Functional role of phosphatidylcholine‐specific phospholipase C in regulating CD16 membrane expression in natural killer cells

Abstract

CD16, the low-affinity FcIgG receptor (FccRIIIA), is predominantly expressed in human NK cells. Our recent findings indicate that CD16 expression on the outer membrane surface of NK cells is correlated with the membrane expression of phosphatidylcholinespecific phospholipase C (PC-PLC). In the present study we analyzed the trafficking of CD16 from the plasma membrane to cytoplasmic regions, after stimulation with specific mAb. The CD16 receptor is internalized, likely degraded and newly synthesized; its endocytosis is independent of ATP, but requires an integral and functional actin cytoskeleton. Antibody-mediated CD16 cross-linking results in an approximately twofold increase in PC-PLC enzymatic activity within 10 min. Analysis of PC-PLC and CD16 distribution in NK cell plasma membrane demonstrates that the proteins are physically associated and partially accumulated in lipid rafts. Pre-incubation of NK cells with a PC-PLC inhibitor, D609, causes a dramatic decrease both in CD16 receptor and PC-PLC enzyme expression on the plasma membrane. Interestingly, among phenotype PBL markers, only CD16 is strongly down-modulated by D609 treatment. CD16-mediated cytotoxicity is also reduced after D609 incubation. Taken together, these data suggest that the PC-PLC enzyme could play an important role in regulating CD16 membrane expression, the CD16-mediated cytolytic mechanism and CD16-triggered signal transduction.

Phosphatidylcholine-Specific Phospholipase C Activation in Epithelial Ovarian Cancer Cells

“…2A), in agreement with previous reports in other mammalian cells (15,16,(25)(26)(27)33). By densitometric analysis, the intensity of the corresponding protein band in most of the investigated cancer cell lines (OVCAR3, SKOV3, and IGROV1) was 1.9 F 0.5 times higher than that observed in nontumoral cells.…”

“…PC-PLC subcellular distribution in either unfixed or fixed and permeabilized cells was characterized by indirect immunofluorescence and confocal laser scanning microscopy (CLSM) analysis, as previously described (16,(25)(26)(27).…”

“…Protein concentration was determined by Bio-Rad protein assay according to the manufacturer's protocol (Bio-Rad Laboratories, Inc.) and total cell lysates or purified membranes (30 Ag proteins) were analyzed by Western blotting, as previously described (27). Densitometry analyses of protein bands were performed with a Bio-Rad apparatus (Bio-Rad Laboratories Srl) using the Quantity One software.…”

“…In fact, this enzyme has been implicated in cellular processes crucial for cell signaling, such as (a) mitogendriven and oncogene-driven extracellular signal-regulated kinase phosphorylation and activation of gene transcription factors in fibroblasts (16,17), (b) agonist stimulation of cells transfected with receptor proteins (18), (c) neuronal and endothelial cell differentiation (19)(20)(21), (d) programmed cell death (22,23), and (e) activation of cells of the immune system (24)(25)(26)(27)(28)(29)(30).…”

Immune Surveillance Properties of Human NK Cell-Derived Exosomes

“…Cytotoxic assay. Cytolysis was assessed, measuring the cell uptake of propidium iodide (PI) by flow cytometry analysis, as previously described with some modifications (28,29). Briefly, purified exosomes were used as effectors and mixed with different target cells, either PBMC or tumor cell lines, at the ratios of 60 mg NK-derived exosome (NKEXO) and LAKEXO or 300 mg plasma-derived exosomes to 50 3 10 3 target cells in complete medium at 37˚C in 5% CO 2 .…”