Subcellular localization and dynamics of Mac-1 (alpha m beta 2) in human neutrophils.

TLDR

Mac‑1 subcellular localization in human neutrophils was mapped by nitrogen cavitation, two‑layer Percoll fractionation, high‑voltage free‑flow electrophoresis, and ELISA using anti‑CD11b antibodies. In resting neutrophils, most Mac‑1 resides in specific granules, with a smaller fraction in secretory vesicles and plasma membrane; upon stimulation, Mac‑1 rapidly translocates from secretory vesicles (and to a lesser extent from specific granules) to the plasma membrane, producing 6–10‑fold surface increases with most inflammatory mediators and a 25‑fold rise with ionomycin, confirming secretory vesicles as the primary Mac‑1 reservoir.

Abstract

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.