ROLE OF THE FC-GAMMA-R SUBCLASSES FC-GAMMA-RII AND FC-GAMMA-RIII IN THE ACTIVATION OF HUMAN NEUTROPHILS BY LOW AND HIGH VALENCY IMMUNE-COMPLEXES

Two Fc gamma receptor (Fc gamma R) subclasses on human neutrophils, Fc gamma RII and Fe gamma RIII, activate different cellular functions. T o examine the involvement of each receptor subtype in polymorphonuclea r leukocyte activation, Fab and F(ab')(2) fragments of subclass-specif ic monoclonal antibodies ([mAbs] mAb IV.3 against Fc gamma RII and mAb 3G8 against Fc gamma RIII, respectively) were used to block the bindi ng of low valency immune complexes (LICs) and high valency immune comp lexes (HICs). Flow cytometry then permitted the simultaneous quantitat ion of antibody and ligand binding, the elicited intracellular Ca2+ co ncentration (Delta[Ca2+](int)), initiation of the oxidative burst, and /or the phospholipase A activation in the same cell. We have previousl y demonstrated that subsaturating dosages of HIC bind uniformly to all the cells but elicit an ''all-or-none'' (i.e., dose independent) maxi mal Delta[Ca2+](int) in a dose-dependent subpopulation of the cells. I n contrast, both the proportion of cells responding and the magnitude of the Delta[Ca2+](int) transient depend on the subsaturating dose of LIC, even though it too binds uniformly to all the cells, nonrespondin g as well as responding. These earlier findings have here been extende d by single cell flow cytometric analysis to demonstrate that F(ab')(2 ) Fc gamma RIII is the major Fc gamma R involved in HIC binding (and [ Ca2+](int) mobilization), as well as in oxidative burst and phospholip ase A activation. In contrast, both receptor subclasses must be availa ble for LIC-elicited Delta[Ca2+](int), as blockage by either of the mA b Fab or F(ab')(2) fragments abrogates this response, even though LIC binding to the receptors is not decreased. Furthermore, LIC elicited l ittle oxidative burst activity and failed to activate phospholipase A but crosslinking to achieve multivalency, previously shown to induce [ Ca2+](int) and oxidative burst responses, elicited phospholipase A act ivity via Fc gamma RIII. Fc gamma RIIIs role appears to be modulation of the small, late Ca2+ influx observed at >1 min, whereas Fc gamma RI II modulates all the earlier larger events. Thus, simultaneous observa tion of receptor identity, receptor occupancy, and consequent activati on parameters in the same cell by flow cytometry permits use to demons trate that Fc gamma RII is necessary for the small signal transduction elicited by LIC; it plays a relatively small role in polymorphonuclea r leukocyte stimulation by HIC. Fc gamma RIII is the main receptor res ponsible for immune complex-elicited polymorphonuclear leukocyte respo nses; its efficacy is greatly enhanced when the receptors are cross-li nked, either by preequilibrated multivalent complexes or by in situ cr oss-linking of bound LIC with excess antibody.