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 β [1.10 gm/ml; vitamin B 12 binding protein (vit B 12 B.P.)‐rich] or pre‐γ (1.07 gm/ml; vit B 12 B.P.‐poor) granular fractions, respectively, whereas 24% was associated with the plasma membrane‐rich γ (1.06 gm/ml) fractions. N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP) stimulation (10 −8 M, 15 min, 37°C) significantly diminished Mac‐1 in pre‐γ (‐18% of total, P < 0.05) but not β fractions (+6% of total). Under these conditions, the content of Mac‐1 in γ 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.