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I Protein Kinase C during Fc
R-Mediated Phagocytosis in Microglia1
* Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe, Japan;
Center for Cell Biology and Cancer Research, Albany Medical College, Albany, NY 12208;
Department of Anatomy and Cell Biology, Kochi Medical School, Kochi, Japan;
Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan;
¶ Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan;
|| Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Japan; and
# CREST JST (Japan Science and Technology)
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Abstract |
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R-mediated phagocytosis, we used live, digital fluorescence imaging of mouse microglial cells expressing GFP-tagged molecules. 
I PKC, 
PKC, and diacylglycerol kinase (DGK) dynamically and transiently accumulated around IgG-opsonized beads (BIgG). Moreover, the accumulation of p47phox, an essential cytosolic component of NADPH oxidase and a substrate for I PKC, at the phagosomal cup/phagosome was apparent during BIgG ingestion. Superoxide (O2–) production was profoundly inhibited by Gö6976, a cPKC inhibitor, and dramatically increased by the DGK inhibitor, R59949. Ultrastructural analysis revealed that BIgG induced O2– production at the phagosome but not at the intracellular granules. We conclude that activation/accumulation of I PKC is involved in O2– production, and that O2– production is primarily initiated at the phagosomal cup/phagosome. This study also suggests that DGK plays a prominent role in regulation of O2– production during FcR-mediated phagocytosis. |
Introduction |
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, complement, scavenger, mannose, phosphatidylserine, and TLR (1). Over the past few years, phagocytosis in microglia, especially FcR-mediated phagocytosis, has attracted a great deal of attention in the context of potential therapy for Alzheimer’s disease (2).
Protein kinase C (PKC)3 comprises a family of 10 isoforms (3). The conventional isoforms (cPKC; 
, I, II, and ) are Ca2+ and diacylglycerol (DAG)-dependent, the novel isoforms (nPKC; 
, , 
, and 
) are also DAG-dependent, but Ca2+ independent, and the atypical isoforms (
and 
) are nonresponsive to Ca2+ or DAG. Phenotypes for various PKC knockout mice have shown a role for a specific isoform in the immune system. Mice deficient in PKC have a marked immunodeficiency (4), and show a reduced production of the superoxide (O2–), a precursor of microbicidal oxidants, during FcR-mediated phagocytosis in neutrophils (5 ). In the absence of PKC, host defense against bacterial infection is severely compromised (6). The signaling capacity of DAG can be terminated by its conversion to phosphatidic acid through the action of diacylglycerol kinase (DGK). DGK family is composed of nine mammalian subtypes and is grouped into five classes; type I DGK (, , and ) has Ca2+-binding motifs (7). Because excessive O2– production appears to be harmful to normal cells and tissues (8), it is implicated that O2– production involved in PKC has a tight regulation by DGK isoform.
FcR-mediated phagocytosis is a spatiotemporally regulated signaling cascade with two rapid responses, remodeling of the cytoskeleton and activation of the respiratory burst. Phospholipase C (PLC) , a key enzyme for actin remodeling, is activated upon engagement of FcRs (9). DAG and inositol-1,4,5-triphosphate (IP3) from PLC activation exert their effects by stimulating PKC or by changing intracellular Ca2+ concentration ([Ca2+]i), respectively. The respiratory burst is initiated by the phagocyte NADPH oxidase, which is dormant in resting cells, but becomes activated during phagocytosis to produce O2– (10). NADPH oxidase is a multiprotein complex that is assembled from a membrane-spanning cytochrome b558 (gp91phox and p22phox) and four main cytosolic factors (p47phox, p67phox, p40phox, and Rac) that translocate to the cytochrome b558 to generate the active enzyme. Various protein kinases, including PKC, MAPK, PKA, the p21-activated kinases, phosphatidic acid-regulated protein kinases, and Akt (protein kinase B) have been reported to activate NADPH oxidase through phosphorylation of p47phox (11, 12). In neutrophils stimulated by PMA, the intracellular granules are proposed as the initial O2–-producing sites, then these granules fuse with the plasma membrane, resulting in the delivery of O2– into the extracellular milieu (13, 14). However, PMA is a nonphysiological stimulus and results obtained using PMA may not accurately reflect the cell response to a physiological stimulus.
