Hepatic injury induced by ischemia and reperfusion is an important clinical problem after liver resection or transplantation. Neutrophils are known to mediate the organ injury, but the precise mechanisms leading to hepatic neutrophil recruitment are undefined. Two CXC chemokines, macrophage inflammatory protein-2 (MIP-2) and KC, are potently chemotactic for neutrophils in vitro and have been reported to be involved in neutrophil-dependent inflammatory tissue injury. The objective of the present study was to determine the roles of MIP-2 and KC in the induction of hepatic ischemia/reperfusion injury. C57BL/6 mice were subjected to 90 minutes of partial hepatic ischemia followed by reperfusion. Hepatic injury was associated with neutrophil sequestration, edema, and elevated serum levels of hepatic transaminases. The expression of MIP-2 messenger RNA (mRNA) was induced within 3 hours after reperfusion, before any detectable increase in neutrophil accumulation, and was also increased to a greater extent in the ischemic lobe after 9 hours of reperfusion. These data suggest that MIP-2 may be involved in the initial recruitment of neutrophils to the ischemic lobe. In contrast, KC mRNA expression was not increased after 3 hours of reperfusion but after 9 hours increased equivalently in both ischemic and nonischemic lobes, suggesting a more generalized role in neutrophil recruitment. Neutralization of MIP-2 or KC resulted in significant decreases in hepatic neutrophil accumulation, edema, and hepatocellular injury. These data suggest that the local expression of MIP-2 and KC are important mediators involved in neutrophil-dependent hepatic injury induced by ischemia and reperfusion in mice. (HEPATOLOGY 1998;27:507-512.)
Hepatic injury caused by ischemia and reperfusion during surgical resection or transplantation of the liver may lead to both local and systemic organ dysfunction. The local hepatic injury is comprised of two phases, with the initial injury being mediated by activated Kupffer cells. Neutrophils are primed during this initial period and play a central role in the ensuing hepatic injury. The temporal involvement of Kupffer cells and neutrophils has been reported by studies in which neutropenic rats incurring hepatic ischemia and reperfusion experienced early injury but were protected from subsequent hepatic damage. The accumulation of neutrophils may result in hepatic hypoperfusion, which is caused by sinusoidal occlusion, 4 and the release of reactive oxygen and proteases by neutrophils may also promote progressive hepatocellular damage. 5 However, the mechanisms by which ischemia and reperfusion induce neutrophil accumulation and the subsequent hepatic injury are undefined.
Increased production of tumor necrosis factor-โฃ is associated with neutrophil-dependent liver injury after hepatic ischemia and reperfusion. The local production of tumor necrosis factor-โฃ is increased after hepatic ischemia and reperfusion; and while tumor necrosis factor-โฃ itself does not induce neutrophil chemotaxis, recent studies show that tumor necrosis factor-โฃ stimulates the local hepatic production of the CXC chemokine ENA-78, which mediates the chemotaxis of neutrophils in a rat model. 6 However, the involvement of other CXC chemokines in hepatic injury induced by ischemia and reperfusion has not been extensively studied.
Two potent CXC chemokines, macrophage inflammatory protein-2 (MIP-2) and KC, have been shown to cause neutrophil activation, including the induction of the respiratory burst and Mac-1 (CD11b) upregulation. Furthermore, the expression of MIP-2 and KC are reported to increase during inflammation of the kidney, liver, and lung. In the present study we investigate the roles of MIP-2 and KC in the local tissue injury induced by hepatic ischemia and reperfusion. The data show that the hepatic expression of MIP-2 and KC messenger RNA (mRNA) are increased in temporally distinct patterns after hepatic ischemia and reperfusion. Additionally, passive immunization against MIP-2 or KC caused reductions of neutrophil sequestration and liver injury. These findings suggest that the CXC chemokines MIP-2 and KC contribute significantly to hepatic neutrophil accumulation and ensuing tissue injury induced by ischemia and reperfusion.
MATERIALS AND METHODS
Animals
Male C57BL/6 mice (Charles Rivers Laboratories, Wilmington, MA) (range, 20-25 g) were used in all experiments. This project was approved by the University of Louisville Animal Care and Use Abbreviations: MIP-2, macrophage inflammatory protein-2; mRNA, messenger RNA; RT-PCR, reverse-transcription polymerase chain reaction; MPO, myeloperoxidase.