Th1/Th2 cytokine balance as a determinant of acetaminophen-induced liver injury

Abstract

Inflammation is an important pathophysiological event in drug-induced liver injury, which is subsequent to metabolic activation and covalent binding of the reactive metabolites to target proteins. Cytokines are recognized as pro- and anti-inflammatory mediators involved in the progression and regression of the toxicity. We thus hypothesized that disturbed balance of Th1/Th2 cytokines exacerbated the drug-induced hepatotoxicity. Acetaminophen-induced liver injury was investigated in two mouse strains, C57BL/6 and BALB/c, which develop predominantly Th1 and Th2 responses, respectively. More severe liver injury after intraperitoneal administration of acetaminophen was observed in C57BL/6 mice than in BALB/c mice. There was no strain difference in metabolism of acetaminophen into its reactive metabolite, N-acetyl-p-benzoquinone imine, which was assessed by early glutathione consumption. Liver mRNA expression of tumor necrosis factor-α (TNF-α) and IL-6 were measured as pro- and anti-inflammatory cytokines, respectively. TNF-α was highly induced 24 h after administration of acetaminophen in C57BL/6 mice, whereas no change in BALB/c mice. On the other hand, liver IL-6 mRNA expression in BALB/c mice was higher than C57BL/6 mice 24 h after the administration. In addition, treatment of CD-1 mice, another susceptible strain, with an anti-inflammatory polyphenol, resveratrol, protected mice against the acetaminophen-induced liver injury, and the mice with attenuated toxicity revealed lower expression of TNF-α and higher expression of IL-6. It is therefore suggested that acetaminophen-induced liver injury is associated with Th1-dominant response in Th1/Th2 cytokine balance, and TNF-α may play a pathological role in the toxicity.

Introduction

Acetaminophen is a commonly used analgesic that is usually safe in therapeutic doses, but an overdose of the drug causes liver injury in experimental animals and humans. The toxicity is initiated by cytochrome P450 metabolism to N-acetyl-p-benzoquinone imine (NAPQI) [1], [2]. The high reactivity of NAPQI with sulfhydryl groups results in depletion of glutathione in hepatocytes, followed by covalent binding to intracellular proteins [3], [4]. Although it has been shown that the relative amount of covalent binding correlated with the development of the toxicity [3], it is also suggested that the covalent binding is not sufficient for the toxicity [5], [6], [7], [8].

Factors other than the covalent binding of NAPQI to critical proteins should contribute to determining the severity of the toxicity. A simple and reasonable theory is classification of stage I and stage II in the acetaminophen-induced liver injury [9]. Stage I includes metabolic activation, covalent binding and mitochondrial dysfunction. On the other hand, stage II represents subsequent processes of adaptation or failure of response to modification of essential cellular processes [9], which may be partially mediated by liver nonparenchymal cells, Kupffer cells and/or infiltrated macrophages [10], [11], [12]. Hepatocytes initially injured by acetaminophen would release factors that chemoattract and subsequently activate these liver nonparenchymal cells, which might contribute to progressing hepatocellular damage by releasing cytotoxic mediators [10], [11], [12], while more recent studies have argued that the nonparenchymal cells may not play an essential role or may play a protective role in acetaminophen-induced hepatotoxicity [13], [14].

A mediator(s) responsible for the progression of acetaminophen-induced liver injury has not been identified, while recent evidence suggests that cytokines are possible candidates that determine severity of the liver toxicity. The major focus has been on tumor necrosis factor-α (TNF-α), which mediates acute-phase response of hepatocytes during inflammatory reactions. The role of TNF-α in pathogenesis of acetaminophen-induced liver injury is controversial. Namely, TNF-α has been reported to be involved in pathogenesis of the acetaminophen-induced liver injury [15], [16], while other studies suggested that TNF-α was not involved in the toxicity [17], [18], or TNF-α may participate in defense systems against the toxicity and/or tissue repair systems [19], [20]. The discrepancy suggests that time and extent of exposure of livers to TNF-α and related mediators, which vary in individual experiments, determine whether TNF-α aggravates or alleviates the liver toxicity induced by acetaminophen. It thus may be a typical example of pleiotropic effects of a cytokine on tissue inflammation. There are many findings that cytokines other than TNF-α, e.g., IL-1α[15] and INF-γ[21] play pathogenic role in acetaminophen-induced liver injury, while other cytokines, such as IL-6 [22] and IL-10 [23] play protective role in the liver injury, which can also be influenced by individual experimental conditions.

Cytokines are categorized into Th1- and Th2-type cytokines, which are secreted from different helper T cell populations [24]. Although it is difficult to explain the above-described stage II toxicity by a few particular cytokines, it seems that Th1 cytokines behave as proinflammatory mediators involved in the pathogenesis of the liver toxicity, which has been demonstrated in immune-mediated liver injury induced by LPS [25] and concanavalin A [26]. Although it remains unclear whether T cells themselves are responsible for the acetaminophen hepatotoxicity, severity of the hepatotoxicity can be associated with balance of Th1/Th2 cytokines probably secreted from hepatocytes and/or nonparenchymal cells, and therefore, acetaminophen-induced liver disease can be regarded as disturbance of Th1/Th2 cytokine balance into Th1-dominant state.

The purpose of the present study is to investigate whether Th1/Th2 cytokine balance is involved in the progression or suppression of pathogenesis of acetaminophen-induced liver injury. First, susceptibility to acetaminophen-induced liver injury was compared between two mouse strains, C57BL/6 and BALB/c, which developed predominantly Th1 and Th2 responses, respectively [25], [26]. Second, mice were treated with a polyphenol, resveratrol, which has anti-inflammatory property [27] and may modulate Th1/Th2 cytokine balance, and its effects on acetaminophen-induced liver injury, and change in cytokine expression were tested. The results thus obtained provide the theory that acetaminophen-induced liver injury is associated with Th1-dominant response in Th1/Th2 balance.