Because nitric oxide (NO) can act both as a regulatory and as a toxic molecule, we have studied N-formyl-methionyl-leucyl-phenylalanine (fMLF) -stimulated responses of human neutrophils (PMNs) during various conditions of NO modulation in unprimed and bacterial lipopolysaccharide (LPS) -primed cells. Effects of various NO modulators were assessed on stimulated superoxide (O2-) generation, granule exocytosis, homotypic aggregation, and rises in intracellular free Ca2+ ([Ca2+]i). Significant differences in the effects of various NO modulators on inflammatory responses of PMNs kept in stirred suspension versus those kept under static and/or adherent conditions, were observed. L-arginine, the physiological substrate for NO synthase (NOS), and NG-nitro-L-arginine methyl ester, an inhibitor of NOS, both caused a 40-50% inhibition of LPS-induced priming of O2- generation in PMNs in stirred suspension, but not in LPS-primed PMNs under static or adherent conditions. The NO donors, sodium nitroprusside and S-nitroso-N-acetylpenicillamine, completely abrogated the LPS-induced priming of 02- generation in PMNs in suspension, while causing only a 40-50% inhibition in PMNs under static or adherent conditions. The Ca2+ ionophore, A23187, prevented the LPS-induced priming of 02-generation without affecting 02- generation in unprimed PMNs. LPS priming of PMNs induced about a twofold increase in fMLF-stimulated homotypic aggregation, exocytosis of secondary granules, and rises in [Ca2+]i. In related studies, we also provide definitive evidence for enzymatic formation of NO in human PMNs and demonstrate a significant decrease in NO levels in LPS-primed PMNs. Taken together, these findings indicate that NO modulates PMN inflammatory responses and plays a protective role in priming and activation processes of inflammatory PMNs.
Studies of human peripheral blood neutrophils (PMNs) demonstrated that botulinum neurotoxin D (BT-D) ADP-ribosylates a 22-kDa PMN G protein (G22k) and inhibits the exocytosis of both specific and azurophilic granules stimulated by FMLP. Furthermore, this inhibition of PMN exocytosis by BT-D was found to be correlated with the degree of irreversible ADP-ribosylation of G22k by BT-D and to require modification of at least 85% of PMN G22k before significant inhibition of secretion is observed. Although both pertussis toxin and BT-D inhibited exocytosis in FMLP-stimulated PMNs, the inhibitory effects of the two toxins were found to be additive. Pertussis toxin and BT-D also inhibited Ca2+/GTP/GTP gamma S-induced secretion in digitonin-permeabilized PMNs, but there were distinct differences between the inhibitory effects of the two toxins. In contrast to BT-D, the exotoxin botulinum C3 was found to ADP-ribosylate primarily a 24- to 25-kDa PMN protein, and it was not found to inhibit Ca(2+)- and GTP-induced secretion in permeabilized PMNs. Ultrastructural studies of BT-D-treated PMNs showed an accumulation of distinct membrane-bound organelles in the periphery of the cells after FMLP stimulation, suggestive of a toxin-induced block in organelle-plasma membrane fusion. Taken together, these findings indicate that BT-D-sensitive G22k has a functional role in stimulated exocytosis of PMNs.
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