We now face the challenge of defining 1) the PKC involved signal transduction pathway regulated by DGK, 2) the isoform-specific function of PKC, and 3) the intracellular site of O2– production, during a FcR-mediated respiratory burst at the cellular level. Unfortunately, primary neutrophils have a severely limited lifetime ex vivo, and both primary and cultured neutrophils are difficult to be transfected. Monocytes have a markedly reduced capacity for O2– production compared with neutrophils. To overcome these problems, we used our microglial cells and visualized the accumulation of I and PKC, DGK and p47phox to the phagosome using real-time confocal laser scanning fluorescence microscopy. In this study, we show that I PKC contributes to O2– production, and that DGK plays an important role in regulation of O2– production, probably in control of excessive O2– production. Finally, we demonstrate that the O2–-producing site during FcR-mediated phagocytosis is at the phagosomal cup/phagosome.
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Two-micrometer glass beads and carboxylated latex beads were obtained from Duke Scientific (Palo Alto, CA) and Polysciences (Warrington, PA), respectively. Eagle’s MEM and endotoxin-free FBS were from Invitrogen Life Technologies (Carlsbad, CA). Bovine insulin and PMA were from Sigma-Aldrich (St. Louis, MO). Mouse GM-CSF was from Genzyme (Cambridge, MA). U73122 and U73343 were from Biomol (Plymouth Meeting, PA), and Gö6976 and R59949 were from Calbiochem (San Diego, CA). Fura 2-AM was from Dojindo (Kumamoto, Japan).
Cell culture
The 6-3 microglial cells are generated by spontaneous immortalization of primary microglia from op/op mice which are deficient of M-CSF-derived macrophages and severely monocytopenic (15, 16). The 6-3 cells closely resemble primary microglia with respect to microglia-specific gene expression and high migrating activity to the brain (15, 17 ). The 6-3 cells were maintained in Eagle’s MEM supplemented with 10% endotoxin-free FBS, 5 µg/ml insulin, 0.2% glucose, and 0.2 ng/ml GM-CSF. For confocal imaging, Ca2+ measurements, phagocytosis assays, and ultrastructural studies, cells were seeded on 35-mm glass-bottom dishes (MatTek, Ashland, MA) without GM-CSF, and used after 48 h.
Construction of plasmids
The PKC-GFP (, I, II, , , , , and ) and GFP-DGK ( and ) constructs were previously described (3, 18). Myristoylated alanine-rich C kinase substrate (MARCKS)-GFP and mutant MARCKS-GFP, whose all three putative PKC phosphorylation sites in the effector domain were substituted to Ala, were prepared as reported (19). DGK with the EcoRI site was produced by PCR, and was cloned into the EcoRI site in pEGFP-C1 (Clontech, Palo Alto, CA), and named as GFP-DGK. The C1 domain of PKC (aa 159–280) with EcoRI/BglII sites was amplified by PCR, and was cloned into the EcoRI/BglII sites of BS 354 (19), and named as PKC(C1)-GFP. The constructs encoding human p47phox, p67phox, and p47phox(W193R) were previously described (20, 21). p47phox and p47phox(W193R) was cloned into the BglII/EcoRI sites of pEGFP-C1, and named as GFP-p47 phox and p47phox(W193R), respectively.
Phagocytosis targets
IgG-opsonized glass beads (BIgG), fluorescently labeled BIgG, and control beads (BBSA) were prepared as described (3). Carboxylated latex beads were opsonized with IgG using the Carbodiimide kit (Polysciences).
Phagocytosis assay
The culture medium was replaced with HBSS++ (3) and targets (50 per cell) were added. After incubation at 37°C for 20 min, cells were fixed with 4% PFA in 0.1 M phosphate buffer (pH 7.4). In inhibitor experiments, cells were preincubated in HBSS++ containing the inhibitor. The number of completely engulfed targets per cell was counted in 50 cells using phase-contrast microscopy. The measurements were made in triplicate on at least three separate experiments, and results are presented as means ± SD.
Synchronized phagocytosis and cell fractionation
Synchronized phagocytosis and cell fractionation were performed as described with modifications (3, 22). The 6-3 cells in 10-cm dishes were treated with ice cold HBSS++ (with or without inhibitor) containing BIgG (50 per cell). After 10 min on ice for target binding, the cells were warmed to 37°C for synchronized phagocytosis. At the indicated times, the cells were scraped and sonicated (3 x 10 s) in 400 µl of lysis buffer (18). The beads were allowed to settle 10 min on ice, and then the settled beads were sonicated again (2 x 10 s) to gain the phagosomal membrane. The beads were settled for 1 h, and the supernatant was centrifuged for 30 min at 100,000 x g. The pellet was solubilized in lysis buffer containing 1% Triton X-100. The samples were centrifuged again as above. The supernatant was designated as the membrane fraction containing the phagosomal membrane. After protein assay with bicinchoninic acid (Pierce, Rockford, IL), equal amounts of the membrane fraction (micrograms per lane) were subjected to SDS-PAGE and Western blotting (18).
