ISCHEMIA ACTIVATES NEUTROPHILS BUT INHIBITS THEIR LOCAL AND REMOTE DIAPEDESIS

Hindlimb ischemia and reperfusion results in local limb and distant lung injury. This study tests whether the mechanism of injury is by ischemia mediated polymorphonuclear leukocyte (PMN) activation and diapedesis. Anesthetized rabbits were subjected to three hours of hindlimb ischemia (n = 8) or sham ischemia (n = 4). PMN derived solely from the reperfused ischemic limb and assayed flow cytometrically displayed an oxidative burst of 135 ± 8 fentamoles dichlorofluorescein (fmDCF)/cell compared to preischemic levels of 74 ± 14 fmDCF/cell (p < 0.05). Additional aliquots of isolated neutrophils were treated with phorbol myristate acetate (PMA) 10-7M. In contrast to a 162% increase in oxidative burst before ischemia, neutrophils at ten minutes of reperfusion had an enhanced response to PMA of 336% (p < 0.05). Plasma collected from the ischemic hindlimb at ten minutes of reperfusion when introduced into an abraded skin chamber or intratracheally induced diapedesis in nonischemic animals. PMN accumulations in the skin chamber' were 1636 ± 258 PMN/mm3 after three hours (n = 8) compared to 63 ± 18 PMN/mm3 induced by sham plasma (n = 4, p < 0.05). Introduction of ischemic plasma intratracheally into a lobar bronchus (n = 4) induced PMN accumulations after three hours, measured by bronchoalveolar lavage fluid of 19 ± 2 × 104 PMN/mm3 compared to 5 ± 1 × 104 PMN/mm3 with sham plasma (n = 4, p < 0.05). Diapedesis was completely prevented (0-3 PMN/ mm3, p < 0.05) by introducing ischemic plasma into skin chambers in animals whose hindlimbs had been made ischemic (n = 6) or into chambers located on skin regions that had been previously made ischemic (n = 6). Similarly after hindlimb ischemia, lavage of the lung with ischemic plasma yielded few PMN 0-3/mm3 (p < 0.05). These data indicate that ischemia and reperfusion lead to generation of a circulating component in plasma that causes an oxidative burst in PMN and inhibits their diapedesis but promotes diapedesis when applied extravascularly to a naive animal.