Elsevier

Journal of Hepatology

Volume 34, Issue 2, February 2001, Pages 248-253
Journal of Hepatology

Hepatocyte apoptosis is a pathologic feature of human alcoholic hepatitis

https://doi.org/10.1016/S0168-8278(00)00089-1Get rights and content

Abstract

Background/Aims: The pathogenesis of alcoholic hepatitis (AH) remains poorly understood. Although apoptosis is now recognized as a mechanism of liver injury, the extent and mechanisms of apoptosis in human AH remain unknown. Thus, our aims were to quantify hepatocyte apoptosis in patients with AH, correlate it with disease severity, and identify the mechanisms of apoptosis induction.

Methods: Hepatocyte apoptosis was assessed in 26 patients with AH and 27 controls without liver disease using the TUNEL assay and immunohistochemistry for activated caspase 3. Liver specimens were also graded for disease severity. The expression of the death receptors, Fas and tumor necrosis factor-α receptor 1 (TNF-R1), was assessed by immunohistochemistry.

Results: In contrast to normal livers, TUNEL- and caspase 3-positive hepatocytes were readily observed in the livers of patients with AH. In the AH group, hepatocyte apoptosis was significantly higher in patients with a serum bilirubin of >3 mg/dl. Apoptosis was also greater in grade 4 steatohepatitis. The Fas receptor was strongly expressed in hepatocytes in AH, but not in normal livers; the TNF-R1 expression was comparable in both groups.

Conclusions: The present results demonstrate that hepatocyte apoptosis is significantly increased in human AH and justify therapeutic strategies aimed at inhibiting apoptosis in this disease.

Introduction

Alcoholic hepatitis (AH) is a toxic liver disease resulting from excessive alcohol use. Although the clinical spectrum of the disease includes both non-icteric and icteric presentations, an estimated mortality of more than 50% occurs in severe forms of icteric AH [1]. Moreover, AH, even as a mild, non-icteric disease, predisposes to the development of cirrhosis [2]. Due to the continued use of alcohol in most cultures, AH remains a significant public health problem. In alcoholics with liver disease, the incidence of uncomplicated AH has been estimated to be up to 33% in the US. However, the true incidence might be higher, since the diagnosis requires biopsy confirmation and is therefore often missed, especially in milder forms of AH [3]. Treatment options for AH remain limited, in part, because the pathophysiology of AH remains poorly understood. Insight into the mechanisms of disease could therefore lead to new therapeutic approaches to prevent the short- and long-term sequelae of AH.

Apoptosis is a form of cell death characterized by stereotypic morphological and biochemical features [4]. Apoptosis is now recognized as one of the initiating events in toxic liver injury, and is increasingly recognized as a key mechanism in tissue inflammation and fibrosis. For example, in kidney and lung tissues, the inhibition of apoptosis abrogates the inflammatory response following ischemia/reperfusion injury and prevents drug-induced fibrosis, respectively [5], [6]. In experimental models of liver injury, hepatocyte apoptosis is associated with an increase in hepatocyte derived enzymes, a clinical indicator of liver injury [7]. If the magnitude of liver cell apoptosis is sufficient to overwhelm the removal process, a loss of tissue architecture and organ failure can occur [8], [9]. Morphological evidence of apoptosis has been identified in such diverse clinical liver diseases as viral hepatitis, cholestasis, and copper overload states [10]. Thus, apoptosis appears to be a key event in many pathophysiological processes affecting the liver, and the inhibition of apoptosis holds promise as a therapeutic strategy for human liver diseases.

Recently, it has been suggested that hepatocyte apoptosis is also a feature of alcohol-induced liver disease [11]. Chronic alcohol administration in mice results in a significant time-dependent increase of apoptotic bodies in hepatocytes, especially around terminal hepatic venules. Similar to human AH, these changes were potentially reversible after the discontinuation of alcohol [12]. Although these experimental data demonstrate that apoptosis plays role in alcoholic liver injury, only one study suggests that apoptosis occurs in AH [13]. However, this study was limited in the number of patients examined (n=5), did not quantitate the magnitude of apoptosis, nor was apoptosis correlated with disease severity. Moreover, the studies used the TUNEL assay to identify apoptosis (an assay based on DNA damage) and did not determine if caspase activation occurred (an essential feature of apoptosis). Thus, the purpose of the present study was to quantitate hepatocyte apoptosis in patients with biopsy-proven AH, to determine if caspase activation occurs and to correlate the rates of apoptosis with the disease severity. In addition, we determined the expression of the Fas receptor and tumor necrosis factor-α receptor 1 (TNF-R1) because these death receptors have been associated with hepatocyte apoptosis in other diseases.

Section snippets

Patient groups

This is a retrospective study approved by the Mayo Clinic Institutional Review Board, employing archived liver biopsy specimens which were fixed and embedded in paraffin by standard procedures. All patients had undergone a liver biopsy to confirm the clinical diagnosis of AH. Liver biopsy material was obtained from 26 patients with AH. Histologically normal liver specimens were obtained from the 27 patients who underwent hepatic resection for colorectal metastases. The diagnosis of AH was based

Is hepatocyte apoptosis increased in AH?

To address this question, hepatocyte apoptosis was assessed in patients with AH and in controls using two different techniques. First, TUNEL-staining was used to identify apoptosis in liver specimens. In normal livers, only rare TUNEL-positive cells were identified. In contrast, TUNEL-positive cells were readily observed in the tissue of patients with AH (Fig. 1a). The quantitation of these TUNEL-positive cells showed a six-fold increase in the AH patient group (2.0±0.31 apoptotic cells/×400

Discussion

In the present study, we have demonstrated that hepatocyte apoptosis is a prominent feature of AH and is significantly increased in patients with this disease. Moreover, the number of apoptotic cells appears to correlate with the severity of AH, as hepatocyte apoptosis was most pronounced in patients with high bilirubin- and AST-levels, and grade 4 steatohepatitis. Collectively, these data suggest that apoptosis plays an important role in the pathophysiology of AH. In addition, Fas, but not

Acknowledgements

This work was supported by grants from the National Institutes of Health (DK41876, GJ Gores), the Deutsche Forschungsgemeinschaft (Ru742/1-1, C Rust), the Gainey Foundation, St. Paul, MN, and by the Mayo Foundation, Rochester, MN.

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