Confocal imaging
The 6-3 cells were transfected using Superfect (Qiagen, Valencia, CA). After 24–30 h, the culture medium was replaced with 800 µl of HBSS++, and cells were imaged using an LSM 510 invert (Carl Zeiss, Jena, Germany) confocal laser scanning fluorescence microscope with a heated stage and objective (x40 oil). Two hundred microliters of HBSS++-containing targets (5 per cell) was added to each plate. To treat cells with inhibitor, the culture medium was replaced by a HBSS++-containing inhibitor and preincubated for the indicated time. The images were collected at 3, 5, or 10 s intervals for 10 min. The time of addition of BIgG was chosen as time 0. In a 1-day experiment, >3 independent cell preparations (dishes) in each group were performed. In one dish, 1–3 cells which engulfed 1–5 BIgG for 10 min were selected for analysis in the study.
Ca2+ measurement
The 6-3 cells were loaded with 5 µM Fura 2-AM in HEPES buffer (18). After a 1-h incubation, the cells were resuspended in HEPES buffer. Fluorescence was monitored using the ARGUS/HiSCA system (Hamamatsu Photonics, Hamamatsu, Japan) using a dual wavelength excitation (340 and 380 nm) and at emission (510 nm). HEPES buffer containing targets (1 per cell) was added, and the images were collected at 1.5 s intervals for 5 min. In inhibitor experiments, cells were further incubated for the indicated time with HEPES buffer containing Fura 2-AM and the inhibitor. Cells were maintained at 37°C with a heated objective (x40 oil). Data are presented as the background-corrected 340:380 ratio of Fura 2 fluorescence.
O2– production assay
O2– production was determined as superoxide dismutase-inhibitable chemiluminescence (Diogenes; National Diagnostics, Atlanta, GA) as described previously (21). After the addition of the luminol-based substrate, the prewarmed cells (1.0 x 104) in HBSS++ were stimulated with BIgG (1.0 x 106). The chemiluminescence was assayed for 20 min using a luminometer (Auto Lumat LB953; EG & G Berthold, Bad Wildbad, Germany).
In inhibitor experiments, cells were preincubated in an HBSS++-containing inhibitor. The 6-3 cells incubated with stimulus in the absence of the inhibitor served as the positive control. Results are presented as the percent of the positive control (means ± SD).
Ultrastructural detection of an oxidant-producing site
The O2–-producing site in 6-3 cells was detected using the cerium-based cytochemical method (23) with modifications (24). The reaction for the detection of H2O2 proceeds as follows: NADPH + 2O2 
NADP+ + H+ + 2O2–, 2O2– + 2H+ H2O2 + O2, H2O2 + CeCl3 Ce(OH)2OOH, Ce(OH)3OOH. The final cerous reaction products can be detected in the electron microscope as a precipitate. Treatment with the O2– scavenger, p-benzoquinone, causes complete abolishment of the reaction precipitate (24). The cells stimulated with either BIgG (latex, 5 per cells) or 1 µM PMA were incubated for 15 min at 37°C in HEPES buffer containing 1 mM CeCl3, 1 mM NaN3, and 20 mM tricine. NaN3 was added to prevent the degradation of H2O2 by endogenous catalase. Tricine was used to protect Ce3+ in the reaction buffer from nonenzymatic precipitation. Exogenous NADPH and FAD are not necessary for the oxidant production in this method. The cells were fixed with 2% glutaraldehyde in HEPES buffer. After a cytochemical reaction, the cells were postfixed and then dehydrated and embedded in epoxy resin. Semithin sections (0.5 µm in thickness) were observed under a JEM-100S electron microscope (JEOL, Tokyo, Japan).
Supporting information
The supplemental movies show the oscillatory accumulation of I PKC-GFP (Video 14) and the accumulation of DGK-GFP (Video 2) during phagocytosis of fluorescently labeled BIgG. Video 3 shows the oscillatory increase of [Ca2+]i during phagocytosis of BIgG. Each movie represents >15 independent experiments.
