Immunocomplexes stimulate different signalling events to chemoattractants in the neutrophil and regulate L-selectin...

Neutrophils express receptors for numerous phlogistons which, when occupied, trigger distinct signal-transduction pathways. Previous studies have shown that stimulation of neutrophils with chemoattractants induces shedding of the adhesive molecule L-selectin and increased expression of the beta 2-integrin CD11b/CD18. We determined the effect of ligation of classic, G-protein-linked chemoattractant receptors [C5a, interleukin-8 (IL-8), formylmethionyl-leucylphenylalanine (FMLP) and substance P], receptors for the Fc portion of IgG (Fc gamma receptors) and receptors for transforming growth factor beta (TGF beta) on expression of adhesive molecules by neutrophils and the stimulus-transduction mechanisms thought to mediate these changes. We were surprised to observe that occupancy of Fc gamma receptors by immunocomplexes (BSA-anti-BSA) stimulated increased expression by neutrophils of CD11b/CD18 at concentrations which did not affect L-selectin expression (EC50 9 micrograms/ml versus 350 micrograms/ml respectively, P < 0.00001, n = 5). In contrast, similar to previous studies, recombinant C5a, recombinant IL-8 and FMLP all stimulated increased expression of CD11b/CD18 (170-260% of basal, P < 0.001, n = 5) and shedding of L-selectin (56-75% reduction from basal, P < 0.001, n = 5) at similar concentrations and with similar potencies (EC50 = 2, 5, and 3 nM respectively). In contrast, neither TGF beta 1 nor, surprisingly, substance P affected expression of CD11b/CD18 or L-selectin. The regulation of expression of CD11b/CD18 or L-selectin in response to FMLP or immunocomplexes was unaffected by cytochalasin B (5 micrograms/ml) or the tyrosine kinase inhibitor tyrphostin-25 (25 microM). Although occupancy of both chemoattractant (FMLP) and Fc gamma receptors stimulated increments in the second messenger diacylglycerol, disruption of actin microfilaments by cytochalasin B enhanced diacylglycerol generation in response to FMLP but not in response to ligation of Fc gamma receptors. Moreover, both FMLP and immune aggregates provoked fluxes of intracellular Ca2+ concentration which differed with respect to both magnitude and kinetics and did not correlate well with regulation of adhesive-molecule expression. As upregulation of CD11b/CD18 is tightly linked to exocytosis of specific granules, these results suggest that shedding of L-selectin by activated neutrophils is not linked to exocytosis. These studies provide further evidence that receptors for chemoattractants and immunocomplexes on the neutrophil are linked to multiple signalling pathways.