Enhanced aggregation of human neutrophils by MnCl2 or DTT differentiates the roles of L-selectin and beta(2)-integrins

MnCl2 and dithiothreitol (DTT) enhance the adhesive functions of beta(2)-integrins. We have used these agents and flow cytometry to distinguish the contributions of beta(2)-integrins and L-selectin to neutrophil aggregation. Although neither compound induced aggregation, they prolonged N-formyl-methionyl-leucyl-phenylalanine -induced aggregation and produced larger aggregates. Because activated polymorphonuclear granulocytes (PMN) shed L-selectin in the presence of MnCl2, but not DTT, we could evaluate the role of L-selectin in the early and late stages of aggregation. Blocking L-selectin sites with DREG200 Fab and/or beta(2)-integrin sites with IB4 Fab indicated that aggregation under all conditions remained beta(2)-integrin- and L-selectin-dependent. Disaggregation was integrin-dependent whether L-selectin was present or shed. The disaggregation kinetics suggested that integrin bonds turned over at a slower rate in MnCl2-treated cells. Enhanced aggregation due to DTT and MnCl2 retired sustained energy output, suggesting intracellular rather than strictly conformational control. These results provide evidence that PMN aggregation, like leukocyte-endothelial cell adhesion, utilizes L-selectin to form intercellular contacts that are maintained through activated integrins.