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We identified the PKC and type I DGK isoforms expressed in 6-3 microglial cells. Proteins of , I, , , , and PKC and mRNAs for DGK , , and were detected by Western blotting and RT-PCR, respectively (data not shown); II and PKC were not detected in 6-3 cells. Expression of gp91phox, p22 phox, p47 phox, p67 phox, and p40 phox in 6-3 cells was confirmed by Western blotting; and expression of Rac1 and Rac2, but not Rac3, were confirmed by RT-PCR (data not shown). PMA-stimulated O2– production in 6-3 cells was about one-tenth of that observed in neutrophils (data not shown).
Isoform-specific accumulation of PKC-GFP and GFP-DGK in Fc-mediated phagocytosis
Of the six PKC isoforms (, I, , , , and PKC) expressed, only I and PKC-GFP showed the localized translocation to the phagosomal cup/phagosome during phagocytosis of BIgG in 6-3 cells (Fig. 1, A and B, and Video 1). GFP-DGK was mainly localized on the plasma membrane before stimulation. Among type I DGK, only GFP-DGK accumulated without oscillation at the phagosomal cup, but not at the closed phagosome, during ingestion of BIgG. Accumulation of GFP-DGK briefly persisted at the plasma membrane where BIgG was ingested after closure of the phagosome (Fig. 1C and Video 2). More than 85% of the cells showed the accumulation of I PKC-GFP, PKC-GFP, and GFP-DGK (I PKC-GFP < PKC-GFP). Control beads (BBSA) were rarely phagocytosed by 6-3 cells (BBSA, 0.5 ± 0.1 per cell; BIgG, 8.4 ± 1.3 per cell), and did not cause any accumulation of above-mentioned molecules.
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I and PKC-GFP in FcR-mediated phagocytosis
The time courses and patterns of the accumulation of I PKC-GFP and PKC-GFP were quite different. Localization times were calculated and defined as T1, time from the first concentration of PKC-GFP at the phagosomal cup until closure of the phagosome; T2, time from closure of the phagosome until the signal returned to cytosolic levels; and Tt, T1 + T2. A repetitive accumulation of I PKC-GFP was observed at the phagosomal cup and subsequently at the phagosome (19 of 22 cells; Fig. 1A and Video 1). The oscillatory accumulation of I PKC-GFP occurred from 1 to 4 times (1, n = 2; 2, n = 6; 3, n = 7; and 4, n = 4) and was divided into two patterns; 1) accumulation only at the phagosomal cup, and 2) accumulation at both the cup and the phagosome. In accumulation pattern 2, T1, T2, and Tt were 24 ± 5, 21 ± 4, and 46 ± 7 s, respectively (n = 10). In contrast, PKC-GFP accumulated at the phagosomal cup/phagosome without oscillation, and persisted longer (T1, 26 ± 6 s; T2, 93 ± 19 s; Tt, 119 ± 19 s; n = 10; Fig. 1B).
We used MARCKS-GFP as an indicator of the active PKC. MARCKS-GFP is constitutively present on the plasma membrane in an unphosphorylated form and is released from the membrane upon phosphorylation by PKC (19). In the present study, the loss of membrane-associated MARCKS-GFP was observed only at regions of the plasma membrane containing BIgG (Fig. 1D). Although not definitive, this evidence is consistent with the presence of active PKC in the forming phagosome. This conclusion is strengthened by the use of a mutant MARCKS-GFP that cannot be phosphorylated by PKC (19). This mutant MARCKS-GFP accumulated and was retained at the phagosomal cup and the forming phagosomal membrane during internalization of BIgG (Fig. 1E).
To validate that the GFP constructs mimic their endogenous counterparts, we measured the translocation of endogenous I and PKC during FcR-mediated phagocytosis in 6-3 cells. I and PKC increased in the membrane fraction of nontransfected 6-3 cells in a time-dependent fashion (Fig. 2). Similar to the results with GFP conjugates, the increase in membrane-associated PKC was sustained longer than that of I PKC (Fig. 2).
