Direct visualization of formylpeptide receptor binding on rounded and polarized human neutrophils: cellular and receptor heterogeneity.

We have used light microscope autoradiography to visualize binding of the formylhexapeptide, N-formyl-norleucyl-leucyl-phenylalanyl-norleucyl-(125I)tyrosyl-lysine to rounded and spontaneously polarized human polymorphonuclear leukocytes. These cells possess receptors known to bind with high specificity and great avidity to the chemotactic formylpeptides. Cells adherent to glass slides were exposed to (125I)-hexapeptide at 4 degrees C, fixed, and autoradiographed. Hexapeptide binding was studied over the biologically active range of peptide concentrations varying from 0.63 nM to 10 nM and autoradiographic silver grains counted on 200 rounded or 50 polarized cells at each concentration. Examination of histograms plotted from these data revealed for rounded cells: two major peaks at each concentration indicating the existence of two neutrophil subpopulations, the predominant subpopulation binding one-half as much formylpeptide (peak I) as the other (peak II); progressively increasing proportions of cells in peak II as the free hexapeptide concentration increased. Accordingly, at 0.63 nM hexapeptide, peak II comprised only 8% of the total cell number, whereas at 10 nM, this peak represented 35% of the total cells. This suggested that different types of receptors may exist in the two cell subpopulations (high/low affinity or high/low negative cooperativity) and that these receptor types were expressed differentially on these subpopulations. Thus, cellular heterogeneity within the neutrophil population and receptor heterogeneity among hexapeptide receptors on an individual cell were both observed here. Each of these may significantly affect neutrophil functional responses to the chemotactic formylpeptides and may explain, at least in part, the curvilinearity in the Scatchard plots of formylpeptide receptor binding that has recently been reported. At higher concentrations of peptide (greater than or equal to 5 nM), spontaneously polarized PMN bound hexapeptide more or less uniformly over the entire cell surface. However, at lower concentrations, hexapeptide binding was markedly shifted toward the cell anterior. As a group, polarized PMN bound similar total quantities of hexapeptide, as did rounded PMN at each peptide concentration tested. Receptors displaying high- and low-affinity characteristics were, however, distributed asymmetrically over the cell surface, with the high-affinity type receptors predominantly on the anterior one-half of the cell. Such an asymmetric distribution may serve to initiate or perpetuate cell locomotion.