Differential Effects of Trovafloxacin on TNF-α and IL-6 Profiles in a Rat Hepatocyte–Kupffer Cell Coculture System

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 hepatocytes and Kupffer cells (KCs) that can be used to examine chemical-induced inflammatory reactions resulting 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 hepatotoxicity in vivo. Cytokine (IL-6 and TNFα) levels, CYP3A activity, and cell viability (ATP, LDH release) were measured 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-α 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-α production was initially upregulated in TVX/LPS-treated cocultures and KC monocultures before showing a gradual decline. The data from these studies suggest that shifts in cytokine profiles, especially IL-6/TNF-α ratios, may play a role in immune-mediated chemical-induced hepatotoxicity. 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.