Localization and quantitation of the vitamin D binding protein (Gc-globulin) in human neutrophils.
Plasma-derived vitamin D binding protein (DBP) is an important physiologic regulator of the neutrophil chemotactic response to activated complement. A cell-associated form of DBP has been observed in numerous cell types. We now report that mature, circulating human neutrophils also contain cell-associated DBP. Immunofluorescence studies of normal untreated neutrophils showed the presence of DBP on the cell surface. Western blotting of detergent-soluble neutrophil lysates with a polyclonal anti-DBP showed two major immunoreactive bands, one with an apparent molecular weight of 56 Kd (identical to purified plasma-derived DBP) and a second less prominent band at 12 to 14 Kd. Quantitation of the immunoreactive bands by video densitometry indicated that normal human neutrophils contain 1.5 +/- 0.8 ng DBP/10(6) cells (n = 9). Immunoprecipitation of detergent-soluble lysates with the polyclonal anti-DBP showed only the 56-Kd form by Western blotting. In contrast, a monoclonal anti-DBP immunoprecipitated the 12 to 14 Kd form of DBP from lysates of surface-radioiodinated cells. Western blots of subcellular fractions showed that immunoreactive bands were found in the specific (secondary) granule and plasma-membrane fractions. In addition, pretreatment of neutrophils with 10 nmol/L phorbol myristate acetate (PMA) resulted in approximately a 50% reduction in the amount of DBP in both the specific granule and plasma-membrane fractions. Finally, analysis of the cell-free supernates showed that DBP was spontaneously released into the extracellular milieu: moreover, this release was enhanced if the cells were first stimulated with C5a, formyl-norleucyl-leucyl-phenylalanine (fNLP) or PMA.
Kew
R R
RR
Department of Pathology, State University of New York, Stony Brook 11794-8691.
Sibug
M A
MA
Liuzzo
J P
JP
Webster
R O
RO
D015153:Blotting, Western; D003594:Cytoplasmic Granules / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D008970:Molecular Weight; D009504:Neutrophils / Q000378:metabolism*; D011233:Precipitin Tests; D014809:Vitamin D-Binding Protein / Q000097:blood* / Q000276:immunology
D014809:Vitamin D-Binding Protein