Chemotactic factor receptors of human PMN leucocytes. I. Effects on migration of labelling plasma membrane determinants with impermeant covalent reagents and inhibition of labelling by chemotactic factors

Abstract

The mechanism of stimulation of human PMN leucocyte-directed migration by chemotactic factors was studied by pre-labelling plasma membrane determinants with impermeant covalent reagents and assessing the effects of such modification on spontaneous mitration and chemotaxis in modified Boyden chambers. Pre-treatment of PMN leucocytes with 10(-9) to 10(-6) M isethionyl acetimidate, which selectively labels amino groups, enhanced spontaneous migration and concomitantly inhibited chemotaxis to fragments of the fifth component of complement (C5fr), 12-L-OH-5,8,10,14-eicosatetraenoic acid (HETE) and several formyl--methionyl (f--Met) peptides to an extent that was inversely related to the magnitude of the chemotactic response of untreated PMN leucocytes. Para-chloromercuribenzene sulphonate, which selectively labels sulphydryl groups, inhibited chemotaxis to diverse stimuli without substantially influencing spontaneous migration, while the diazonium salt of sulphanilic acid, which labels several types of plasma membrane determinants, altered neither spontaneous nor chemotactic migration. Incubation of PMN leucocytes with various concentrations of [3H]-isethionyl acetimidate labelled from 33,000 amino groups per PMN leucocyte at 10(-6) M to over 800,000 at 10-4) M, a reaction that was substantially inhibited by chemotactic concentrations of C5fr and HETE, but not by f--Met peptides. Subcellular fractionation of PMN leucocytes labelled with [3H]-isethionyl acetimidate localized the radioactivity to membrane-rich fractions. Free amino groups thus appear to be functionally critical determinants of some chemotactic factor receptors on the plasma membrane of PMN leucocytes.