Degranulating stimuli increase the availability of receptors on human neutrophils for the chemoattractant f-met-leu-phe.

Abstract Degranulating stimuli have been reported to modulate the ability of the chemoattractant f-met-leu-phe to bind to human polymorphonuclear leukocytes (PMN) with increased binding of the chemoattractant noted under conditions of limited exocytosis. The following studies were performed to determine if the increased binding was due to increased availability of receptors and/or modified affinity of binding. Human peripheral blood neutrophils, isolated by Hypaque-Ficoll and dextran sedimentation, were incubated for 30 min at 37°C with low concentrations of the secretagogues ionophore A23187 (5 × 10-8 M), Escherichia coli endotoxin-activated serum (1.25% v/v) + cytochalasin B (5 μg/ml), and phorbol myristate acetate (0.5 μg/ml). These conditions resulted in significant release of lysozyme 17 ± 3% vs 6 ± 1% for control cells (p0.05) with no significant release of β-glucuronidase or lactate dehydrogenase. After secretion the cells were washed twice and the binding of varying concentrations of f-met-leu-3Hphe to control (unstimulated) and stimulated cells was assayed. By using Scatchard analysis of the data thus obtained, the calculated number of available receptors for stimulated cells was significantly greater than the number of available receptors for control (unstimulated) cells (19,640 vs 10,250, p0.01). Additionally, by the Scatchard analysis there was a significant decrease in apparent affinity of f-met-leu-3Hphe binding in the group of stimulated cells (Ka = 0.151 nM-1 for control cells vs 0.123 nM-1 for stimulated cells, p0.05). These data indicate that incubation of human PMN with concentrations of degranulating stimuli causing limited granule release increases the receptor availability for f-met-leu-phe and decreases the apparent affinity of binding.