Neutrophil-cytokine interactions in a rat model of sulindac-induced idiosyncratic liver injury

Abstract

Previous studies indicated that lipopolysaccharide (LPS) interacts with the nonsteroidal anti-inflammatory drug sulindac (SLD) to produce liver injury in rats. In the present study, the mechanism of SLD/LPS-induced liver injury was further investigated. Accumulation of polymorphonuclear neutrophils (PMNs) in the liver was greater in SLD/LPS-cotreated rats compared to those treated with SLD or LPS alone. In addition, PMN activation occurred specifically in livers of rats cotreated with SLD/LPS. The hypothesis that PMNs and proteases released from them play critical roles in the hepatotoxicity was tested. SLD/LPS-induced liver injury was attenuated by prior depletion of PMNs or by treatment with the PMN protease inhibitor, eglin C. Previous studies suggested that tumor necrosis factor-α (TNF) and the hemostatic system play critical roles in the pathogenesis of liver injury induced by SLD/LPS. TNF and plasminogen activator inhibitor-1 (PAI-1) can contribute to hepatotoxicity by affecting PMN activation and fibrin deposition. Therefore, the role of TNF and PAI-1 in PMN activation and fibrin deposition in the SLD/LPS-induced liver injury model was tested. Neutralization of TNF or inhibition of PAI-1 attenuated PMN activation. TNF had no effect on PAI-1 production or fibrin deposition. In contrast, PAI-1 contributed to fibrin deposition in livers of rats treated with SLD/LPS. In summary, PMNs, TNF and PAI-1 contribute to the liver injury induced by SLD/LPS cotreatment. TNF and PAI-1 independently contributed to PMN activation, which is critical to the pathogenesis of liver injury. Moreover, PAI-1 contributed to liver injury by promoting fibrin deposition.

Fig. 1. PMN accumulation and activation in rat livers

Rats were treated with two administrations of SLD (50 mg/kg, p.o.) or its vehicle (Veh, 0.5% methyl cellulose) with a 16 hr interval. LPS (8.25 × 10⁵ EU/kg, i.v.) or its saline vehicle was administered half an hour before the second administration of SLD. PMN staining was performed on livers collected 4 hr after the second administration of SLD. (A) PMN numbers in 400 X high power fields (HPF) were counted to evaluate PMN accumulation. (B) HOCl-protein adduct staining was performed on slides of frozen liver collected at 8 hr. Ten randomly chosen fields were photographed for each section, and the fraction of positive pixels was determined. *significantly different from respective group without LPS. #significantly different from Veh/LPS group. P<0.05, n=4-5.

Fig. 2. Concentrations of PMN chemokines in rat plasma

Rats were treated with SLD and LPS or their vehicles (Veh) as described in Fig. 1. At 1, 4 and 12 hr after the second administration of SLD, plasma was collected and concentrations of (A) cytokine-induced neutrophil chemoattractant-1 (CINC-1) and (B) macrophage inflammatory protein-1α (MIP-1α) were evaluated by multiplex ELISA. *significantly different from Veh/Veh group at the same time. P<0.05, n=5.

Fig.3. Effect of PMN depletion on SLD/LPS-induced liver injury

Rats were pretreated with either normal serum (NS) or rabbit anti-rat PMN serum (AS) half an hour before the first administration of SLD. Rats were euthanized 12 hr after the 2^nd administration of SLD, and serum ALT activity was determined. Vehicle (Veh). *significantly different from Veh/Veh/NS group, #significantly different from SLD/LPS/NS group. P<0.05, n=3-6.

Fig.4. Effect of PMN protease inhibition on SLD/LPS-induced liver injury

Rats were treated with SLD and LPS or their vehicles (Veh) as described in Fig. 1. Eglin C or its vehicle (Veh) was administered to rats 4, 6, and 8 hr after the 2nd administration of SLD. Rats were euthanized at 12 hr, and ALT activity in serum was determined. *significantly different from Veh/Veh/Veh. #significantly different from Veh/Veh/Eglin C group. ^asignificantly different from SLD/LPS/Veh group. P<0.05, n=3-6.

Fig. 5. Effect of TNF inhibition on PMN accumulation and activation

Rats administered SLD/LPS were pretreated with etanercept(Etan) or its vehicle (Veh) 1 hr before LPS. (A) PMN staining was performed on livers collected at 8 hr. The accumulation of PMNs in livers was evaluated by averaging PMN numbers in 10, randomly chosen, 400 X fields. (B) Quantification of HOCl-protein adducts in the livers of rats at 8 hr. *significantly different from Veh/Veh/Veh group. #significantly different from Veh/SLD/LPS group. P<0.05, n=3-6.

Fig. 6. Effect of PAI-1 inhibition on liver injury and PMN accumulation and activation

Rats were treated with SLD/LPS as described in Fig.1. PAI-1 inhibitor, PAI039 (6 mg/kg, p.o.), or its vehicle (Veh, 0.5% methyl cellulose) was administered at 1 hr after the second administration of SLD. Rats were euthanized at 12 hr to measure ALT activity (A) or at 8 hr to assess PMN accumulation (B) and activation (C). * significantly different from Veh/Veh/Veh. # significantly different from SLD/LPS/Veh group. P<0.05, n=4-16.

Fig.7

Effect of PAI-1 inhibition on liver histopathology in SLD/LPS-cotreated rats. Rats were treated with Veh/Veh/Veh (A), Veh/SLD/LPS (B) or PAI039/SLD/LPS (C) as described in Fig. 6. Liver sections collected at 12 h were stained with hematoxylin and eosin for histopathological evaluation.. Arrows point to areas of hepatocellular necrosis.

Fig.8. Effect of TNF inhibition on plasma PAI-1 concentration

Rats were treated with etanercept (Etan), SLD and LPS or their vehicles (Veh) as described in the legend to Fig. 5. Plasma active PAI-1 concentration was determined at 8 hr. * significantly different from Veh/Veh/Veh. P<0.05, n=4.

Fig. 9. Effect of TNF or PAI-1 inhibition on fibrin deposition in liver

Rats were treated with SLD/LPS and etanercept (Etan, A) or PAI-1 inhibitor (B) respectively. Fibrin deposition was evaluated at 8 hr. * significantly different from SLD/LPS/Vehicle (Veh). P<0.05, n=4-7.

Fig. 10. Mechanisms of SLD/LPS-induced liver injury

Solid lines indicate interactions observed in current research. Dotted lines indicate interactions that have been identified in previously published research. See text for details.