Brief Communication THE EFFECT OF NITRIC OXIDE AND PEROXYNITRITE ON APOPTOSIS IN HUMAN POLYMORPHONUCLEAR LEUKOCYTES MORGAN G. BLAYLOCK,BRIAN H. CUTHBERTSON,HELEN F. GALLEY, N. RANALD FERGUSON, and NIGEL R. WEBSTER Academic Unit of Anaesthesia & Intensive Care, University of Aberdeen, Aberdeen, United Kingdom (Received 5 January 1998; Revised 13 April 1998; Accepted 13 April 1998) Abstract—In acute lung injury, neutrophil apoptosis may be important in regulating the inflammatory process by controlling neutrophil numbers and thus activity. Exogenous inhaled nitric oxide is now a widely used therapy in patients with acute lung injury, and its effects on apoptosis may be important. We investigated the effect of nitric oxide and peroxynitrite on apoptosis in lipopolysaccharide stimulated polymorphonuclear leukocytes as a model of nitric oxide-treated lung injury. Cells were incubated for up to 16 h with and without 1.7 g/ml lipopolysaccharide and the nitric oxide donor GEA-3162 or the peroxynitrite donor SIN-1. Apoptosis was assessed using flow cytometry following annexin-V staining, after 4, 6, 8, and 16 h. Data were assessed using Kruskal–Wallis analysis of variance or Mann–Whitney U-test as appropriate. Annexin-V staining increased spontaneously over 16 h in untreated cells (p = .0002) and incubation with either 1000 M SIN-1 or 10 M GEA-3162 increased annexin staining at early time points in nonactivated cells. Apoptosis was attenuated when cells were exposed to lipopolysaccharide and both nitric oxide and peroxynitrite dose dependently inhibited this suppression at all time points and was most apparent at 16 h (p = .004 and .001, respectively). Exposure of activated neutrophils to exogenous nitric oxide or peroxynitrite has marked influences on apoptosis. This work has implications for the modulation of neutrophil function within the lung in patients with lung injury who receive inhaled nitric oxide therapy. © 1998 Elsevier Science Inc. Keywords—Nitric oxide, Superoxide, Peroxynitrite, Apoptosis, Neutrophil, Human, Culture, Free radical INTRODUCTION The use of inhaled nitric oxide in the intensive care unit has become widespread in both acute lung injury and acute respiratory distress syndrome, improving oxygen- ation and reducing pulmonary pressure by a selective pulmonary vasodilator action [1]. Polymorphonuclear leukocytes (PMN) are impor- tant in acute lung inflammation. They migrate into the lung parenchyma during inflammation in response to mediators such as interleukin-8 and leukotrienes [2]. Both clinical and in vitro studies have suggested that exogenous nitric oxide may modulate some aspects of the inflammatory response [3,4] and that nitric oxide also plays a role in PMN adherence and migration through regulation of adhesion molecule expression [5]. Apoptosis or programmed cell death represents a set pattern of morphological changes distinguishable from necrosis. These include cell shrinkage, mem- brane blebbing, chromatin condensation, and nuclear fragmentation [6]. Apoptosis occurs spontaneously in unactivated PMN, and is thought to be an important regulatory mechanism in the control of PMN function and thus inflammatory response [7]. Nitric oxide has been shown to induce apoptosis in some cells [8 –12]. However, superoxide production by macrophage cell lines may decrease the sensitivity of nitric oxide- induced apoptosis. This suggests that the balance be- tween local nitric oxide and superoxide anion concen- trations is an important control mechanism in apoptosis. We therefore investigated the effect of ex- ogenous nitric oxide and peroxynitrite in the presence and absence of lipopolysaccharide, on apoptosis in human PMN assessed using annexin-V staining. Address correspondence to: Dr. H. F. Galley, Academic Unit of Anaesthesia & Intensive Care, Institute of Medical Sciences, Forester- hill, Aberdeen, AB25 2ZD, UK; Tel: +44 (0)1224 681818, ext. 52775; Fax: +44 (0) 1224 273066; E-Mail: h.f.galley@abdn.ac.uk. Free Radical Biology & Medicine, Vol. 25, No. 6, pp. 748 –752, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/98 $19.00 + .00 PII S0891-5849(98)00108-7 748