u-75302 and Reperfusion-Injury

Neutrophil migration protects the body against foreign invasion. Sequestration and activation of neutrophils, however, require stringent regulation because they may also cause tissue damage by the release of lysosomal enzymes and reactive oxygen species. The activity of various chemoattractants [e.g., leukotriene B(4) (LTB(4)), interleukin-8, and complements] has been documented by in vitro assays, whereas in vivo data have been limited mostly to histology. To examine in an in vivo model the chemotactic activity and subsequent tissue infiltration and the role of a specific chemoattractant, LTB(4), we used a rat renal ischemia-reperfusion injury model. Fluorescence-labeled Chinese hamster ovary (CHO) cells stably expressing the LTB(4) receptor (CHO-BLT) were able to accumulate along with neutrophils in the postischemic kidney, in contrast to vector control CHO cells. Furthermore, LTB(4) antagonists that protect against the decrease in renal function and diminish the tissue myeloperoxidase activity also led to the marked decrease in the number of CHO-BLT cells and neutrophils. Thus, LTB(4) alone appears sufficient to cause cells to migrate into postischemic tissues, and its dominant role in reperfusion injury has been demonstrated. The utilization of transfectants to pinpoint the role of LTB(4) in these in vivo experiments suggests their potential use with other ligands and/or in other pathological conditions.

Topics: Acute Kidney Injury; Animals; Blood Urea Nitrogen; Cell Movement; Chemotaxis; CHO Cells; Creatinine; Cricetinae; Fatty Alcohols; Glycols; Kidney; Leukocyte Count; Leukotriene Antagonists; Leukotriene B4; Male; Neutrophil Infiltration; Phenylpropionates; Rats; Rats, Sprague-Dawley; Receptors, Leukotriene B4; Reperfusion Injury

Neutrophils and eicosanoid chemoattractants are centrally involved with ischemia-reperfusion (I/R) injury. The CD 18 complex of adhesive glycoproteins, readily up-regulated by chemoattractants in vitro, is required for polymorphonuclear leukocyte (PMN) adherence to endothelium. This study tests whether CD 18 is up-regulated by ischemia in vivo and its role in mediating PMN diapedesis. Anesthetized rabbits underwent 3 h of bilateral hindlimb tourniquet ischemia (n = 16). Ten min after tourniquet release, levels of plasma leukotriene (LT)B4 increased to 390 +/- 62 pg/ml (mean +/- SE), higher than 134 +/- 26 pg/ml in control rabbits (n = 13, p less than 0.01). Aliquots of plasma were added to whole blood from normal rabbits (n = 6) for flow cytometric analysis of neutrophils with the CD 18 mAb R 15.7. Addition of I/R plasma failed to demonstrate an increase in surface expression of CD 18. Similarly, no CD 18 up-regulation was observed in vivo upon reperfusion in ischemic animals pretreated with mAb R 15.7 (n = 3). However, I/R plasma when introduced into plastic chambers taped atop dermabrasion sites in normal rabbits (n = 12) resulted in diapedesis, measured by the accumulation after 3 h of 1130 +/- 125 PMN/mm3 in the chambers relative to 120 +/- 31 PMN/mm3 with control plasma (p less than 0.01). Diapedesis in response to I/R plasma was abolished by pretreatment with mAb R 15.7 (less than 5 PMN/mm3, n = 6), was reduced by U 75,302, an LTB4 receptor antagonist (253 +/- 101 PMN/mm3, n = 6) (both p less than 0.01) and was not protein synthesis dependent. These results demonstrate that PMN diapedesis in response to I/R plasma is exclusively dependent upon the CD 18 glycoprotein complex by an LTB4-dependent mechanism, despite the fact that CD 18 is not up-regulated on circulating PMN in ischemia. These data indirectly indicate the functional importance of conformational changes of CD 18 in determining PMN adhesion.

Topics: Animals; Antibodies, Monoclonal; CD18 Antigens; Cell Adhesion; Chemotaxis, Leukocyte; Dactinomycin; Fatty Alcohols; Glycols; Leukotriene B4; Male; Neutrophils; Rabbits; Receptors, Leukocyte-Adhesion; Reperfusion Injury