DO IMMUNOSUPPRESSANTS DIRECTLY AFFECT NEUTROPHILS, RESULTING IN PROTECTION OF THE LIVER AGAINST ISCHEMIA-REPERFUSION INJURY? (1998)

Ischemia-reperfusion injury in the liver occurs in a biphasic pattern via acute oxygen free radicals and subacute neutrophil-mediated damage. Pretreatment with immunosuppressive drugs such as cyclosporine and tacrolimus is reported to protect against ischemia-induced liver injury (1,2), but the mechanisms involved are still unknown. Recent animal studies have shown that these agents decrease both tissue free radical levels and neutrophil infiltration after liver ischemia-reperfusion (3,4). Lymphocyte-neutrophil interaction is also involved in neutrophil-mediated ischemia-reperfusion injury where CD4+ T lymphocytes regulate an initial inflammatory response after reperfusion of the liver (5). We are very interested in a recent study reported by Garcia-Criado et al. (Transplantation 1997; 64: 594 ). These investigators experimentally confirmed the protective effect of tacrolimus against ischemia-reperfusion injury of the liver. Pretreatment with tacrolimus suppressed cytokine responses, especially the production of interleukin 1 and tumor necrosis factor-α, and decreased neutrophil migration in the liver. The question that remains to be determined is whether tacrolimus directly affects circulatory neutrophils because conventional immunosuppressants strongly block intracellular signal induction of cytokine gene expression on cells in the liver (6). We have focused on the relationship between the protective effect of cyclosporine against liver ischemia-reperfusion injury and functional changes of circulatory neutrophils after administration of this agent. Using adult male Wistar rats, temporal normothermic ischemia of the liver was induced by portal pedical clamping with a minor modification (3,4). The effect of pretreatment with cyclosporine (10 mg/kg for 4 days, oral administration) was examined. A control group (olive oil pretreatment) revealed that serum alanine aminotransferase and asparate aminotransferase levels were transiently elevated and peaked at 6 hr after reperfusion (2002±271 IU/L and 1682±328 IU/L [mean±SE], respectively, n=5). The amount of myeloperoxidase in the liver after reperfusion was significantly greater than that in the control group (0.3±0.1 vs. 2.1±0.3 µmol/mg·protein, P<0.05). By comparison, the serum alanine aminotransferase levels were less elevated 6 hr after reperfusion (1181±116 IU/L, P<0.05) in the cyclosporine-treated group, which is also consistent with what has been previously reported (1,3). The activity of peripheral neutrophils from the euthanized rats at 6 hr after reperfusion was estimated by free radical production to phorbol ester myristate acetate (PMA-stimulated) and control buffer (unstimulated). Data obtained demonstrated that the activity of circulatory neutrophils in the cyclosporine-treated group did not differ from that in the control group (unstimulated: cyclosporine, 3.6±0.6 nmol/µl/hr and control, 5.4±0.8 nmol/µl/hr; PMA-stimulated: cyclosporine, 19.9±4.3 and control, 31.6±5.5 nmol/µl/hr, respectively, n=8). Although there is a possibility that "circulatory" neutrophils differ from the cells that act locally at the inflammatory site (7), these cells in the impaired liver could not be collected using mechanical mincing followed by Ficoll gradient separation (unpublished data). Neutrophils have been observed to adhere to the vascular wall of the liver just after reperfusion using a microcirculation system (8). However, a difference in the adhesive pattern of neutrophils studied between the control and cyclosporine-treated groups was not detected using our system (9). Garcia-Criado et al. demonstrated that tacrolimus suppresses the elevation of proinflammatory cytokines such as interleukin 1 and tumor necrosis factor-α in peripheral blood (4). However, neutrophils produce these cytokines through the activation of the interleukin 8 gene, which in turn inflammatory site (10). Based on this experimental evidence (4,10), the primed neutrophils might produce these initial cytokines after migration from the circulating pool to the site of inflammation within the liver. An explanation for these observation might be that cyclosporine and tacrolimus have no effect on circulatory neutrophils, or that local neutrophils differ from circulatory cells, or both. Further studies are clearly required to understand this mechanism completely. We found the study by Garcia-Criado et al. interesting in that the down-regulation on neutrophil infiltration and cytokine production by tacrolimus was an important factor for preventing liver ischemia-reperfusion injury. The evidence that immunosuppressive drugs have protective effects against ischemia-reperfusion injury supports the usefulness of pretreatment with these agents in recipients. We are therefore looking forward to seeing clinical data on this subject in the near future and further elaboration on the mechanism revealed by this experimental evidence. Masami Ohmori Eiji Kobayashi Kazuhiro Harada Yasuhiko Kitoh Koichi Mizuta Hiroo Uchida Yoshinori Ogino Akio Fujimura Department of Clinical Pharmacology; Jichi Medical School; Tochigi 329-0498, Japan