PMA-ACTIVATED NEUTROPHILS DECREASE ECTOENZYME ACTIVITIES IN RABBIT AORTIC ENDOTHELIAL-CELLS IN CULTURE

We have studied the effects of phorbol 12-myristate 13-acetate (PMA)-activated neutrophils [polymorphonuclear leukocytes (PMN)] on endothelial ectoenzyme [angiotensin-converting enzyme (ACE) and 5'-nucleotidase (NCT)] activities in cultured rabbit aortic endothelial cells (EC) with the use of [H-3]benzoyl-Phe-Ala-Pro and C-14-labeled AMP as substrates, respectively, under first-order reaction conditions. PMA (1-1,000 ng/ml) or PMN alone had no effect on ACE activity. When PMA was incubated together with PMN (PMN/EC = 1.25:1 or 2.5 X 10(5) neutrophils/ml) for 4 h in Earle's salts, a PMA dose-dependent decrease in ACE activity was observed. Threshold PMA concentration was 2 ng/ml. At 8 ng PMA/ml, ACE activity was totally abolished, without any evidence of cytotoxicity, as inferred from release of Cr-51 from prelabeled EC. The decrease in ACE activity was also dependent on PMN concentration and was detectable at PMN/EC values as low as 1.25:10 (0.25 X 10(5) PMN/ml). Inhibition of ACE occurred as early as 1 h after incubation (PMA 10 ng/ml, PMN/EC = 1.25:1). PMA alone caused a small but significant increase in NCT activity, whereas PMA coincubation with PMN produced a significant decrease in NCT activity (20-29%), which was PMA and PMN concentration independent. PMA increased PMN adherence to endothelial monolayers in a concentration-dependent manner. Pretreating PMN with monoclonal antibody 60.3 (raised against the adhesion glycoprotein CD18) or placing a 2-mum filter between PMN and EC, protected the decrease in ACE activity. These results suggest that 1) PMA-activated PMN decrease endothelial ectoenzyme activities in culture, and 2) prevention of adherence of PMN to EC significantly reduces the PMN-mediated decrease in endothelial ACE activities.