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Alterations in cytokine/chemokine expression during organ-to-organ communication established via acetaminophen-induced toxicity

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Abstract

A variety of studies have demonstrated that organ-to-organ communication circuits are established during various disease states. For example, an activated liver may release high levels of cytokines, which are carried to the lung and activate this organ. In the present study, we have examined the inflammation occurring as the liver-lung interact during the initiation of acetaminophen-induced toxicity. An overnight fast followed by an intraperitoneal acetaminophen challenge was required to elicit liver injury. In these animals, lung injury was most pronounced at 24 h post-challenge and was characterized by necrosis, edema and inflammation. Interestingly, the non-fasted/fed animals that received acetaminophen had only minor liver injury, but still presented with significant pathologic changes of the lung. BAL fluid contained increased neutrophils after acetaminophen challenge in the fasted (26%) and the fed (35%) animal groups. A significant vascular leak was found in the fasted, but not the fed, acetaminophen challenged animals. However, lung levels of the chemokine, eotaxin, and the cytokine, IL-12, were significantly elevated in the acetaminophen challenged animals that were fed, but not in the fasted group. The immunoneutralization of eotaxin, but not IL-12 or TNF-α, improved the histological appearance of the lung in fed mice challenged with acetaminophen.

Introduction

Accidental or intentional acetaminophen (APAP) overdose is the most common cause of drug-induced liver injury in developed countries and remains a leading cause of acute hepatic failure. Despite advances in supportive care, the mortality rate of APAP-induced acute hepatic failure still reaches 60%. A liver transplant can ensure patient survival in these cases, but the prognosis of hepatic failure is determined by the injury of secondary organs (Gill and Sterling, 2001). The toxic response of APAP is initiated by the metabolite, N-acetyl-p-benzoquinone imine (Dahlin et al., 1984), which depletes glutathione in hepatocytes (Mitchell et al., 1973), reacts with intracellular proteins (Cohen and Khairallah, 1997), and finally leads to cell damage or cell death. After acute toxic hepatic injury, several cytokines and chemokines generated by injured hepatocytes and inflammatory cells are involved in the inflammatory process and contribute to the destructive and regenerative processes Blazka et al., 1995, Hogaboam et al., 1999.

The liver and the lungs have been identified as organs that can affect each other in various disease states via organ-organ communication (Matuschak, 1994). For example, in the context of sepsis, the gut, liver, and lung circuit is anatomically positioned, such that loss of gut integrity leads to the escape of polymicrobial organisms and products that impact upon the liver. This may serve as a trigger for the dysregulated release of cytokines from both Kupffer cells and hepatocytes. The combination of gut- and liver-derived mediators in turn activates the pulmonary microvasculature-alveolar-capillary wall, resulting in acute lung injury. During the evolution of these responses, a balance of inflammatory and compensatory mediators are expressed in an effort to restore homeostasis. This liver-lung-interaction is mostly pronounced in the systemic inflammatory response syndrome (SIRS), but is likely to act also in toxic liver injury. In fact, acute lung injury in fulminant hepatic failure due to APAP overdose is a common clinical finding, and the degree of lung injury appears to be dependent upon the course of hepatic injury Baudouin et al., 1995, Khanlou et al., 1999. However, the known cytotoxicity of APAP to type II pneumocytes and alveolar macrophages in vitro does not explain this in vivo finding (Dimova et al., 2000).

Although complex cytokine networks, acting in toxic liver injury have been partly elucidated, the pathogenesis of lung injury in this regard remains unclear. Thus, the aim of the present project was to investigate the inflammatory liver-lung interactions in the context of toxic liver injury and to elucidate the cytokines and chemokines that may contribute to organ-to-organ communication. We hypothesized that different degrees of liver inflammation seen after APAP challenge in fasted and non-fasted/fed animals could directly impact the severity of lung inflammation. While prior fasting results in glutathione depletion and subsequent severe liver and lung injury, non-fasted/fed animals show only mild liver damage after challenge with the same dose of APAP. In the present study, we demonstrate that the mild liver damage in non-fasted/fed animals was accompanied by remarkable lung injury. The analysis of protein levels of cytokines and chemokines suggested a role for specific chemokines in this latter model.

Section snippets

Mice

Female C57BL6 mice (age 8–11 weeks) were purchased from Charles River Laboratories (Portage, MI, USA) and maintained under specific pathogen-free conditions in the University Laboratory Animal Medicine facility (University of Michigan Medical School). Prior approval for mouse usage was obtained from University Laboratory Animal Medicine.

Acetaminophen model—experimental protocols

In all experiments, mice were allowed free access to water and food or to only water for 16 to 18 hours prior to an intraperitoneal (i.p.) injection of 300

Non-fasted/fed mice do not show liver injury following APAP challenge

The intraperitoneal injection of 300 mg/kg APAP in fasted and glutathione depleted mice resulted in highly increased serum ALT levels at both 8 and 24 h post APAP challenge (Fig. 1). In contrast the non-fasted/fed APAP group showed no significant increase in circulating ALT levels compared to the control mice. The histological appearance of liver tissues confirmed these results (Fig. 2 ), as the fasted APAP group clearly showed centrilobular necrosis, inflammation and hemorrhagic injury.

Discussion

This study is one of the first to investigate the impact of toxic liver injury on lung pathology. We observed that severe liver injury after APAP challenge in fasted mice revealed inflammatory changes and structural damage of the lung resulting in microvasculature-alveolar-capillary leakage. However, mild liver damage in non-fasted/fed animals could still elaborate mediators that impact the lung and precipitate remarkable injury. Interestingly, the immunoneutralization of eotaxin, but not IL-12

Acknowledgements

This work was supported in part by a grant from the Swiss National Science Foundation and National Institute of Health Grants HL31237 and P50 HL60289.

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