Subcellular localization and dynamics of Mac-1 (alpha m beta 2) in human neutrophils.
The subcellular localization of Mac-1 was determined in resting and stimulated human neutrophils after disruption by nitrogen cavitation and fractionation on two-layer Percoll density gradients. Light membranes were further separated by high voltage free flow electrophoresis. Mac-1 was determined by an ELISA with monoclonal antibodies that were specific for the alpha-chain (CD11b). In unstimulated neutrophils, 75% of Mac-1 colocalized with specific granules including gelatinase granules, 20% with secretory vesicles and the rest with plasma membranes. Stimulation with nanomolar concentrations of FMLP resulted in the translocation of Mac-1 from secretory vesicles to the plasma membrane, and only minimal translocation from specific granules and gelatinase granules. Stimulation with PMA or Ionomycin resulted in full translocation of Mac-1 from secretory vesicles and gelatinase granules to the plasma membrane, and partial translocation of Mac-1 from specific granules. These findings were corroborated by flow cytometry, which demonstrated a 6-10-fold increase in the surface membrane content of Mac-1 in response to stimulation with FMLP, granulocyte-macrophage colony stimulating factor, IL-8, leukotriene B4, platelet-activating factor, TNF-alpha, and zymosan-activated serum, and a 25-fold increase in response to Ionomycin. Thus, secretory vesicles constitute the most important reservoir of Mac-1 that is incorporated into the plasma membrane during stimulation with inflammatory mediators.
Department of Hematology L, University Hospital, Copenhagen, Denmark.
H
H
Sengeløv
L
L
Kjeldsen
M S
MS
Diamond
T A
TA
Springer
N
N
Borregaard
D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D016177:Macrophage-1 Antigen / Q000378:metabolism*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000378:metabolism* / Q000648:ultrastructure; D013347:Subcellular Fractions / Q000737:chemistry
D016177:Macrophage-1 Antigen; D009240:N-Formylmethionine Leucyl-Phenylalanine