Nitric oxide inhibits neutrophil beta 2 integrin function by inhibiting membrane-associated cyclic GMP synthesis.

The aim of this investigation was to identify the mechanism by which nitric oxide inhibits neutrophil beta 2 integrin dependent adherence. Isolated rat neutrophils from blood and peritoneal exudates were exposed for 2 min to nitric oxide generated by diethylamine-NO at rates between 1.6 and 138 nmol/min. Exposure to nitric oxide at rates less than 14 nmol/min had no effect on adherence. Exposure to 14 to 56 nmol nitric oxide/min inhibited beta 2 integrin dependent adherence to endothelial cells, nylon columns, and fibrinogen-coated plates, but higher concentrations had no significant effect on adherence. Adherence by beta 2 integrins could be restored by incubating cells with dithioerythritol, phorbol 12-myristate 13-acetate, or 8-bromo cyclic GMP. Elevations in cellular cyclic GMP concentration were associated with adherence, but this did not occur after cells were exposed to concentrations of nitric oxide that inhibited beta 2 integrin-dependent adherence. Elevations in cyclic GMP did occur after cells were incubated with dithioerythritol or phorbol 12-myristate 13-acetate. Concentrations of nitric oxide that inhibited beta 2 integrin-dependent adherence also inhibited catalytic activity of membrane associated guanylate cyclase and binding of atrial natriuretic peptide, but were insufficient to activate cytosolic guanylate cyclase. Nitric oxide did not inhibit neutrophil oxidative burst or degranulation, nor effect beta 2 integrin expression or adherence that did not depend on beta 2 integrins, nor cause oxidative stress identified in terms of cellular glutathione concentration or protein nitrotyrosine. The results indicate that nitric oxide inhibited beta 2 integrins in a concentration-dependent fashion by inhibiting cell-surface transduction of signals linked to the activity of membrane-bound guanylate cyclase. The inhibitory effect could be overcome by providing cells with cyclic GMP exogenously or by stimulating cytosolic guanylate cyclase.

Functional inhibition of rat polymorphonuclear leukocyte B2 integrins by hyperbaric oxygen is associated with impaired cGMP synthesis.

We hypothesized that hyperbaric oxygen caused a metabolic derangement in polymorphonuclear leukocytes that impaired the function of B2 integrins. Isolated neutrophils from rats that had been exposed to 3 atm oxygen for 45 min failed to exhibit B2 integrin-dependent adherence to nylon columns or to fibrinogen-coated plates. Adherence was restored after cells were incubated with 8-bromo-cGMP, phorbol 12-myristate 13-acetate (PMA) or the reducing agent dithioerythritol. Hyperbaric oxygen was found to inhibit cGMP synthesis that normally occurred when cells were stimulated by passage through nylon columns, and exposure to PMA or dithioerythritol reestablished cGMP synthesis. Cells adherent to plastic plates synthesized cGMP when they were exposed to N-formyl-methionyl-leucine-phenylalanine (FMLP) or PMA. Neutrophils from rats exposed to hyperbaric oxygen synthesized cGMP in response to PMA but failed to respond to FMLP, although hyperbaric oxygen did not alter the affinity of the FMLP receptor or its associated G protein. Dithioerythritol restored the cGMP synthetic ability of adherent neutrophils in response to FMLP. We conclude that hyperbaric oxygen inhibits B2 integrin-dependent adherence because it impairs cGMP synthesis by activated neutrophils.