The two neutrophil plasma membrane markers alkaline phosphatase and HLA class I antigen localize differently in granule-deficient cytoplasts. An ideal plasma membrane marker in human neutrophils is still lacking.
Neutrophil function relies largely on the ability of the cell to mobilize its different granules and vesicles to the cell surface and thereby expose and/or release effector molecules to the surrounding tissue. To properly identify these subcellular compartments is thus a prerequisite for studies of neutrophil physiology. A range of specific markers for the classical granules is available, but finding optimal markers for the secretory vesicles and plasma membrane has historically been more challenging. Latent and non-latent alkaline phosphatase activities are often used to distinguish these two light membrane structures, but the outcome using this technique depends on the level of cellular activation. Therefore, HLA-I was introduced some years ago as a specific, stimulation-independent marker for the plasma membrane. In this study we however report that detailed fractionation studies of neutrophil cytoplasts, lacking secretory vesicles, granules and other dense organelles, reveal that the HLA-I antigen is not only co-localizing with the plasma membrane marker ALP, but is also present in other, more dense organelles. Further, we found the mixed enzyme-linked immunosorbent assay (MELISA), detecting the beta(2)-microglobulin/HLA-I complex, to be negatively influenced by uncomplexed beta(2)-microglobulin present in the specific granules and secretory vesicles, making it difficult to use HLA-I as a plasma membrane marker during maturation of for example phagolysosomes.
The Phagocyte Research Laboratory, Department of Rheumatology and Inflammation Research, University of Göteborg, Sweden. sara.pellme@rheuma.gu.se
Sara
S
Pellmé
Claes
C
Dahlgren
Anna
A
Karlsson
D000469:Alkaline Phosphatase / Q000032:analysis / Q000378:metabolism*; D015415:Biomarkers / Q000032:analysis / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D015395:Histocompatibility Antigens Class I / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D046529:Microscopy, Electron, Transmission; D009504:Neutrophils / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D015388:Organelles / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism; D001613:beta 2-Microglobulin / Q000032:analysis / Q000378:metabolism
D015415:Biomarkers; D015395:Histocompatibility Antigens Class I; D001613:beta 2-Microglobulin; D000469:Alkaline Phosphatase