Tumor necrosis factor alpha is a proximal mediator of synergistic hepatotoxicity from trovafloxacin/lipopolysaccharide coexposure

Abstract

The use of trovafloxacin (TVX), a fluoroquinolone antibiotic, was severely restricted because of an association of TVX therapy with idiosyncratic hepatotoxicity in patients. The mechanisms underlying idiosyncratic toxicity are unknown; however, one hypothesis is that an inflammatory stress can render an individual sensitive to the drug. Previously, we reported that treatment of mice with TVX and lipopolysaccharide (LPS) induced tumor necrosis factor (TNF) alpha-dependent liver injury, whereas TVX or LPS treatment alone was nontoxic. The goal of this study was to elucidate the role of TNFalpha in TVX/LPS-induced liver injury. TNF receptor (TNFR) 1 p55(-/-) and TNFR2 (p75(-/-)) mice were protected from hepatotoxicity caused by TVX/LPS coexposure, suggesting that TVX/LPS-induced liver injury requires both TNF receptors. TNFalpha inhibition using etanercept significantly reduced the TVX/LPS-induced increases in the plasma concentrations of several cytokines around the time of onset of liver injury. However, despite the reduction in chemokines, etanercept treatment did not affect the TVX/LPS-induced hepatic accumulation of neutrophils. In addition, etanercept treatment attenuated TVX/LPS induction of plasminogen activator inhibitor-1, and this was associated with a reduction in hepatic fibrin deposition. Mice treated with TVX and a nontoxic dose of TNFalpha also developed liver injury. In summary, TNFalpha acts through p55 and p75 receptors to precipitate an innocuous inflammatory cascade. TVX enhances this cascade, converting it into one that results in hepatocellular injury.

Fig. 1.

The role of TNF receptors in TVX/LPS-induced liver injury. Wild-type, p55^-/-, and p75^-/- mice were treated with TVX 3 h before LPS as described under Materials and Methods. Mice were killed 15 h after LPS, and plasma ALT activity was measured. n = 5–8 animals/group. *, significantly different from wild-type group; #, significantly different from p55^-/- group. Photomicrographs were taken of livers from representative mice from each group. Wild-type mice treated with TVX/LPS had severe necrosis, whereas necrosis in p55^-/- and p75^-/- mice was mild or nonexistent, respectively.

Fig. 2.

Effect of TNFα inhibition on TVX/LPS-induced increases in plasma cytokines. Mice were treated with vehicles or with TVX/LPS in addition to etanercept or its vehicle as described under Materials and Methods. Mice were sacrificed 4.5 h after LPS administration. Plasma concentrations of IFNγ, IL-6, IL-10, IL-1β, MCP-1, and VEGF were measured as described under Materials and Methods. n = 4–6 animals/group. *, significantly different from respective Veh/Veh group. #, significantly different from TVX/LPS/Veh group.

Fig. 3.

Effect of TNFα inhibition on TVX/LPS-induced increases in plasma chemokines. Mice were treated with vehicles or with TVX/LPS in addition to etanercept or its vehicle as described under Materials and Methods. Mice were sacrificed 4.5 h after LPS administration. Plasma concentrations of MIP-2, KC, and MIP-1α were measured as described under Materials and Methods. n = 4–6 animals/group. *, significantly different from respective Veh/Veh group. #, significantly different from TVX/LPS/Veh group.

Fig. 4.

Effect of TNFα inhibition on TVX/LPS-induced hepatic PMN accumulation. Mice were treated with vehicles or with TVX/LPS in addition to etanercept or its vehicle as described under Materials and Methods. Mice were sacrificed 4.5 h after LPS administration. Paraffin-embedded livers were stained for neutrophils, and the number of neutrophils was quantified as described under Materials and Methods. HPF, high-power field. n = 4–6 animals/group. *, significantly different from respective Veh/Veh group; #, significantly different from TVX/LPS/Veh group.

Fig. 5.

Effect of TNFα inhibition on hemostatic system dysregulation mediated by TVX/LPS coexposure. Mice were treated with vehicles or with TVX/LPS in addition to etanercept or its vehicle as described under Materials and Methods. They were sacrificed 4.5 h after LPS administration. Plasma concentrations of TAT dimers (A) and active PAI-1 (B) were measured as described under Materials and Methods. C, hepatic fibrin was stained immunohistochemically and quantified as described under Materials and Methods. n = 4–6 animals/group. *, significantly different from respective Veh/Veh group; #, significantly different from TVX/LPS/Veh group.

Fig. 6.

TVX/TNFα coexposure-induced liver injury. Mice were treated with TVX 3 h before recombinant murine TNFα as described under Materials and Methods. Mice were killed 15 h after TNFα administration, and plasma ALT activity was measured. n = 4–5 animals/group. *, significantly different from TVX/Veh group; #, significantly different from Veh/TNFα group.

Fig. 7.

Histopathology of TVX/TNFα-induced liver injury. Mice were treated with TVX 3 h before recombinant murine TNFα as described under Materials and Methods. Mice were killed 15 h after TNFα administration, and photomicrographs were taken of representative livers.