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I PKC-GFP in FcR-mediated phagocytosis
We have reported that PKC, but not cPKC, enhances the rate of BIgG uptake, and that inhibition of PKC causes 
50% inhibition of FcR-mediated phagocytosis (3). Therefore, we focused on clarifying the accumulation/activation mechanism and the functional role of I PKC in the present study. The accumulation of I PKC-GFP was abolished by 2 µM U73122, an inhibitor of PLC (9), but not its inactive analog, U73343 (Fig. 3A). Pretreatment with R59949, an inhibitor of type I DGK, enhanced the accumulation of I PKC-GFP, and induced the translocation of I PKC-GFP to the plasma membrane (Fig. 3B). This result was confirmed by fractionation experiments. Treatment with R59949 increased the amount of membrane-associated I PKC (Fig. 3, C and D).
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I PKC-GFP oscillations, [Ca2+]i increase, and the production of DAG during phagocytosis were examined. One phagocytic event resulted in 1–4 [Ca2+]i oscillations as determined by an increase in the 340:380 ratio of Fura 2 fluorescence (Fig. 4A and Video 3). Pretreatment with 2 µM U73122, but not U73343, significantly suppressed the amplitude of [Ca2+]i increase (Fig. 4B). PKC(C1)-GFP, a DAG indicator (9), transiently accumulated at the phagosomal cup/phagosome without oscillation (Fig. 4C). U73122 inhibited the accumulation of PKC(C1)-GFP in dose-dependent manner (data not shown). Both Ca2+ and DAG are necessary for the full-activation of cPKC on the membrane (25). Taken together, these results are consistent with a model in which the oscillations of I PKC-GFP during BIgG ingestion are due to parallel [Ca2+]i oscillations in cooperation with localized DAG production at the phagosomal cup/phagosome.
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R-mediated phagocytosis
We measured O2– production in 6-3 cells during FcR-mediated phagocytosis using a superoxide dismutase-inhibitable luminal-based detection system (21).
O2– production was abolished by 100 nM Gö6976, a selective inhibitor of cPKC (1.64 ± 0.1% of uninhibited controls) or 2 µM U73122, a PLC inhibitor (0.1 ± 0.0%). In contrast, 10 µM R59949 increased O2– production 4.37-fold (Table I). O2– production was inhibited by Gö6976 (0–300 nM) and enhanced by R59949 (0–10 µM) in a dose-dependent manner (Fig. 5). As I PKC is the only cPKC associated with phagosomes in 6-3 cells, and Gö6976 effectively blocks O2– production, this data supports a model in which I PKC mediates O2– release. The fact that U73122 mimics the Gö6976 effect with respect to the inhibition of O2– release is consistent with the activation of I PKC via PLC-derived DAG and IP3-mediated rise in [Ca2+]i. However, Gö6976 did not affect the accumulation of I PKC (Table I), providing evidence that I PCK activity, rather than localization, is necessary for O2– production.
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R-mediated phagocytosis
PKC phosphorylates p47phox, acting as the major activating kinase for the NADPH oxidase (11). We tested the hypothesis that accumulation of I PKC corresponds with the temporal localization of p47phox. In 6-3 cells expressing GFP-p47phox and p67phox, GFP-p47phox accumulated at the phagosomal cup/phagosome without oscillation (Tt, 65 ± 7; n = 5; Fig. 6A), but not on nontarget-associated plasma membranes. GFP-p47phox(R193W), which does not bind to p22phox nor produce O2– (20), did not accumulate at the phagosomal cup/phagosome (Fig. 6B). The accumulation of GFP-p47phox was not enhanced by pretreatment with R59949 (data not shown).
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I PKC-GFP and GFP-p47phox accumulated at the phagosomal cup/phagosome during phagocytosis of BIgG, we hypothesized that NADPH oxidase could be activated only at the site of PKC accumulation. We used electron microscopy to visualize the intracellular site of H2O2 production (dismutated from O2– generated by NADPH oxidase) in response to either BIgG or PMA. In 6-3 cells, the reaction product stimulated by BIgG was detected only at the phagosome (Fig. 7A), while that by PMA was at the intracellular granules (Fig. 7B). No reaction precipitate was found in cells incubated in the absence of CeCl3, BIgG, or PMA (data not shown). These results show that oxidant production during FcR-stimulated phagocytosis initiates at the phagosomal cup/phagosome, but not at the intracellular granules.
