genotypes 2 and 3 compared with wild-type carriers; a weaker association was found for genotype 1 [75]. Consistent with these findings, two other studies [76, 77] showed that this variant was associated with HCV positivity in 206 and 649 patients, respectively. These data suggest that decreased BSEP expression in patients harbouring the p.A444V variant may lead to increased intracellular bile acid levels, which in turn may enhance viral replication via FXR. Furthermore, bile acids have been shown to reduce the activity of 2′,5′-oligoadenylate synthetase, a protein involved in the antiviral activity of interferon [78]. Accordingly, there is a need to investigate whether bile acid levels and genetic variants of bile acid transporters also affect response rates in patients receiving antiviral triple therapy comprising peginterferon, ribavirin and direct antiviral agents, such as the protease inhibitors boceprevir or telaprevir.

A specific subtype of HCV infection with a progressive cholestatic course has been described in patients after liver transplantation [79-81]. This cholestatic HCV disease has also been noticed in some cases after renal, heart and bone marrow transplantation and outside the transplant setting in HCV/human immunodeficiency virus coinfection. In liver transplant recipients, recurrence of HCV in the hepatic allograft leads to cholestatic hepatitis with histological features of hepatocyte ballooning predominantly in the perivenular zone analogous to features observed in patients with fibrosing cholestatic hepatitis B. Typically, serum bilirubin levels, AP and γ-GT activities are increased, and serum HCV-RNA levels are very high. Independent predictors of cholestatic HCV disease are donor age, previous rejection and total bilirubin [82]. Without treatment, progression to liver failure and graft loss is usually seen within 1 year after transplantation. Next to reduction in immunosuppressive medication, patients should be considered for antiviral therapy. First successful treatments with regimes including the recently introduced directly antiviral agents have been reported [83, 84].

In summary, cholestatic presentation is infrequently seen in chronic HCV infection. Most patients with cholestasis display advanced disease, present with pruritus, and show bile duct injury in the setting of advanced fibrosis.

Granulomatous liver diseases

Granulomatous hepatitis is a chronic exudative and proliferative inflammatory reaction of liver tissue to antigenic stimuli that occurs in association with infections, systemic diseases or drugs. Histological features include nodular inflammatory infiltrates (granulomas) composed of macrophages, epithelioid cells, lymphocytes and fibroblasts. Of all liver biopsies, 0·7–15% contain hepatic granulomas [85], but in many cases, they are not accompanied by hepatitis, and no specific disease is detected during the course of further investigations. The presence of clinical or laboratory signs of advanced liver disease and portal hypertension is suspicious for schistosomiasis, sarcoidosis or PBC. A variable 3–37% range of patients are diagnosed with idiopathic granulomatous hepatitis associated with fever, fatigue, abdominal pain, weight loss and liver dysfunction [86].

Elevated cholestatic enzymes may be the predominant change in patients with granulomas related to sarcoidosis, PBC or drug-induced granulomas. In contrast, in granulomatous hepatitis that does not generally affect the bile ducts, such as schistosomiasis or drug-induced liver injury, the granulomas are found in the setting of abnormal aminotransferase levels or even normal liver chemistry results. Specific stains, culture biopsies and PCR for acid-fast bacilli or other pathogens, including CMV, EBV, fungi, Brucella abortus, Tropheryma whipplei or Bartonella henselae, should be performed in cases of granulomatous hepatitis of unknown origin, but the sensitivity of many tests is low [85].

Liver biopsy confirms the diagnosis of granulomatous hepatitis. In PBC and sarcoidosis, granulomas are more often observed in portal spaces and closely associated with damaged or absent septal and interlobular bile ducts (Fig. 3b). The formation of granulomas results from the complex interaction of macrophage activation and potent chemotactic mediators engaged in the recruitment, differentiation, proliferation and accumulation of monocytes and T lymphocytes into the portal tract granulomas. Biochemical or clinical manifestations of cholestasis subsequently develop as a consequence of damage to bile ducts by the granulomatous inflammatory reaction. The granulomatous injury and destruction of the bile ducts might cause ductopenia (Fig. 3b) and a histological picture similar to PBC [87]. In chronic granulomatous liver disease, nodular regenerative hyperplasia secondary to the inflammation and phlebitis of portal and hepatic veins might represent a major mechanism leading to portal hypertension [88, 89].

In summary, the degree of cholestasis in granulomatous liver diseases is variable and related clinical symptoms range from none to intractable pruritus. This is reflected by the morphological alterations causing intrahepatic cholestasis, which include periportal granulomatous infiltrates associated with damaged bile ducts, nodular regenerative hyperplasia and ductopenia.

