Rho Is Involved in Superoxide Formation during Phagocytosis of Opsonized Zymosans

Phagocytosis is accompanied by the production of superoxide by the NADPH oxidase complex, for which GTP-bound Rac is essential. We wanted to determine whether Rho is also involved in the production of superoxide during phagocytosis. Inhibition of Rho by Tat-C3 exoenzyme (Tat-C3) blocked superoxide formation and curtailed the phagocytosis of serum- (SOZ), C3bi- (COZ), and IgG-opsonized zymosan (IOZ) particles. Tat-C3 did not affect superoxide formation in response to phorbol myristate acetate (PMA), formyl Met-Leu-Phe (fMLP), or macrophage colony-stimulating factor (M-CSF). Superoxide formation was also reduced in J774 cells transfected with a cDNA expressing dominant-negative form of RhoA (N19RhoA). However, purified prenylated recombinant RhoA did not activate NADPH oxidase in vitro, suggesting that Rho does not interact directly with NADPH oxidase. Tat-C3 inhibited the activity of RhoA, but did not affect that of Rac in vitro or in vivo. It also inhibited the phosphorylation of p47PHOX, one of the cytosolic components of NADPH oxidase. Taken together, these results suggest that Rho plays an important role in superoxide formation during phagocytosis of SOZ, COZ, and IOZ via phosphorylation of p47PHOX. Phagocytosis is accompanied by the production of superoxide by the NADPH oxidase complex, for which GTP-bound Rac is essential. We wanted to determine whether Rho is also involved in the production of superoxide during phagocytosis. Inhibition of Rho by Tat-C3 exoenzyme (Tat-C3) blocked superoxide formation and curtailed the phagocytosis of serum- (SOZ), C3bi- (COZ), and IgG-opsonized zymosan (IOZ) particles. Tat-C3 did not affect superoxide formation in response to phorbol myristate acetate (PMA), formyl Met-Leu-Phe (fMLP), or macrophage colony-stimulating factor (M-CSF). Superoxide formation was also reduced in J774 cells transfected with a cDNA expressing dominant-negative form of RhoA (N19RhoA). However, purified prenylated recombinant RhoA did not activate NADPH oxidase in vitro, suggesting that Rho does not interact directly with NADPH oxidase. Tat-C3 inhibited the activity of RhoA, but did not affect that of Rac in vitro or in vivo. It also inhibited the phosphorylation of p47PHOX, one of the cytosolic components of NADPH oxidase. Taken together, these results suggest that Rho plays an important role in superoxide formation during phagocytosis of SOZ, COZ, and IOZ via phosphorylation of p47PHOX. Phagocytosis by macrophages is essential for the uptake and degradation of pathogens. It also triggers immune responses and participates in development and tissue remodeling. Phagocytosis can be triggered by the macrophage receptors FcγRs, which recognize the Fc domain of immunoglobulin G (IgG) (1Aderem A. Underhill D.M. Annu. Rev. Immunol. 1999; 17: 593-623Crossref PubMed Scopus (2149) Google Scholar). Another type of receptor through which phagocytosis occurs is the complement receptor (CR), 1The abbreviations used are: CR, complement receptor; COZ, C3bi-opsonized zymosan; DCFH-DA, 2′,7′-dichlorofluorescein diacetate; ERK1/2, extracellular signal-regulated kinase 1/2; FITC, fluorescein isothiocyanate; IPTG, isopropyl-1-thio-β-d-galactopyranoside; IOZ, IgG-opsonized zymosan; luminol, 5-amino-2,3-dihydroxy-1,4-phtalazinedione; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; MAPK, mitogen-activated protein kinase; MV, methylviologen; PA, phosphatidic acid; PD98059, 2′-amino-3′-methoxyflavone; PMA, phorbol 12-myristate 13-acetate; SB203580, 4-(4-fluorophenyl)-2-(4-methyl-sulfinylphenyl)-5-(4-pyridyl)1H-imidazole; SOD, superoxide dismutase; SOZ, serum-opsonized zymosan; PMSF, phenylmethylsulfonyl fluoride; TRITC, tetramethylrhodamine B isothiocyanate; GST, glutathione S-transferase; GFP, green fluorescent protein; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; PKC, protein kinase C; γS-GTP, guanosine 5′-3-O-(thio)triphosphate; HIV-1, human immunodeficiency virus-1; fMLP, formyl Met-Leu-Phe; M-CSF, macrophage colony-stimulating factor; SRBC, sheep red blood cells. which recognizes C3b/C3bi fragments. CR1 is thought to participate mainly in particle binding, whereas CR3 (also referred to as CD11b/CD18, Mac-1, and αMβ2) and CR4 (CD11c/CD18 and αXβ2), which are heterodimers of integrin, are responsible for internalizing particles (1Aderem A. Underhill D.M. Annu. Rev. Immunol. 