Neutrophil aggregation is beta 2-integrin- and L-selectin-dependent in blood and isolated cells.
Neutrophil aggregation in response to formyl peptide was analyzed in blood and isolated cells by fluorescence flow cytometry. The isolated leukocyte aggregates and the leukocytes in blood were identified with the vital nucleic acid stain LDS-751. This method enabled us to discriminate nucleated cells from other blood cells and to detect granulocyte aggregates without isolation or E lysis. Cells isolated in the absence of endotoxin retained the characteristics of cells in blood and exhibited similar aggregation kinetics and dose-response to formyl peptide. We show that it is possible to analyze epitope expression in blood with homogeneous flow cytometric assays and that carefully isolated neutrophils retain the expression characteristics of those in blood. The expression of CD18 was at its lowest levels in unstimulated cells, while the rate of formyl peptide stimulated aggregation was most rapid in these cells. Aggregation in isolated cells as well as blood preceded an increase in receptor expression. After stimulation, L-selectin expression decreased in both blood and isolated cells over a time frame similar to disaggregation. The aggregation response in blood was blocked by pretreatment with antibody to CD18 over a concentration range consistent with the amount of antibody bound. Aggregation was also blocked in isolated cells and blood by antibodies DREG-200 and DREG-56 to L-selectin, but not by isotype controls or anti-LFA-1. The results are discussed in terms of the roles of adhesive receptor expression and recognition in neutrophil aggregation. The methods validated here permit linkage between isolated cells and in vivo studies.
Simon
S I
SI
Department of Pathology, University of New Mexico School of Medicine, Albuquerque 87131.
Chambers
J D
JD
Butcher
E
E
Sklar
L A
LA
D015703:Antigens, CD / Q000032:analysis / Q000502:physiology; D018821:CD18 Antigens; D015815:Cell Adhesion Molecules / Q000502:physiology*; D002449:Cell Aggregation; D002478:Cells, Cultured; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D016023:Integrins / Q000502:physiology*; D019041:L-Selectin; D009504:Neutrophils / Q000502:physiology*; D009842:Oligopeptides / Q000494:pharmacology; D015854:Up-Regulation
D015703:Antigens, CD; D018821:CD18 Antigens; D015815:Cell Adhesion Molecules; D007140:Immunoglobulin Fab Fragments; D016023:Integrins; D009842:Oligopeptides; D019041:L-Selectin; C021642:F-chemotactic peptide