Drug-induced cholestasis

Drug-induced cholestatic injury has been defined by an isolated elevation of serum AP > 2 × upper limit of normal (ULN) or an ALT/AP ratio (both elevated above ULN) <2 [2]. Several hundred drugs and other compounds have been reported to induce drug-induced cholestasis. For many drugs, the reported prevalence of drug-induced liver injury (DILI) is between 1 in 10 000 and 1 in 100 000 patients, and about 30% of cases with DILI are cholestatic. The outcome of drug-induced cholestasis after withdrawal of the drug is generally good and few patients who develop DILI show abnormal liver tests and persistent histological evidence of liver damage at follow-up [2]; in a recent study, mortality rates were about 8% as compared to 13% for hepatocellular DILI [90]. A long-term Spanish study reported that patients with drug-induced cholestatic or mixed DILI (18 of 194 cases, 9%) were more prone to persistent biochemical abnormalities than patients with hepatocellular injury (10 of 240 cases, 4%; P = 0·03) [91]. In a recent Swedish registry study of 712 survivors of idiosyncratic DILI with jaundice, up to 1% of all patients with DILI subsequently developed liver cirrhosis [92]. The prototype drug chlorpromazine causes cholestasis lasting > 6 months (Table 4).

Table 4. Selected drugs causing chronic intrahepatic cholestasis
  1. Modified from Desmet (1997) [101], Lilly (2012) [121], Mohi-ud-din and Lewis (2004) [122] and Stickel et al. (2005) [123].

Antibiotic agentsAmoxicillin–clavulanic acid
 Organic arsenicals
 Erythromycin and its derivatives
 Flucloxacillin
 Terbinafine
 Trimethoprim–sulphamethoxazole
Psychotropic agentsCarbamazepine
 Chlorpromazine
 Tricyclics
 Sulpiride
 Phenothiazines
 Haloperidol
MiscellaneousAjmaline
 Ibuprofen
 Cyamemazine
 Oral contraceptives
Herbals Cascara sagrada
  Chaparral

Drug-induced cholestasis is based on two major mechanisms and sites of action (Table 4): (i) the inhibition of hepatocellular transporter expression and/or function and (ii) the induction of an idiosyncratic or hypersensitive reaction at the bile ductular/cholangiocellular level with ductular/ductal cholestasis. The multifactorial, complex pathogenesis includes exposure to the drug or, more frequently, its metabolites, which cause initial DILI through direct cell stress, mitochondrial inhibition or specific immune reactions after major histocompatibility complex class II presentation of haptenised peptides to T cells [93]. DILI may lead to mitochondrial permeability transition, leading to apoptosis or necrosis of liver cells. Genetically determined variations of hepatobiliary biotransformation enzymes and transporter expression and function are risk factors for a patient's susceptibility to drug-induced cholestasis [94]. Lang et al. [95] demonstrated that the ABCB11 variant p.A444V was significantly more frequent in patients with drug-induced cholestasis (76%) compared with drug-induced hepatocellular injury (50%) and healthy controls (59%, P < 0·05) and concluded that genotyping of selected patients with acquired cholestasis might help to identify individuals with a genetic predisposition. Of note, the first genome-wide studies of DILI identified genetic risk factors for hepatotoxicity associated with flucloxacillin [96], lumiracoxib [97] and ximelagatran [98]. In the small but seminal genome-wide association study (51 patients), flucloxacillin-induced liver injury was associated with variants within the HLA complex (DRB1*5701), which are unrelated to the HLA haplotypes associated with autoimmune hepatitis, as were lumiracoxib- and ximelagatran-induced liver injury [96]. Although the risk allele confers an 80-fold increased risk of developing liver injury upon treatment with flucloxacillin, the incidence of this form of liver damage in carriers of the risk allele who are treated with flucloxacillin is only 1 : 500–1 : 1000. Hence, screening for this allele before drug treatment is not feasible, but the test might be clinically helpful in polypharmacy-associated severe DILI. However, these findings point to immune-mediated DILI, with immune reactions against specific protein adducts forming haptens in genetically susceptible individuals. A potential benefit of corticosteroid therapy in cases of immune-mediated DILI has been reported and may be particularly expected in hypersensitivity-induced cholestasis [99], but no relevant controlled trials have been conducted.

A severe, progressive and prolonged course requires liver biopsy to obtain additional information on the type of liver injury and to exclude other causes of liver cholestasis. The morphological features of chronic cholestasis include periportal cholate stasis (referring to the foamy appearance of hepatocytes), copper retention and the presence of Mallory bodies. In a recent standardised study of 35 liver biopsies from patients with DILI, portal neutrophils and intracellular (hepatocellular) cholestasis were more prevalent in drug-induced cholestasis (P < 0·02) [100]. The combination of portal inflammation with prominent portal neutrophils and plasma cells, intracellular cholestasis and fibrosis yielded an area under the curve of 0·91 in predicting drug-induced cholestasis vs. autoimmune hepatitis.