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R-mediated respiratory burst at the cellular level in microglia. There are many reports that multiple PKC isoforms (, , , and PKC) involved in phagocytosis (22, 27). In 6-3 microglial cells, PKC isoforms , I, , , , and PKC were expressed, and only I and PKC-GFP, but not , , , or PKC-GFP, accumulated at the phagosomal cup/phagosome, but not on the plasma membrane, during phagocytosis of BIgG. These findings are intriguing that receptor ligation not only controls which PKC isoform translocates but also the membranes to which they localize. There is a report that dominant negative PKC impaired FcR-mediated phagocytosis in RAW macrophages (28). However, we have reported that cPKC mediates the FcR-stimulated respiratory burst, and that nPKC is necessary for phagocytosis in RAW cells (22). More recently, we have demonstrated that PKC, but not cPKC, enhances the rate of BIgG uptake, and that inhibition of PKC causes 50% inhibition of FcR-mediated phagocytosis (3). In microglia, no accumulation of PKC-GFP was observed during BIgG ingestion, and a selective inhibitor of cPKC did not change the phagocytosis (Table I). Taken together, FcR efficiently couples to I PKC to facilitate O2– production in microglia. In addition, accumulation of PKC had a good correlation with BIgG uptake in microglia as macrophages (our unpublished data). In the present study, we also showed the apparent differences of the accumulation patterns between I PKC-GFP and PKC-GFP. Two signals, one the locally produced DAG at the phagosomal cup/phagosome by PLC and the other the [Ca2+]i oscillations by PLC, acting in concert are required for activation of I PKC during FcR-mediated phagocytosis. In an earlier study, stepwise phosphorylation of p47phox by PMA was indeed reported: although the final phosphorylation event of p47phox initiating O2– production occurs at membrane components, the first phosphorylation of p47phox promoting its translocation to membrane components primarily occurs in cytosol (29). We showed that accumulation of p47phox was not oscillatory, supporting a model that phosphorylated p47phox tightly associates with membrane (29). Recently, Dewitt et al. (30) reported that the first [Ca2+]i increase is not sufficient for O2– production. The possibility exists that the oscillatory signal by I PKC between the phagosomal cup/phagosome and cytosol are used for both initialization of membrane translocation of p47phox in cytosol and tight association of p47phox to membrane and full activation of the NADPH oxidase at membrane. However, the physiological contributions of the oscillatory accumulation of I PKC is presently unclear.
In vitro, p47phox is phosphorylated on 8–10 serine residues within its C-terminal region by different types of protein kinases; one or more PKC family members are indicated to play a major role (31). Phosphopeptide mapping of p47phox showed that , , and PKC phosphorylated all major peptides, although PKC was less active toward some peptides; phosphorylation by PKC was only a few peptides (11). Using neutrophils from PKC null mice, Dekker et al. (5) reported that the lack of PKC decreased the amount of O2– production by 50% stimulated by BIgG and PMA, and they also demonstrated a significant inhibition of O2– production stimulated by PMA and IgG-opsonized bacteria in human neutrophils treated with a PKC-specific inhibitor (5). It was also reported that a similar degree of inhibition of O2– production by PMA, immune complex, and fMLP in human HL60 cells using an antisense method (32 ). In contrast, PKC, but not PKC, is involved in O2– production by BIgG (22) and by opsonized zymosan (33), in monocytes. Although apparently disparate, these results suggest that PKC contributes to the respiratory burst in neutrophils while PKC performs this function in monocytes. The present study is consistent with microglia using a neutrophil-like pathway for PKC regulation of O2– production. Furthermore, recent reports show that PKC and PKC bind to p47phox in intact neutrophils stimulated by PMA with different time courses (34), and PKC participates in the fMLP-induced, but not PMA-induced, respiratory burst (35). It was also reported that PKC accumulated on the phagosome during ingestion of Helicobacter pylori, but not IgG-opsonized H. pylori (36), in monocytes. Thus, it is likely that different receptors are coupled to the specialized signaling networks and use different PKC isoforms. Taken together, use of PKC isoform likely is determined by both the cell type and the stimulus.
