Regulation of alternative macrophage activation in the liver following acetaminophen intoxication by stem cell-derived tyrosine kinase

Abstract

Stem cell-derived tyrosine kinase (STK) is a transmembrane receptor reported to play a role in macrophage switching from a classically activated/proinflammatory phenotype to an alternatively activated/wound repair phenotype. In the present studies, STK^−/− mice were used to assess the role of STK in acetaminophen-induced hepatotoxicity as evidence suggests that the pathogenic process involves both of these macrophage subpopulations. In wild type mice, centrilobular hepatic necrosis and increases in serum transaminase levels were observed within 6 h of acetaminophen administration (300 mg/kg, i.p.). Loss of STK resulted in a significant increase in sensitivity of mice to the hepatotoxic effects of acetaminophen and increased mortality, effects independent of its metabolism. This was associated with reduced levels of hepatic glutathione, rapid upregulation of inducible nitric oxide synthase, and prolonged induction of heme oxygenase-1, suggesting excessive oxidative stress in STK^−/− mice. F4/80, a marker of mature macrophages, was highly expressed on subpopulations of Kupffer cells in livers of wild type, but not STK^−/− mice. Whereas F4/80⁺ macrophages rapidly declined in the livers of wild type mice following acetaminophen intoxication, they increased in STK^−/− mice. In wild type mice hepatic expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-12, products of classically activated macrophages, increased after acetaminophen administration. Monocyte chemotactic protein-1 (MCP-1) and its receptor, CCR2, as well as IL-10, mediators involved in recruiting and activating anti-inflammatory/wound repair macrophages, also increased in wild type mice after acetaminophen. Loss of STK blunted the effects of acetaminophen on expression of TNFα, IL-1β, IL-12, MCP-1 and CCR2, while expression of IL-10 increased. Hepatic expression of CX3CL1, and its receptor, CX3CR1 also increased in STK^−/− mice treated with acetaminophen. These data demonstrate that STK plays a role in regulating macrophage recruitment and activation in the liver following acetaminophen administration, and in hepatotoxicity.

Introduction

Acetaminophen is a commonly used over the counter analgesic and the major cause of acute liver failure following accidental and intentional overdose (Bower et al., 2007). Acute intoxication results in centrilobular hepatic necrosis due to covalent binding of the reactive acetaminophen metabolite, N-acetyl-p-benzoquinone-imine (NAPQI), generated via cytochrome P450 (Cyp) mediated metabolism, to critical cellular targets in these regions of the liver (Cohen and Khairallah, 1997). Evidence suggests that activated macrophages contribute to the pathogenic response to acetaminophen (reviewed in Laskin, 2009). However, the precise role of these cells in hepatotoxicity depends on the timing of their appearance in the liver and the mediators they encounter in the tissue microenvironment which dictate their phenotype and functional activities. Thus, following acetaminophen intoxication, macrophages are initially classically activated and release proinflammatory/cytotoxic mediators which promote tissue injury; subsequently, as toxicity progresses, macrophages become alternatively activated and release mediators which downregulate inflammation and initiate wound repair. The mechanisms regulating the switch of liver macrophages from a pro- to an anti-inflammatory/wound repair phenotype have not been established.

Stem cell-derived tyrosine kinase (STK) is a 180 kDa transmembrane receptor present on mouse macrophages which has been reported to negatively regulate inflammatory responses (Correll et al., 1997, Laskin et al., 2010a, Morrison et al., 2004, Stuart et al., 2011, Zhou et al., 2002). Ligand binding to STK initiates cellular signaling leading to inhibition of ΙκB kinase and decreased expression of proinflammatory mediators (Chen et al., 1998, Leonis et al., 2002, Ray et al., 2010, Stuart et al., 2011, Wilson et al., 2008). Previous studies have shown that targeted disruption of the STK gene results in increased sensitivity of mice to endotoxin-induced liver injury, a process thought to be due to an inability of hepatic macrophages to become alternatively activated (Correll et al., 1997). This is supported by findings that upregulation of macrophage STK results in suppression of classical activation and induction of alternative activation in these cells (Ray et al., 2010). In the present studies, we used STK^−/− mice to evaluate its role in liver macrophage activation and acetaminophen-induced hepatotoxicity. Our findings that STK^−/− mice are hypersensitive to acetaminophen and that this correlates with aberrant alternative macrophage activation are consistent with a role of STK in regulating the phenotype and function of macrophages in the liver during the pathogenesis of hepatotoxicity.