1999; 17: 593-623Crossref PubMed Scopus (2149) Google Scholar, 2Fallman M. Andersson R. Andersson T. J. Immunol. 1993; 151: 330-338PubMed Google Scholar). A third receptor involved in phagocytosis is the mannose receptor that recognizes mannose and fucose saccharides in the capsule or on the lipopolysaccharide of invading bacteria (1Aderem A. Underhill D.M. Annu. Rev. Immunol. 1999; 17: 593-623Crossref PubMed Scopus (2149) Google Scholar, 3Brown E.J. BioEssay. 1995; 17: 109-117Crossref PubMed Scopus (142) Google Scholar, 4Chimini G. Chavrier P. Nat. Cell Biol. 2000; 2: E191-E196Crossref PubMed Scopus (266) Google Scholar). Rho family GTPases are essential for the actin changes needed for phagocytosis and engulfment (4Chimini G. Chavrier P. Nat. Cell Biol. 2000; 2: E191-E196Crossref PubMed Scopus (266) Google Scholar). Cdc42/Rac regulates the macrophage phagocytosis mediated by FcγR, whereas Rho is thought to regulate that mediated by CR3 (5Caron E. Hall A. Science. 1998; 282: 1717-1721Crossref PubMed Scopus (819) Google Scholar, 6Massol P. Montocourrier P. Guillemot J.C. Chavrier P. EMBO J. 1998; 17: 6219-6229Crossref PubMed Scopus (206) Google Scholar, 7Cox D. Chang P. Zhang Q. Reddy P.G. Bokoch G.M. Greenberg S. J. Exp. Med. 1997; 186: 1487-1494Crossref PubMed Scopus (373) Google Scholar). Nevertheless it has been reported that C3 exoenzyme abrogates FcγR-mediated phagocytosis (8Hackam D.J. Rotstein O.D. Schreiber A. Zhang W. Grinstein S. J. Exp. Med. 1997; 186: 955-966Crossref PubMed Scopus (148) Google Scholar). After phagocytosis in macrophages, there is an abrupt increase in superoxide formation, known as the oxidative burst, which is catalyzed by the membrane-associated NADPH oxidase enzyme complex, which generates superoxide (O2−) by the one-electron reduction of oxygen, using NADPH as electron donor (9Babior B.M. Blood. 1999; 93: 1464-1476Crossref PubMed Google Scholar). The redox core of the phagocyte NADPH oxidase is a membrane-spanning heterodimeric flavocytochrome b558 composed of p22PHOX and gp91PHOX. It is present in the membranes of secretory granules that fuse with the plasma membrane upon phagocytosis (10Rotrosen D. Yeung C.L. Katkin J.P. J. Biol. Chem. 1993; 268: 14256-14260Abstract Full Text PDF PubMed Google Scholar). The other components, p40PHOX, p47PHOX, and p67PHOX form a cytoplasmic complex (11Wientjes F.B. Panayotou G. Reeves E. Segal A.W. Biochem. J. 1996; 317: 919-924Crossref PubMed Scopus (77) Google Scholar). p47PHOX is phosphorylated by protein kinases (12Park J.W. 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Google Scholar). be as to C3bi-opsonized zymosan particles induced superoxide production in macrophages a and superoxide formation was in The be to the needed for to to the of the of cells with Tat-C3 reduced superoxide of the macrophages with Tat-C3 a as as for blocked superoxide formation induced by particles the other did not superoxide formation it was macrophages In addition, did not affect superoxide formation to C3 exoenzyme and for which did not the did superoxide formation suggesting that the domain is not involved in the of superoxide C3 exoenzyme can be a of it is used a of I. 1995; PubMed Scopus Google Scholar). Tat-C3 also superoxide by in macrophages In addition, Tat-C3 blocked superoxide formation by and IOZ in human superoxide formation by zymosan particles in cells. macrophages and human with or