Evidence for a third component in neutrophil aggregation: potential roles of O-linked glycoproteins as L-selectin counter-structures.

The homotypic aggregation of neutrophils requires the participation of L-selectin and the beta 2-integrins, but it has not been clear whether the two receptors recognize one another as counter-structures or whether other adhesion molecules are involved. We have examined aggregation of live neutrophils with target populations, manipulated to alter expression of adhesive epitopes, using flow cytometry. A target population depleted of both L-selectin and activatable beta 2-integrin displayed an ability to aggregate with live neutrophils, suggesting that these two molecules are not counter-structures. We also found that an O-sialoglycoprotease (GCP) from Pasteurella haemolytica is capable of inhibiting homotypic aggregation. Neutrophils treated with GCP lose O-glycosylated proteins but retain L-selectin and activatable beta 2-integrin. One or more of the GCP substrates appears to function in L-selectin-dependent binding but not in beta 2-integrin-dependent binding. Together the data suggest a mechanism of aggregation that is analogous to leukocyte-endothelial cell adhesion in which a low-affinity carbohydrate-dependent interaction precedes a high-affinity integrin-dependent adhesion.