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Quantitation of Intracellular Mac‐1 (CD11b/CD18) Pools in Human Neutrophils
Author(s) -
Jones Douglas H.,
Anderson Donald C.,
Burr Bean L.,
Rudioff Helen E.,
Smith C. Wayne,
Krater Sharon S.,
Schmalstieg Frank C.
Publication year - 1988
Publication title -
journal of leukocyte biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.819
H-Index - 191
eISSN - 1938-3673
pISSN - 0741-5400
DOI - 10.1002/jlb.44.6.535
Subject(s) - intracellular , biology , percoll , chemotaxis , cd18 , n formylmethionine leucyl phenylalanine , integrin alpha m , stimulation , microbiology and biotechnology , neutrophile , biochemistry , flow cytometry , endocrinology , centrifugation , in vitro , receptor
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.