Cellular Localization and Functional Role of Phosphatidylcholine-Specific Phospholipase C in NK Cells

Abstract

Although several classes of phospholipases have been implicated in NK cell-mediated cytotoxicity, no evidence has been reported to date on involvement of phosphatidylcholine-specific phospholipase C (PC-PLC) in NK activation by lymphokines and/or in lytic granule exocytosis. This study demonstrated the expression of two PC-PLC isoforms (M(r) 40 and 66 kDa) and their IL-2-dependent distribution between cytoplasm and ectoplasmic membrane surface in human NK cells. Following cell activation by IL-2, cytoplasmic PC-PLC translocated from the microtubule-organizing center toward cell periphery, essentially by kinesin-supported transport along microtubules, while PC-PLC exposed on the outer cell surface increased 2-fold. Preincubation of NK cells with a PC-PLC inhibitor, tricyclodecan-9-yl-xanthogenate, strongly reduced NK-mediated cytotoxicity. In IL-2-activated cells, this loss of cytotoxicity was associated with a decrease of PC-PLC exposed on the cell surface, and accumulation of cytoplasmic PC-PLC in the Golgi region. Massive colocalization of PC-PLC-rich particles with perforin-containing granules was found in the cytoplasm of NK-activated (but not NK-resting) cells; both organelles clustered at the intercellular contact region of effector-target cell conjugates. These newly detected mechanisms of PC-PLC translocation and function support an essential role of this enzyme in regulated granule exocytosis and NK-mediated cytotoxicity.

Expression and role of phosphatidylcholine‐specific phospholipase C in human NK and T lymphocyte subsets

“…In our previous study [12], we reported evidence of the localization of PC-PLC inside PFN-containing granules of NK cells and of the involvement of the enzyme in PFN transportation towards the immunological synapse. To investigate whether PFN molecules also translocate to the CD8 + T immunological synapse together with PC-PLC enzyme, we performed CLSM analyses that did not indicate any correlation between the localization of PC-PLC (red) and PFN molecules (green) in resting or PHAactivated CD8 + T cells (Fig.…”

“…We recently reported that a PC-PLC inhibitor, D609, strongly reduced PFN-dependent NK-mediated cytolytic activity and induced alterations in intracellular PC-PLC and PFN traffic [12].…”

“…To verify a possible analogy with NK cells, in which PC-PLC has been found to undergo IL-2-dependent translocation from cytoplasmic areas to the membrane surface [12], we investigated the effect of cell activation on PC-PLC expression in CD8 + and CD4 + T lymphocytes. As shown in Fig.…”

“…A major gap in our knowledge of agonist-stimulated PC hydrolysis mechanisms is linked to the fact that mammalian neutral-active PC-PLC has not yet been cloned and fully characterized, although direct evidence clearly demonstrates the expression of PC-PLC isoforms in mammalian cells [9][10][11][12][13].…”

“…Recent studies in our laboratory characterized the subcellular localization and suggested a functional role of PC-PLC in human NK-mediated cell killing [12].…”