Quantitation of intracellular Mac-1 (CD11b/CD18) pools in human neutrophils.
The adhesive glycoprotein Mac-1 (CD11b/CD18) of the CD11/CD18 complex contributes to multiple neutrophil inflammatory functions. Activation of neutrophils by chemotactic stimuli results in a rapid, protein synthesis-independent increase in surface Mac-1 derived from incompletely defined intracellular compartments. Therefore, we developed a novel quantitative lectin immunoblot technique to define intracellular pools of Mac-1 in subcellular neutrophil fractions resolved on discontinuous Percoll gradients. In cavitates of unstimulated neutrophils, 30% and 26% of total Mac-1 was identified in beta [1.10 gm/ml; vitamin B12 binding protein (vit B12 B.P.)-rich] or pre-gamma (1.07 gm/ml; vit B12 B.P.-poor) granular fractions, respectively, whereas 24% was associated with the plasma membrane-rich gamma (1.06 gm/ml) fractions. N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation (10(-8) M, 15 min, 37 degrees C) significantly diminished Mac-1 in pre-gamma (-18% of total, P less than 0.05) but not beta fractions (+6% of total). Under these conditions, the content of Mac-1 in gamma fractions increased 13% in association with four- to eightfold increase in surface Mac-1 expression (OKM-1 binding). These findings suggest that chemotactic stimuli increase plasma membrane and/or surface Mac-1 on human neutrophils by mobilizing a novel intracellular granule pool.
D000328:Adult; D000943:Antigens, Differentiation / Q000032:analysis*; D000954:Antigens, Surface / Q000032:analysis*; D018845:CD11 Antigens; D018821:CD18 Antigens; D006801:Humans; D008562:Membrane Glycoproteins / Q000032:analysis*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000032:analysis* / Q000648:ultrastructure; D014155:Transcobalamins / Q000032:analysis
D000943:Antigens, Differentiation; D000954:Antigens, Surface; D018845:CD11 Antigens; D018821:CD18 Antigens; D008562:Membrane Glycoproteins; D014155:Transcobalamins; D009240:N-Formylmethionine Leucyl-Phenylalanine