Subcellular localization of CD66, CD67, and NCA in human neutrophils

Abstract

CD66 and CD67 are granulocyte-specific activation antigens; their surface expression is up-regulated when neutrophils are activated. CD66 antibodies recognize an approximately 180-kd neutrophil surface protein that is also recognized by anti-carcinoembryonic antigen (CEA) antibodies and is therefore a nonspecific cross-reacting antigen (NCA). CD67 antibodies recognize an approximately 100-kd neutrophil surface protein that is attached to the membrane via a glycosyl-phosphatidylinositol anchor. To identify an intracellular pool from which CD66 and CD67 could be up-regulated, the subcellular distribution of proteins recognized by CD66 and CD67 monoclonal antibodies and polyclonal anti-CEA was studied. Neutrophil plasma membranes, granules, and cytoplasm were prepared by nitrogen cavitation and differential centrifugation and then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Most of the 180-kd protein recognized by CD66 antibodies and the 100-kd protein recognized by CD67 antibodies were located in the secondary granule fraction, with lesser amounts detectable in the plasma membrane fraction. Several NCA species ranging from approximately 40 to 200 kd were identified, and the distribution of these NCAs was different in the primary granules, secondary granules, and plasma membrane fractions. The major NCAs in the plasma membrane fraction were of approximately 95 to 100 and approximately 180 to 200 kd; the secondary granule fraction contained major NCAs of approximately 42, 85, 95 to 100, and 180 to 200 kd. NCAs were also detected in the primary granule fraction, the most prominent being of approximately 90-100 kd; no NCA of approximately 180 to 200 kd was detected in the primary granules. The presence of CD66, CD67, and NCAs in the secondary granules suggests secondary granules as a likely source from which these antigens could be recruited to the cell surface with activation. The potential role for NCAs in the primary granules is unknown.