Immunochemical Analysis of Carcinoembryonic Antigen (CEA)-related Antigens Differentially Localized in Intracellular Granules of Human Neutrophils

Subcellular localization and antigenic properties of eight carcinoembryonic antigen (CEA)-related proteins (nonspecific cross-reacting antigens, NCAs) in neutrophils, including CD66 antigens, were examined with a panel of CD66 monoclonal antibodies (MAbs), whose specificities were characterized with CHO cells expressing CEA gene family recombinant proteins. Intracellular granules of neutrophils were isolated by cell lysis followed by centrifugation on a sucrose gradient. SDS-PAGE analysis of immunoprecipitates from the granules revealed that NCA-95 (CD66c, NCA-50/90) exists predominantly in the azurophil granule-enriched fraction. NCA-90 was identified in the same fraction and was suggested to be a molecule closely-related to NCA-95. NCA-26 was identified in the specific granule fraction together with NCA-160 (CD66a, BGP), and was likely a splicing variant of NCA-160. NCA-100 (CD66b, CGM6) was also identified in this fraction. NCA-80, -58 and -30, which are detectable in culture medium of neutrophils with polyclonal anti-NCA-50/90, were not recognized by any of the CD66 MAbs tested. These findings indicate that NCA-160, -100, -95, -90 and -26 are the products of the CEA gene family and stored separately in azurophil and specific granules. It remains to be clarified whether or not the other three secretory antigens, NCA-80, -58 and -30, are still unknown members of the CEA family.