Possible tacrolimus action mechanisms in its protector effects on ischemia-reperfusion injury.

Abstract

The leukocyte-endothelium interaction phenomena are absolutely important in the ischemia-reperfusion injury mechanism. The CD18 receptor activity modulation will regulate the neutrophil-dependent adhesion. The influence of different inflammatory mediators, such as oxygen free radicals (OFRs), leukotrienes, thromboxanes, platelet-activating factor, and complement, will regulate the endothelium-dependent adhesion in its early stage while the cytokines and cellular adhesion molecules (CAMs) will do it in its later stage (1,2). We agree with Ohmori et al., who affirm that the OFRs and the neutrophil infiltration mainly mediate the ischemia-reperfusion injury. It has been demonstrated in several studies that pretreatment with tacrolimus protects against ischemia-reperfusion damage in different organs; a minor grade of organ injury, better organ function, and major survival rates have been observed (3-5). The possible action mechanisms of this drug are becoming better known. From this point of view, we have to take into account that important regulation factors, during the ischemic stage, can be very interesting in regard to activation of multiple injury mechanisms in reperfusion. It has been demonstrated that tacrolimus decreases the mitochondrion Ca2+ concentration during ischemia and reperfusion, thereby maintaining the mitochondrion function and regulating the activation of multiple enzymatic systems which produce different inflammatory mediators (6). In the same way, it has been proven that the ATP levels are conserved in ischemic hepatic injury of tacrolimus-treated animals (7). The production of OFRs, which is increased in reperfusion and responsible of the tissular damage, is decreased significantly in ischemia-reperfusion models pretreated with tacrolimus (3,8,9). This decrease can be due to minor Kupffer cell activation (8) or to a decrease in neutrophil infiltration (3,10,11), both of which are important producers of OFRs. On the other hand, in a study of tacrolimus modulation in proinflammatory cytokine liberation, a decrease in tumor necrosis factor-α production (3,12,13) and of other cytokines, such as interleukin-1 and interleukin-6 (3,13) was demonstrated. In this way, we can consider that the cytokines, especially tumor necrosis factor-α, are powerful OFR stimulators and that, at the same time, the OFRs (superoxide anion) are important activators of the NF-κB transcriptional protein. In this way, the exert important control in CAM expression, nitric oxide synthase activation, and cytokine production. On the basis of the results in previous studies (3), we began a new study using the same experimental design that was previously described (3). We evaluated the expression of two CAMs: P-selectin, which belongs to the selectin family and is involved in the initial leukocyte-endothelium interaction mechanisms (rolling, no postischemic reflux, etc.), and intracellular adhesion molecule-1, which belongs to the immunoglobulin superfamily and is related to the hard adhesion of the leukocyte to the endothelium and antigenic recognition and lymphocytic activation mechanisms. We used hepatic tissue samples to perform Western blot and immunohisto-chemistry (frozen tissue) analyses at 6 hr after reperfusion for P-selectin and at 24 hr after reperfusion for intracellular adhesion molecule -1. Our results demonstrate a decrease in CAM expression in the tacrolimus-pretreated animals in relation to the ischemic control group in both the Western blot studies (Fig. 1) and in the immunohistochemistry analyses.Figure 1: Intracellular adhesion molecule-1 (ICAM-1) and P-selectin Western blot analyses of hepatic samples. In both cases, we observed a decrease in CAM in the tacrolimus-pretreated group in comparison with the ischemic control group.The causes of this modulation can be primary, such as control of NF-κB transcriptional protein activation (in study), or secondary, through regulation of the inflammatory response, as has already been demonstrated; thus, the decrease in cytokine production is an important factor in the regulation of CAM expression. The consequences of minor expression of these CAMs is minor leukocyte-endothelium interaction with minor leukocyte migration and consequently minor postreperfusion injury. Taking into account these results, we designed another study in a mesenteric ischemic model (clamping the superior mesenteric artery for 60 min) using male Sprague-Dawley rats to study the rolling and adhesion stages by means of intravital microscopy techniques. We observed a significant leukocyte decrease in the rolling and adhesion stages in comparison to the ischemic control group (Fig. 2).Figure 2: (A) Rolling stage leukocytes. We observed a significant rolling decrease in the treated group in comparison with the ischemic control group (P<0.05) from the first minute after reperfusion. (B) Adhesion stage leukocytes. We observed a significant adhesion decrease in the treated group in comparison with the ischemic control group (P<0.05) from the first minute after reperfusion. (B)In conclusion, we believe that the tacrolimus action mechanism is better known at present, and we agree with Ohmori et al. about the usefulness of pretreatment of recipients with tacrolimus. Francisco Javier Garcia-Criado Francisco Lozano-Sanchez Javier Fernandez-Regalado Joaquin Jose Valdunciel-Garcia Felipe Parreno-Manchado Isabel Silva-Benito Yolanda Zambrano-Cuadrado Alberto Gomez-Alonso Department of Surgery; Faculty of Medicine; University of Salamanca; Salamanca, Spain