Drugs may induce chronic cholestasis with vanishing bile duct syndrome (VBDS) [101, 102]. In a review of more than 2000 cases with small duct biliary diseases seen at the Mayo Clinic, VBDS was present in 0·5% of patients [103]. The histopathological hallmark of the chronic stage in VBDS is a decrease in the number of interlobular bile ducts, termed ductopenia when they are lost in more than 50% of portal tracts [101]. The syndrome might begin with hypersensitivity syndrome including skin rash. In many cases, jaundice subsides, although sometimes as long as several years after its onset [104]. In some patients, however, progressive bile duct loss leads to secondary biliary cirrhosis, permanent jaundice and liver failure, and these patients with end-stage VBDS have to be considered for liver transplantation [101, 102]. In a sequential histological analysis of drug-induced prolonged cholestasis in adults, hepatocellular injury and cholangitis developed acutely, whereas at the chronic phase, degeneration of the ducts with periductal fibrosis and the absence of interlobular bile ducts in at least 50% of small portal tracts were detected in all patients [105]; the degree of ductopenia and the chronicity of the disease might be directly related to the severity of the early acute DILI. Consequently, in patients with severe drug-induced cholestasis, monitoring of early morphological signs of acute cholangitis and then of ductopenia seems to be critical. Understanding the pathophysiology of cholangiopathies represents the beginning of the development of new therapeutic concepts [106]. Because the dogma of irreversibility of cirrhosis has been eroded recently, new ways of modulating the plasticity of the biliary epithelium might also help to reverse the changes seen in VBDS in the future.

Genetic variants contributing to intrahepatic cholestasis

Hereditary defects of the hepatobiliary transporters ATP8B1, ABCB11 and ABCB4 represent ‘primary’, that is, monogenic hereditary forms of cholestasis. Dependent on the underlying gene variant of these transporters, disease severity ranges from severe cholestatic liver disease in early childhood to mildly elevated cholestatic enzymes in adults [107]. Of note, γ-GT is normal in patients suffering from ATP8B1 and ABCB11 but not ABCB4 mutations [108]. ‘Mild’ heterozygous variants of these transporters, even without an own clinical phenotype, can predispose to cholestasis in combination with exogenous factors such as chronic alcohol consumption or viral hepatitis.

In particular, impaired bile formation is a typical feature of intrahepatic cholestasis of pregnancy [109]. Here, pregnancy might uncover a formerly unknown chronic liver disease by the cholestatic presentation, which is potentially triggered by hormonal load, metabolic stress or the inflammatory response to acute infections, all of which interact with the gene variants to decrease the expression and/or function of hepatobiliary transporters [110]. Whether variants of these transporters are also mediators of a secondary cholestatic course in chronic liver diseases in general is an interesting field for future research.

Other genetic liver diseases such as Wilson disease can also manifest with profound cholestasis and jaundice, typically in the setting of (sub)acute liver failure [111]; hence, serum bilirubin levels are included in a prognostic index for survival in acute Wilson disease [112]. Interestingly, the clinical presentation of this disease is highly variable and ranges from mildly elevated liver enzymes, chronic hepatitis with fibrosis to liver cirrhosis [113].

Outlook

This review illustrates that intrahepatic cholestasis represents a clinical subphenotype of many chronic liver diseases and not only the hallmark of the primary cholestatic liver diseases PBC and primary sclerosing cholangitis (PSC). To discriminate the different types of cholestasis, we have proposed the term ‘SIC’ for cholestasis caused by chronic, originally noncholestatic liver diseases (Fig. 1). Indeed, SIC correlates with disease severity in alcoholic liver diseases and viral hepatitis but can also occur in a subgroup of NAFLD and DILI patients. In the past, very few randomised controlled studies in cholestatic patients with liver disease have been performed [114, 115]. Ursodeoxycholic acid (UDCA) had no beneficial effect on survival in jaundiced patients with alcoholic liver cirrhosis [116]. Only recently, clinical trials with new promising agents have been initiated in patients with primary cholestatic liver disease. Obeticholic acid, an agonist of the nuclear receptor FXR, is tested in a phase III trial in patients with PBC, and norUDCA, a side chain–modified derivate of UDCA, is under investigation in a phase II study in patients with PSC. These observations might open new avenues for therapeutic interventions that reverse SIC, with the aim not only to relieve the clinical symptoms but to modulate disease progression. Additional molecular studies are needed to gain more knowledge about the intracellular effects of SIC such as signalling effects, mitochondrial damage or effects on hepatobiliary transporter expression. In fact, the recent identification of molecular mechanisms and genetic risk factors predisposing to (drug-induced) cholestasis might guide the design of studies for patients at highest risk of SIC.

Address

Department of Medicine II, Saarland University Medical Center, 66421 Homburg, Germany (C. Jüngst, F. Lammert); Clinic of Gastroenterology and Rheumatology, Division of Hepatology, Leipzig University Hospital, 04103 Leipzig, Germany (T. Berg); Liver Center of Beijing Ditan Hospital, Capital Medical University, Beijing 100015, China (J. Cheng); Division of Hepatology, Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA (R. M. Green); Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China (J. Jia); Center of Excellence in Gastrointestinal Inflammation and Immunity Research, University of Alberta, Edmonton, Alberta T6G 2X8, Canada (A. L. Mason).

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