Tat-C3 for and superoxide formation induced by and and IOZ particles was by as in the to Rho in Superoxide to form superoxide in response to PMA, fMLP, and M-CSF, which are not The of superoxide in these was that by SOZ, but formation was by Tat-C3 that Tat-C3 superoxide formation with not that of or of Tat-C3 Phagocytosis of by phagocytosis of particles by J774 cells and the of Tat-C3 on these Tat-C3 inhibited phagocytosis of whereas did not of the cells with of C3 exoenzyme a small on phagocytosis not C3 exoenzyme on small of C3 exoenzyme to be cells a of and I. 1995; PubMed Scopus Google Scholar). Tat-C3 to an a as as Tat-C3 inhibited not and phagocytosis of particles to a suggesting that and phagocytosis of the particles are It is important to that whereas phagocytosis was reduced by superoxide formation was by the of Tat-C3 Superoxide Phagocytosis of the that superoxide plays a role as a for phagocytosis. and oxidase P. Pick E. J. Biol. 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Immunol. 2001; 2: PubMed Scopus Google Scholar). that GTP-bound RhoA did not superoxide production by NADPH whereas Rac2 prenylated RhoA does not NADPH oxidase in a Tat-C3 of Superoxide by and IOZ to the and of phagocytosis and superoxide formation, phagocytosis of and IOZ particles and superoxide formation in or IOZ particles by the macrophages, particles as Tat-C3 inhibited the phagocytosis of phagocytosis of particles was inhibited that of IOZ particles is in with the that uptake of particles by macrophages is mediated by RhoA (5Caron E. Hall A. Science. 1998; 282: 1717-1721Crossref PubMed Scopus (819) Google Scholar). Phagocytosis of IOZ particles to the production of superoxide that of and Tat-C3 also inhibited superoxide formation in The of superoxide IOZ and particles did not that with IOZ particles induced a of superoxide and it was inhibited by Tat-C3 In addition, of and by and on to macrophages a of superoxide that was also inhibited by Tat-C3 However, superoxide formation during phagocytosis of and IOZ particles was that by and Tat-C3 Superoxide by to that reduction of superoxide formation by Tat-C3 is not a of reduced cells with by serum-opsonized IOZ to protein through the that are to be zymosan particles to be with and as particles to to the of not with the not the but of which can be in response to superoxide However, cells with Tat-C3 did not it of of the cells to the that superoxide formation induced by and IOZ occurs of phagocytosis. 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Cell 1997; PubMed Google Scholar). was inhibited by suggesting that Tat-C3 does not affect Rac activity in and that of Rho to of membrane The in response to and M-CSF, and the on it of Tat-C3 and the results are in an inhibitor of Rho kinase also did not affect membrane induced by and not RhoA and Rac during Phagocytosis of and the of Rho GTPases in phagocytosis and superoxide formation, of RhoA, and in RhoA, and Rac2 during phagocytosis of and IOZ particles. with and IOZ the of and for and whereas Rac2 was for with and RhoA was Rac1 and Rac2 in response to SOZ, Rac1 and Rac2 RhoA in response to Tat-C3 inhibited of RhoA but not that of Rac1 and Rac2 with and IOZ particles Tat-C3 of to the of Rho with NADPH whether of Rho by Tat-C3 the phosphorylation of p47PHOX, one of the cytosolic components of NADPH oxidase in of cells and IOZ the phosphorylation of p47PHOX, of the cells with inhibitor of or inhibitor of kinase kinase inhibited and been reported to p47PHOX Benna J. J. J.W. E. Babior B.M. Biochem. 