Differential Requirement for Classic and Novel PKC Isoforms in Respiratory Burst and Phagocytosis in RAW 264.7 Cells

Abstract

The binding of Ab (IgG)-opsonized particles by FcγRs on macrophages results in phagocytosis of the particles and generation of a respiratory burst. Both IgG-stimulated phagocytosis and respiratory burst involve activation of protein kinase C (PKC). However, the specific PKC isoforms required for these responses have yet to be identified. We have studied the involvement of PKC isoforms in IgG-mediated phagocytosis and respiratory burst in the mouse macrophage-like cell line, RAW 264.7. Like primary monocyte/macrophages, their IgG-mediated phagocytosis was calcium independent and diacylglycerol sensitive, consistent with novel PKC activation. Respiratory burst in these cells was Ca2+ dependent and inhibited by staurosporine and calphostin C as well as by the classic PKC-selective inhibitors Gö 6976 and CGP 41251, suggesting that classic PKC is required. In contrast, phagocytosis was blocked by general PKC inhibitors but not by the classic PKC-specific drugs. RAW 264.7 cells expressed PKCs α, βI, δ, ε, and ζ. Subcellular fractionation demonstrated that PKCs α, δ, and ε translocate to membranes during phagocytosis. In Ca2+-depleted cells, only novel PKCs δ and ε increased in membranes, and the time course of their translocation was consistent with phagosome formation. Confocal microscopy of cells transfected with green fluorescent protein-conjugated PKC α or ε confirmed that these isoforms translocated to the forming phagosome in Ca-replete cells, but only PKC ε colocalized with phagosomes in Ca2+-depleted cells. Taken together, these results suggest that the classic PKC α mediates IgG-stimulated respiratory burst in macrophages, whereas the novel PKCs δ and/or ε are necessary for phagocytosis.

Cytosolic Phospholipase A2 Translocates to Forming Phagosomes during Phagocytosis of Zymosan in Macrophages

“…Synchronized Phagocytosis and Immunofluorescence MicroscopyThe protocol for synchronized phagocytosis was adapted from Larsen et al (38). Briefly, macrophages plated in Plastek dishes and either infected or not with AdGFP-cPLA 2 ␣ were washed in cold HHBSS and cooled on ice for 5 min prior to addition of zymosan.…”

Recognition of apoptotic cells by macrophages activates the peroxisome proliferator-activated receptor-γ and attenuates the oxidative burst

“…For human monocytes and RAW264.7 macrophages, it has been shown that the primary event, that is, phosphorylation of p47 phox , is facilitated by protein kinase Ca (PKCa). 7,8 Recently, we demonstrated that downregulation of PKCa or expression of a kinase-dead mutant attenuates the PMA-induced oxidative burst. 9 However, the impact of AC on the oxidative burst remains unknown.…”

The coordination of signaling during Fc receptor-mediated phagocytosis

“…Various PKC isoforms are recruited to forming phagosomes [49,50,66]. PKCε appears to regulate the rate of phagosome formation, as overexpression of PKCε increased rates of phagocytosis [50].…”

“…PLC hydrolyzes PI(4,5)P 2 to diacylglycerol and inositol trisphosphate, which can activate PKC and increase cytosolic-free calcium levels, respectively. Although increases in calcium are not necessary for phagocytosis by macrophages [88,89], local activation of PKC via diacylglycerol is important for phagosome formation [66] and activation of the NADPH oxidase complex [9]. PLA 2 , PLA-D 1 , and PLA-D 2 are also necessary for phagocytosis, perhaps because of their roles in membrane fusion reactions [46,68,69,90].…”