Functional, physical, and ultrastructural localization of CD15 antigens to the human polymorphonuclear leukocyte secondary granule.
A murine monoclonal IgM antibody, M3, which interferes with both polymorphonuclear leukocyte (PMN) phagocytosis and bactericidal activity, was used to examine the subcellular location of antigens bearing 3-fucosyllactosamine (CD15 antigens) within this cell type. Percoll gradient-separated secondary granule fractions were rich in CD15 antigens, with at least seven antigens recognizable in SDS-PAGE/electroblot studies. Sonication/sedimentation experiments using secondary granule fractions showed that both soluble and sedimentable CD15 antigens were present. Exposure of purified PMN to the secondary granule secretagogue phorbol myristate acetate caused extracellular release of two or three CD15 antigens, which could be purified by immunoprecipitation using antibody M3. Triton X-114 phase-partition experiments showed that secondary granule fraction CD15 antigens could be partitioned into hydrophilic (aqueous phase) and hydrophobic (detergent phase) antigens, suggesting that several of these antigens were integral secondary granule membrane components. Ultrastructurally, PMN intracellular granules showed two patterns of CD15 expression, localization over both granule matrix/granule membrane and localization to only granule membrane. Colocalization studies showed that lactoferrin and CD15 antigens were both present in a subset of intracellular granules, confirming a secondary granule location for these antigens.
Buescher
E S
ES
Department of Pediatrics, University of Texas Medical School, Houston 77030.
Livesey
S A
SA
Linner
J G
JG
Skubitz
K M
KM
McIlheran
S M
SM
D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D000943:Antigens, Differentiation / Q000032:analysis* / Q000378:metabolism / Q000502:physiology; D000954:Antigens, Surface / Q000032:analysis / Q000378:metabolism / Q000502:physiology; D002458:Cell Fractionation / Q000379:methods; D003594:Cytoplasmic Granules / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D016256:Lewis X Antigen; D008854:Microscopy, Electron; D009504:Neutrophils / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D011233:Precipitin Tests; D014465:Ultrasonics
D000911:Antibodies, Monoclonal; D000943:Antigens, Differentiation; D000954:Antigens, Surface; D016256:Lewis X Antigen; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40