Differential Effects of Trovafloxacin on TNF-a and IL-6 Profiles in a Rat Hepatocyte–Kupffer Cell Coculture System Jessica A. Bonzo, 1 Kelly Rose, 2 Kimberly Freeman, 3 Erica Deibert, 3 Kirsten B. Amaral, 3 Stephen S. Ferguson, 3 Melvin E. Andersen, 2 Rafal P. Witek, 1 and Edward L. LeCluyse 2 Abstract Immune-mediated chemical-induced hepatotoxicity is often overlooked as a potential mode of action due to the lack of appropriate in vitro models. For this reason, we have established a coculture system of rat primary he- patocytes and Kupffer cells (KCs) that can be used to examine chemical-induced inflammatory reactions result- ing in acute hepatocellular toxicity. Cocultures were maintained for 24 or 48 hr with various concentrations of lipopolysaccharide (LPS) and trovafloxacin (TVX), a compound associated with immune-mediated hepatotox- icity in vivo. Cytokine (IL-6 and TNFa) levels, CYP3A activity, and cell viability (ATP, LDH release) were mea- sured to monitor endotoxin- and TVX-induced changes in metabolic capacity and cell integrity. LPS-treated cocultures showed marked downregulation of CYP3A activity, which correlated with upregulation of IL-6 and TNF-a production. TVX/LPS-treated cocultures exhibited concentration-dependent hepatocellular injury, represented by decreased ATP content and increased LDH leakage. The IL-6-induced downregulation of CYP3A was impaired in cocultures cotreated with TVX/LPS, which coincided with a concentration-dependent decline in IL-6 production. However, TNF-a production was initially upregulated in TVX/LPS-treated cocul- tures and KC monocultures before showing a gradual decline. The data from these studies suggest that shifts in cytokine profiles, especially IL-6/TNF-a ratios, may play a role in immune-mediated chemical-induced hep- atotoxicity. Coculture of hepatocytes and KC represents a valuable in vitro tool to assess adverse liver effects resulting from indirect adaptive immune responses during chemical exposure. Key words: coculture, hepatocytes, in vitro model systems, Kupffer cells, trovafloxacin. Introduction H epatotoxicity continues to be one of the main ad- verse events observed in humans during clinical trials, after environmental exposure to chemicals, or due to interac- tions with dietary supplements. 1–3 In this regard, the mode of action for many types of chemical- or drug-induced hepato- toxic responses often involves multiple cell types and pertur- bation of pathways involving both the parenchymal cells (i.e., hepatocytes) and resident immune cells [i.e., Kupffer cells (KCs)]. 1,4–6 Immune-mediated responses that are asso- ciated with reactive metabolites or therapeutic proteins, or that occur upon exposure to gut endotoxins require interac- tions between hepatocytes, endothelial cells, and KCs. 1,4,6,7 KCs are the largest population of resident macrophages in the liver and thus play a critical role in immune-mediated hepatotoxicity and liver injury. 8 They play a very important role in immune surveillance of the host and are involved in modulating systemic responses to severe infections and con- trolling concomitant immune responses via antigen presenta- tion and suppression of the activation and proliferation of T- cells. 9 In their primary scavenger role, KCs endocytose for- eign particles and bacterial endotoxins, which causes their activation and subsequent production of a number of modu- lators of cell signaling pathways, such as oxygen-derived free radicals, nitric oxide, eicosanoids, peptide leukotrienes, prostaglandins, and various cytokines, including TNF-a, TGF-b, IL-1, and IL-6. 9 During drug- or chemical-induced liver injury, injurious stimuli and stress signals also can cause an activation of KCs, and a release of a plethora of ag- gressive mediators, which are involved in modulating cell death by apoptosis of hepatocytes and other cell types. 8–10 1 Cell Biology and 3 Primary & Stem Cell Systems, Thermo Fisher Scientific (Life Technologies), Frederick, Maryland. 2 The Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences, Research Triangle Park, North Carolina. APPLIED IN VITRO TOXICOLOGY Volume 1, Number 1, 2015 ª Mary Ann Liebert, Inc. DOI: 10.1089/aivt.2014.0004 45