Sequential Chemotactic and Phagocytic Activation of Human Polymorphonuclear Neutrophils

Human polymorphonuclear neutrophils (PMN) chemotax to a foreign entity. When the chemoattractants' origins are reached, specific receptors bind to the invader's surface, initiating phagocytosis, phagosome formation, and fusion with granule membranes, generating the bactericidal oxidative burst, and releasing lytic enzymes, specific peptides, and proteins. We explored the initial signaling involved in these functions by observing naïve, unprimed PMN in suspension using fluorescent indicators of cytoplasmic signals (Δ[Ca²⁺]i and ΔpHi) and of bactericidal entities (oxidative species and elastase) exposed to N-formyl-methionyl-leucyl-phenylalanine (fMLP) and/or multivalent immune complexes (IC). fMLP and IC each initiate a rapid transient rise in [Ca²⁺]i, mostly from intracellular stores, simultaneously with a drop in pHi; these are followed by a drop in [Ca²⁺]i and a rise in pHi, with the latter being due to a Na⁺/H⁺ antiport. The impact of a second stimulation depends on the order in which stimuli are applied, on their dose, and on their nature. Provided that [Ca²⁺]i is restored, 10⁻⁷ M fMLP, previously shown to elicit maximal Δ[Ca²⁺]i but no bactericidal functions, did not prevent the cells' responses with Δ[Ca²⁺]i to a subsequent high dose of fMLP or IC; conversely, cells first exposed to 120 μg/ml IC, previously shown to elicit maximal Δ[Ca²⁺]i and bactericidal functions, exhibited no subsequent Δ[Ca²⁺]i or ΔpHi to either stimulus. While exposure to 10⁻⁷ M fMLP, which saturates the PMN high-affinity receptor, did not elicit bactericidal release from these naïve unprimed PMN in suspension, 10⁻⁵ M fMLP did, presumably via the low-affinity receptor, using a different Ca²⁺ source.