ENDOTOXIN RECEPTORS (CD14) ARE FOUND WITH CD16 (FC-GAMMA-RIII) IN AN INTRACELLULAR COMPARTMENT OF NEUTROPHILS THAT CONTAINS ALKALINE-PHOSPHATASE

CD14 is a glycosylphosphatidylinositol (CPI)-anchored protein on the s urfaces of monocytes and polymorphonuclear leukocytes (PMN) that binds and initiates cellular responses to bacterial LPS. PMN also contain a n intracellular pool of CD14 that can be deployed rapidly to the cell surface in response to stimulation with a variety of agonists. To dete rmine which of the well-characterized subcellular compartments of PMN contains CD14, cells were cavitated and fractionated on Percoll gradie nts. The gradient fractions were assayed for CD14 by ELISA and Western blot and for the marker proteins beta-glucuronidase (azurophil granul es), vitamin B12 binding protein (specific granules), alkaline phospha tase (secretory vesicles and plasma membrane), and HLA (plasma membran e). Approximately one-half of the CD14 ran with plasma membrane fracti ons and one-half with intracellular membranes of light density. Both i ntracellular and cell surface CD14 were associated tightly with membra ne, and both forms showed identical electrophoretic mobility. The intr acellular CD14 was clearly not present in azurophil granules or specif ic granules, but ran precisely with alkaline phosphatase, a marker for secretory vesicles. Parallel studies showed that an additional GPI-li nked protein, Fc gamma RIII (CD16), also fractionated precisely with C D14 and alkaline phosphatase. Association of CD14 with secretory vesic les was confirmed by studies on cells stimulated with the formyl pepti de fNLLP for 20 min at 37 degrees C before fractionation. This treatme nt caused translocation of CD14 from intracellular fractions to plasma membrane fractions. No release of the specific granule marker vitamin B12 binding protein was observed under these conditions, whereas two other GPI-anchored proteins, alkaline phosphatase and CD16, moved coin cidentally with CD14 to comigrate with the plasma membrane; Time cours e studies of CD14 and CD16 surface expression confirmed the rapid and coordinate up-regulation of these proteins. Thus, the intracellular co mpartment containing CD14 and CD16 had the properties of secretory ves icles. These vesicles may represent a specialized membrane domain of P MN enriched in GPI-anchored proteins.