The role of damage associated molecular pattern molecules in acetaminophen-induced liver injury in mice
Introduction
Drug-induced liver injury (DILI) is the leading cause of acute liver failure in the United States, contributing to approximately half of all cases (Gunawan and Kaplowitz, 2007, Ostapowicz et al., 2002). In addition, DILI represents the primary reason for drug termination during clinical development as well as withdrawal of FDA-approved drugs from the market, with both major medical and economical consequences (Kaplowitz, 2005, Lee and Senior, 2005). The current detection of DILI remains difficult during the early stages of drug development due to the lack of screening methods, the relatively low incidence of these reactions and the limited knowledge regarding the underlying mechanisms (Lee, 2003). Therefore, a better understanding of the molecular and cellular mechanisms of DILI is imperative in order to identify susceptibility factors and develop successful strategies in the treatment and prevention of DILI.
Acetaminophen (APAP) is a widely used analgesic and antipyretic known to be effective and safe when consumed at therapeutic doses (1–4 g/day) (Kaplowitz, 2001, Rumack, 2004). However, severe liver injury resulting in liver failure can occur in some cases following an acute or cumulative overdose (10–15 g) (Kaplowitz, 2001). APAP overdose accounts for more than 56,000 emergency room visits, 2600 hospitalizations and an estimated 458 deaths due to acute liver failure each year within the United States (Lee, 2004). Overdose of APAP is also known to cause liver injury in laboratory animals with similar characteristics as those found in patients. The murine model of APAP-induced hepatotoxicity represents the most widely used model to study the pathogenesis of DILI. The initiation of APAP-induced liver injury (AILI) results from the metabolism of APAP into a reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI) (Nelson, 1990, Raucy et al., 1989). Ample evidence supports that depletion of hepatic glutathione (GSH) and the covalent binding of NAPQI to cellular macromolecules contributes to protein modification and mitochondrial dysfunction with ATP depletion, culminating in massive centrilobular necrosis (Kaplowitz, 2004, Lee et al., 1996, Pumford et al., 1997).
In addition to direct hepatic cellular dysfunction and death, the pathogenesis of APAP-induced hepatotoxicity also involves the release of a variety of inflammatory mediators that may influence individual susceptibility. It has been demonstrated that macrophage migration inhibitory factor (MIF) (Bourdi et al., 2002b), interferon (IFN)-γ (Ishida et al., 2002), and tumor necrosis factor (TNF)-α (Blazka et al., 1996) contribute to the severity of AILI, while interlukin (IL)-6 (Masubuchi et al., 2003), cyclooxygenase (COX)-2 (Reilly et al., 2001) and IL-10 (Bourdi et al., 2002a) have been shown to promote resolution and regeneration following the initial hepatic damage. Recent studies have also reported the activation and involvement of various innate immune cells, including natural killer (NK) cells and NK cells with T cell receptor (NKT) (Liu et al., 2004, Masson et al., 2008), hepatic macrophages (Ju et al., 2002, Laskin and Laskin, 2001, Michael et al., 1999), and neutrophils (Cover et al., 2006, Ishida et al., 2006, Liu et al., 2006) during APAP-induced hepatotoxicity. However, the exact role of these cells in the pathogenesis of injury remains controversial. Importantly, it remains unknown as to the specific factors that trigger the recruitment and activation of these cells within the liver.
It is possible that damaged hepatocytes themselves can trigger inflammation through the release of various mediators. One hypothesis proposes the critical role of damage associated molecular pattern (DAMP) molecules in this process. Numerous studies have demonstrated that apoptotic and necrotic cells release DAMP molecules such as high mobility group box-1 (HMGB1), S-100 proteins, heat-shock proteins (HSPs), hyaluronan, surfactant protein, interferon-alpha, uric acid, fibronectin, beta defensin, and cardiolipin, which act as endogenous stimulators of immune responses following tissue damage (Asea et al., 2002, Biragyn et al., 2002, Lotze and Tracey, 2005, Okamura et al., 2001, Peitsch et al., 1988, Seong and Matzinger, 2004, Shi et al., 2003, Termeer et al., 2002, Wallin et al., 2002, Wang et al., 1999). The function of HMGB1 and HSP-70 as pro-inflammatory mediators has been extensively studied in recent years. HMGB1 is a nuclear binding protein that can be passively released from necrotic cells or secreted by activated macrophages (Shi et al., 2003). HMGB1 has been demonstrated to function as both a chemokine and cytokine, thereby playing a critical role in the initiation of inflammation (Lotze and Tracey, 2005, Wang et al., 1999). HSP-70 is an inducible stress-response protein that can undergo translocation to the cell surface or release into the extra-cellular milieu during periods of cellular stress or necrotic death (Wallin et al., 2002). It has been revealed that extra-cellular HSP-70 can stimulate dendritic cells and macrophages to promote immune responses and inflammation (Asea et al., 2002, Hickman-Miller and Hildebrand, 2004, Vabulas et al., 2002). However, the release of HMGB1 and HSP-70 by damaged hepatocytes and their specific role in the pathogenesis of DILI have not been investigated.
The objectives of the present study were to examine: (i) whether the DAMP molecules HMGB1 and HSP-70 are released from damaged hepatocytes following APAP-challenge in vivo and in vitro, and (ii) whether these molecules can trigger the activation of hepatic macrophages, Kupffer cells (KC). Our results provide evidence that APAP-challenged hepatocytes do release HMGB1 and HSP-70. In addition, recombinant HSP-70 (rHSP-70) can activate isolated primary liver non-parenchymal cells (NPC), as well as purified KC, to produce pro-inflammatory mediators.
Section snippets
Cell lines and culture conditions
RAW 264.7, a murine macrophage cell line [obtained from American Type Culture Collection (Manassas, VA)] was maintained in RPMI 1640 media supplemented with 10% fetal bovine serum, 10 mM HEPES and 1× penicillin/streptomycin. TAMH, a hepatocyte cell line [provided by Christopher C. Franklin, Department of Pharmaceutical Sciences, University of Colorado Denver (UCD), CO, USA] was maintained in Dulbecco's modified Eagle's medium/Hams's F12 (Gibco) supplemented with 5 μg/mL insulin, 5 μg/mL
Macrophage activation following APAP treatment
To investigate whether APAP treatment can activate hepatic NPC, these cells were isolated from the liver of mice at 1 h post-APAP-challenge. Compared with cells isolated from saline-treated control mice, liver NPC obtained from APAP-challenged mice exhibited a significant increase in the mRNA expression of pro-inflammatory cytokines, including TNF-α, IL-6 and IL-1β (Fig. 1). Hepatic NPC consist of both KC and LSEC. To identify which cell population represents the predominant source of these
Discussion
APAP overdose represents a major cause of acute liver failure in the United States each year. While hepatocyte damage is initiated by the formation of NAPQI, studies using the murine model of AILI have suggested a role of inflammatory responses in the progression of hepatic injury (Blazka et al., 1995, Blazka et al., 1996, Bourdi et al., 2002a, Bourdi et al., 2002a, Ishida et al., 2002, Laskin and Laskin, 2001, Liu et al., 2004, Liu et al., 2006, Masubuchi et al., 2003, Michael et al., 1999).
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgements
This work was supported by U.S. National Institutes of Health grant Ruth L. Kirschstein National Service Award F31DK082269 (to Brittany Martin-Murphy) and R01ES012914 (to Cynthia Ju).
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