O2– can be produced either extracellularly or within the phagosome during phagocytosis. Extracellular O2– is thought to originate from either the phagosomal cup or the plasma membrane. In the present study, during the ingestion of BIgG, I PKC-GFP and GFP-p47phox translocated to the phagosomal cup/phagosome, but not to the plasma membrane. These results indicate that the extracellular O2– primarily originates from the phagosomal cup. DAG is one of the key lipid mediators in O2– production (37, 38). Inhibition of DGK with R59949 significantly enhanced O2– production and induced the translocation of I PKC-GFP to the plasma membrane in addition to the enhanced accumulation at the phagosomal cup/phagosome. However, the accumulation of GFP-p47phox was not enhanced by pretreatment with R59949, this result suggests that DAG may not be primarily associated with the translocation of p47phox and enhanced O2– production may be primarily from the enhanced accumulation of I PKC at the phagosomal cup/phagosome. Furthermore, DGK accumulated on the forming phagosome, but not at the closed phagosome, while I PKC and p47phox accumulated both at the phagosomal cup and phagosome. Thus, DGK which is localized on the plasma membrane in resting states, accumulates on the forming phagosome, and briefly stays at the phagocytosis-involved plasma membrane after closure of the phagosome, likely plays an important role in control of excessive secretion of O2– to the extracellular milieu through regulation of I PKC activation during phagocytosis rather than termination of O2– production. This is consistent with a report that termination of O2– production correlated with dissociation of p47phox/p67phox complex from the phagosome (39). Because I PKC accumulated at the phagosomal cup from initiation of phagocytosis and inhibition of I PKC showed a profound decrease of O2– production, I PKC most likely plays a key role for initiation of respiratory burst, at least. Other pathways, such as PI3K pathway, likely contribute to the later stage of O2– production (40). In agreement with this idea, GFP-tagged PX domain of p40phox, a phosphatidylinositol 3-phosphate indicator, accumulated at the phagosome only after closure of the phagosome (our unpublished data).
It has been reported that cytochrome b558 is primarily localized on intracellular granules (80%) in neutrophils (41). Recently, it was reported that O2– production stimulated by PMA initially takes place at intracellular granules, followed by fusion of these granules with the plasma membrane, resulting in the delivery of O2– into the extracellular milieu (13, 14). However, because I PKC-GFP and GFP-p47phox accumulated at the phagosomal cup/phagosome in this study, we hypothesized that NADPH oxidase could be activated primarily at the phagosomal cup/phagosome during FcR-mediated phagocytosis in microglia. Our present results using electron microscopy supported that oxidant production during FcR-stimulated phagocytosis initiates at the phagosomal cup/phagosome where DAG is locally formed during BIgG ingestion, but not at the intracellular granules. In contrast, consistent with previous reports, PMA caused the oxidant production at the intracellular granules. We propose that O2– production during FcR-stimulated phagocytosis begins at the phagosomal cup/phagosome through phosphorylation of p47phox by cPKC. Although microglia use PKC as neutrophils do, and macrophages use PKC, the basis of this model may be adapted to all phagocytes in FcR-stimulated phagocytosis. In support of our results, it was reported that, based on stoichiometry, O2– is formed at the phagosomal cup/phagosome during phagocytosis of opsonized zymosan in neutrophils (42). When phagocytosis is blocked by cytochalasin B, O2– is formed at the phagosomal cup (cell-zymosan interface) only and secreted into the extracellular milieu (S. Kanegasaki, Tokyo University, unpublished observation). Microglia had only about one-tenth capacity for O2– production compared with neutrophils. Further elucidation, especially about the assembly of NADPH oxidase complex during FcR-stimulated phagocytosis at the cellular level, is required.
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Acknowledgments |
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Footnotes |
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1 This work was supported by grants from the 21st Century Center of Excellence Program of the Ministry of Education, Culture, Sports, Science, and Technology of Japan, Core Research for Evolutional Science and Technology, the Ministry of Education, Culture, Sports, Science, and Technology in Japan, a Grant-in-Aid for Scientific Research on Priority Areas (C)-Advanced Brain Science Project from the Ministry of Education, Culture, Sports, Science, and Technology in Japan, the Uehara Memorial Foundation, and the Sankyo Foundation of Life Science. 
2 Address correspondence and reprint requests to Dr. Naoaki Saito, Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Rokkodai-cho 1-1, Nada-ku, Kobe 657-8501, Japan. E-mail address: naosaito{at}kobe-u.ac.jp
3 Abbreviations used in this paper: PKC, protein kinase C; DGK, diacylglycerol kinase; MARCKS, myristoylated alanine-rich C kinase substrate; c, conventional isoform; n, novel isoform; DAG, diacylglycerol; O2– superoxide; PLC, phospholipase C; IP3, inositol-1,4,5-triphosphate; [Ca2+]i, intracellular Ca2+ concentration; BIgG, IgG-opsonized beads.
4 The on-line version of this article contains supplemental material.
Received for publication April 5, 2004. Accepted for publication July 16, 2004.
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