Relationship of C5a receptor modulation to the functional responsiveness of human polymorphonuclear leukocytes to C5a

The relationship of C5a receptor expression on human polymorphonuclear leukocytes (PMN) to the functional response of these cells to C5a was studied using flow cytometry. C5a receptor expression was determined with a fluorescein conjugate of C5a and oxidative burst activity was monitored by conversion of dichlorofluorescein to dichlorofluorescein (DCF) as a measure of H2O2 production. These studies showed that after incubation of PMN with increasing concentrations of C5a, and allowing for internalization of bound ligand, more than 40% of the cell surface C5a receptors were internalized before the DCF response to optimal concentrations of C5a was decreased below the levels for untreated control cells. Although C5a responsiveness was lost after preincubation with 10(-8) M C5a, cells remained responsive to formyl peptide. In other studies, cells were preincubated with unlabeled C5a under conditions that provided for internalization of nearly all C5a receptors. PMN were then cultured for up to 90 min and monitored for C5a receptor reexpression and return of cell function. In these studies, the DCF response of PMN to C5a returned to 100% much earlier than the cells regained full expression of C5a receptors. The DCF response to formyl peptide remained intact throughout the period of C5a receptor reexpression. These studies showed that once > 40% of the original population of C5a receptors are reexpressed on the PMN, that these cells regain 100% of their functional responsiveness to C5a in the DCF assay. Evaluation of the affinity and number of C5a receptors using 125I-labeled C5a after receptor reexpression showed that maximal receptor reexpression was approximately 73% of that obtained with control cells and the Kd of reexpressed receptors was 0.60 vs 0.94 nM for control cells. These studies demonstrate that only a portion of the total C5a receptors expressed on PMN are essential to stimulate a 100% functional response in PMN and that the reexpressed receptors are capable of transducing a signal that activates the oxidative burst in these cells.