Differentiation of cellular processes involved in the induction and maintenance of stimulated neutrophil adherence

Neutrophil adherence stimulated by phorbol myristate acetate (PMA) was investigated by quantitating the attachment of 51Cr-labeled neutrophils to plastic surfaces and to the endothelium of umbilical veins mounted in compartmentalized Lucite chambers. PMA-induced adherence could be functionally separated into an induction phase requiring cellular metabolism and a Mg++ dependent maintenance phase that was independent of cellular metabolism. Thus, metabolic inhibitors (N-ethylmaleimide, 2- deoxyglucose) blocked adherence when added to neutrophils prior to PMA, but did not cause detachment of cells adhering as a consequence of prior exposure to PMA. PMA failed to induce adherence of neutrophils incubated at low (0.4 degree C) temperature, but temperature reduction, even for prolonged periods, did not cause detachment of adherent cells. Thus, the attractive forces that mediate stimulated adherence persist independently of any sustained metabolic response to the inducing stimulus. However, removal of Mg++ from the media above adherent cells resulted in immediate detachment, indicating that the cation was required for the persistent expression or maintenance of the attractive forces involved. The extent of stimulated adherence correlated well with the extent of degranulation when rates were varied by limiting the incubation time or stimulus concentration. This correlation was not absolute; in the absence of Mg++, PMA induced degranulation normally but failed to enhance adherence. To explain these findings, we investigated the possibility that PMA-stimulated adherence was maintained by Mg++-dependent cellular adherence molecules released during exocytosis. Supernatants of stimulated neutrophils were devoid of adherence-promoting activity, and only weak activity was recovered in supernatants of mechanically disrupted neutrophils. PMA effectively stimulated the tight adherence of degranulated neutrophil cytoplasts to plastic surfaces and did so in the absence of stimulated granule enzyme release. Thus, conditions have been identified under which degranulation occurs in the absence of adherence (removal of Mg++) and adherence occurs without concurrent degranulation. Since neutrophil cytoplasts do contain some granule products and granule material can be identified on cytoplast membranes, it is possible that degranulation or granule products may be involved in the adherent response. However, hyperadherence was shown to develop in the absence of de novo degranulation.(ABSTRACT TRUNCATED AT 400 WORDS)