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 required 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.
Lynam
E B
EB
Department of Pathology, University of New Mexico School of Medicine, Albuquerque 87131, USA.
Rogelj
S
S
Edwards
B S
BS
Sklar
L A
LA
D000918:Antibody Specificity; D018821:CD18 Antigens / Q000502:physiology*; D002448:Cell Adhesion / Q000187:drug effects; D002449:Cell Aggregation / Q000187:drug effects; D002712:Chlorides / Q000494:pharmacology*; D004229:Dithiothreitol / Q000494:pharmacology*; D006801:Humans; D017895:Manganese Compounds / Q000494:pharmacology*; D009504:Neutrophils / Q000166:cytology* / Q000187:drug effects* / Q000502:physiology; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
D018821:CD18 Antigens; D002712:Chlorides; D017895:Manganese Compounds; D013439:Sulfhydryl Reagents; C025340:manganese chloride; D004229:Dithiothreitol