1996; PubMed Scopus Google Scholar). Tat-C3 of and of macrophages in response to not Rho in Superoxide by the present that superoxide induced by and IOZ particles and and that with the dominant-negative cDNA reduced superoxide formation whereas RhoA cDNA to of it Tat-C3 is that RhoA is involved in superoxide Tat-C3 inhibited phagocytosis of by and the a on phagocytosis In addition, Tat-C3 reduced phagocytosis of IOZ by However, Tat-C3 superoxide formation induced by and IOZ and reduced superoxide production phagocytosis. results suggest that superoxide formation by and IOZ is not on phagocytosis. the other inhibited phagocytosis and superoxide production in response to SOZ, and the of the in In superoxide formation to be to phagocytosis. phagocytosis of the actin (4Chimini G. Chavrier P. Nat. Cell Biol. 2000; 2: E191-E196Crossref PubMed Scopus (266) Google Scholar), and the also plays a role in the that NADPH oxidase J. H. M. M. C. 1999; PubMed Scopus Google Scholar, M. M. H. T. A. J. Clin. Immunol. Full Text Full Text PDF PubMed Scopus Google Scholar). In superoxide is not for phagocytosis of in cells with Tat-C3 blocked superoxide that of superoxide formation by Tat-C3 is not a of a reduction in phagocytosis. RhoA is not directly with the NADPH oxidase complex it participate in superoxide the of RhoA triggered by and IOZ particles participate in phagocytosis and superoxide formation via of p47PHOX The that phagocytosis and superoxide formation are through was by the that the inhibitor superoxide formation, but phagocytosis of serum-opsonized particles T. H. M. 2000; PubMed Scopus Google Scholar). We also that Tat-C3 did not superoxide formation in response to PMA, fMLP, and suggesting that Tat-C3 is not and that Rho is not involved in superoxide formation in response to the of the it does not superoxide formation in response to NADPH oxidase G. Pick E. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). It is also by the that production of S. S. J.P. J. Cell PubMed Google of protein does not In to the of on superoxide formation, it PKC, and in p47PHOX by A. J. Benna J. A. J. Immunol. 2001; 166: PubMed Scopus Google Scholar). In addition, of p47PHOX, and Rac1 to membranes T. Bokoch G.M. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). fMLP, on the other was reported to activate through receptor to form phosphatidic which in protein in of p22PHOX by phosphorylation S. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). induced superoxide formation in J774 and Tat-C3 did not it not suggesting that Rho is not involved in the via to activate NADPH oxidase. to the of Tat-C3 on phagocytosis and superoxide formation, that it inhibited the phagocytosis of particles that of IOZ However, Rho was also for the FcγR-mediated of phagocytosis and superoxide formation as as for A and It also to be involved in to superoxide formation through CR3 and not to phagocytosis However, superoxide formation induced by PMA, fMLP, and M-CSF, which is to was to Tat-C3 We that Rho is for and FcγR-mediated superoxide which are by mannose receptors J. P. R. M. J. P. J. Biol. 1993; PubMed Scopus Google Scholar), and CR3 Rev. Immunol. 2000; PubMed Google Scholar), also induced phagocytosis and superoxide formation the other mannose phagocytosis is not accompanied by superoxide production C. J. 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Science. 1996; PubMed Scopus Google Scholar, Blood. 2000; PubMed Google Scholar, P.G. J. Curnutte J.T. Biochem. J. 1995; PubMed Scopus Google Scholar, T. H. T. H. M. Biochem. Scopus Google Scholar). regulates superoxide and the phosphorylation and of p47PHOX P.G. J. Curnutte J.T. Biochem. J. 1995; PubMed Scopus Google Scholar). In the present PD98059, and inhibited phosphorylation of p47PHOX that and are involved in the to the phosphorylation of p47PHOX. Tat-C3 of and not suggesting that Rho is involved in the of and Tat-C3 also blocked of RhoA to membranes not suggesting that of RhoA be essential for superoxide Taken together, Rho participates in the of not phagocytosis but also superoxide the with superoxide formation, of Rho regulates these to be We can be that Rho is involved in of superoxide formation via phosphorylation of p47PHOX. We J. W. for the and an of p47PHOX J. and We M. Bokoch The for and with