Related Experiment Videos
Functional, physical, and ultrastructural localization of CD15 antigens to the human polymorphonuclear leukocyte
E S Buescher1, S A Livesey, J G Linner
1Department of Pediatrics, University of Texas Medical School, Houston 77030.
Abstract:
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.
Insights
This study investigated CD15 antigens in polymorphonuclear leukocytes (PMNs). CD15 antigens are primarily located within secondary granules, with some released extracellularly upon stimulation.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Polymorphonuclear leukocytes (PMNs) play a crucial role in innate immunity.
- CD15 antigens (3-fucosyllactosamine) are cell surface molecules found on various immune cells, including PMNs.
- The precise subcellular localization of CD15 antigens within PMNs has not been fully elucidated.
Purpose of the Study:
- To determine the subcellular localization of CD15 antigens within human polymorphonuclear leukocytes (PMNs).
- To investigate the association of CD15 antigens with specific PMN granule populations.
- To explore the release of CD15 antigens from PMNs upon stimulation.
Main Methods:
- Utilized a murine monoclonal IgM antibody (M3) that inhibits PMN phagocytosis and bactericidal activity.
- Employed Percoll gradient centrifugation to isolate PMN secondary granule fractions.
- Applied SDS-PAGE/electroblot, sonication/sedimentation, Triton X-114 phase partitioning, and ultrastructural analysis for antigen characterization.
- Stimulated purified PMNs with phorbol myristate acetate to induce granule secretion.
Main Results:
- Secondary granule fractions were enriched in CD15 antigens, with at least seven distinct antigens identified.
- Both soluble and sedimentable forms of CD15 antigens were detected within secondary granules.
- Extracellular release of two to three CD15 antigens was observed following PMN stimulation.
- Triton X-114 partitioning indicated that some CD15 antigens are integral membrane components of secondary granules.
- Ultrastructural studies revealed CD15 antigen localization on both the granule matrix/membrane and exclusively on the granule membrane.
- Colocalization with lactoferrin confirmed the presence of CD15 antigens in a subset of secondary granules.
Conclusions:
- CD15 antigens are predominantly localized within the secondary granules of polymorphonuclear leukocytes (PMNs).
- These antigens exist in both soluble and membrane-associated forms within the granules.
- PMN activation leads to the release of specific CD15 antigens into the extracellular environment.
- The findings suggest CD15 antigens are important components of PMN secondary granule function and release.