A Requirement for Phosphatidylinositol 3-Kinase in Pseudopod Extension
1999; Elsevier BV; Volume: 274; Issue: 3 Linguagem: Inglês
10.1074/jbc.274.3.1240
ISSN1083-351X
AutoresDianne Cox, Ching-Chun Tseng, Gordana Bjekic, Steven M. Greenberg,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoPhagocytosis requires actin assembly and pseudopod extension, two cellular events that coincide spatially and temporally. The signal transduction events underlying both processes may be distinct. We tested whether phagocytic signaling resembles that of growth factor receptors, which induce actin polymerization via activation of phosphatidylinositol 3-kinase (PI 3-kinase). Fcγ receptor-mediated phagocytosis was accompanied by a rapid increase in the accumulation of phosphatidylinositol 3,4,5-trisphosphatein vivo, and addition of wortmannin (WM) or LY294002, two inhibitors of PI 3-kinase(s), inhibited phagocytosis but not Fcγ receptor-directed actin polymerization. However, both compounds prevented maximal pseudopod extension, suggesting that PI 3-kinase inhibition produced a limitation in membrane required for pseudopod extension. Availability of plasma membrane was not limiting for phagocytosis, because blockade of ingestion in the presence of WM was not overcome by reducing the number of particles adhering to macrophages. However, decreasing bead size, and hence the magnitude of pseudopod extension required for particle engulfment, relieved the inhibition of phagocytosis in the presence of WM or LY294002 by up to 80%. The block in phagocytosis of large particles occurred before phagosomal closure, because both compounds inhibited spreading of macrophages on substrate-bound IgG. Macrophage spreading on IgG was accompanied by exocytic insertion of membrane from an intracellular source, as measured by the dye FM1-43. These results indicate that one or more isoforms of PI 3 kinase are required for maximal pseudopod extension but not phagocytosis per se. We suggest that PI 3-kinase is required for coordinating exocytic membrane insertion and pseudopod extension. Phagocytosis requires actin assembly and pseudopod extension, two cellular events that coincide spatially and temporally. The signal transduction events underlying both processes may be distinct. We tested whether phagocytic signaling resembles that of growth factor receptors, which induce actin polymerization via activation of phosphatidylinositol 3-kinase (PI 3-kinase). Fcγ receptor-mediated phagocytosis was accompanied by a rapid increase in the accumulation of phosphatidylinositol 3,4,5-trisphosphatein vivo, and addition of wortmannin (WM) or LY294002, two inhibitors of PI 3-kinase(s), inhibited phagocytosis but not Fcγ receptor-directed actin polymerization. However, both compounds prevented maximal pseudopod extension, suggesting that PI 3-kinase inhibition produced a limitation in membrane required for pseudopod extension. Availability of plasma membrane was not limiting for phagocytosis, because blockade of ingestion in the presence of WM was not overcome by reducing the number of particles adhering to macrophages. However, decreasing bead size, and hence the magnitude of pseudopod extension required for particle engulfment, relieved the inhibition of phagocytosis in the presence of WM or LY294002 by up to 80%. The block in phagocytosis of large particles occurred before phagosomal closure, because both compounds inhibited spreading of macrophages on substrate-bound IgG. Macrophage spreading on IgG was accompanied by exocytic insertion of membrane from an intracellular source, as measured by the dye FM1-43. These results indicate that one or more isoforms of PI 3 kinase are required for maximal pseudopod extension but not phagocytosis per se. We suggest that PI 3-kinase is required for coordinating exocytic membrane insertion and pseudopod extension. Phagocytosis via Fcγ receptors in macrophages is accompanied by actin assembly, pseudopod extension, and phagosomal closure (1Greenberg S. El Khoury J. Di Virgilio F. Kaplan E.M. Silverstein S.C. J. Cell Biol. 1991; 113: 757-767Crossref PubMed Scopus (145) Google Scholar). FcγR-directed actin assembly is blocked by tyrosine kinase inhibitors (2Greenberg S. Chang P. Silverstein S.C. J. Exp. Med. 1993; 177: 529-534Crossref PubMed Scopus (171) Google Scholar) and requires the participation of Rac1 and Cdc42 (3Cox D. Chang P. Zhang Q. Reddy P.G. Bokoch G.M. Greenberg S. J. Exp. Med. 1997; 186: 1487-1494Crossref PubMed Scopus (372) Google Scholar), two members of the Rho family of GTPases. However, it is not known precisely how enhanced protein tyrosine phosphorylation leads to changes in either the cytoskeleton or the membrane. Signaling by Fcγ receptors shares many elements in common with that of growth factor receptors. For example, both classes of receptors signal directly or indirectly through tyrosine kinases, and ligation of multiple growth factor receptors and FcγRs 1The abbreviations used are: FcγR, receptor for the Fc portion of IgG; EIgG, sheep erythrocytes opsonized with rabbit IgG; PI 3-kinase, phosphatidylinositol 3-kinase; thio-macrophages, mouse macrophages elicited after intraperitoneal injection of thioglycollate broth; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PDGF, platelet-derived growth factor; BSA, bovine serum albumin; PLA2, phospholipase A2; GTPγS, guanosine 5′-3-O-(thio)triphosphate. culminates in net actin assembly and plasma membrane-based protrusions (1Greenberg S. El Khoury J. Di Virgilio F. Kaplan E.M. Silverstein S.C. J. Cell Biol. 1991; 113: 757-767Crossref PubMed Scopus (145) Google Scholar, 4Salmon J.E. Brogle N.L. Edberg J.C. Kimberly R.P. J. Immunol. 1991; 146: 997-1004PubMed Google Scholar, 5Motto D.G. Ross S.E. Jackman J.K. Sun Q. Olson A.L. Findell P.R. Koretzky G.A. J. Biol. Chem. 1994; 269: 21608-21613Abstract Full Text PDF PubMed Google Scholar). Studies of the PDGF receptor indicate a prominent role for PI 3-kinase in the generation of F-actin-rich membrane ruffles. Phosphotyrosine residues within the kinase insert region of the cytosolic domain of the PDGF receptor bind the p85/p110 isoform of PI 3-kinase, and mutation of these residues abolishes membrane ruffling induced by this receptor (6Kundra V. Escobedo J.A. Kazlauskas A. Kim H.K. Rhee S.G. Williams L.T. Zetter B.R. Nature. 1994; 367: 474-476Crossref PubMed Scopus (403) Google Scholar, 7Wennstrom S. Siegbahn A. Yokote K. Arvidsson A.-K. Heldin C.-H. Mori S. Claesson-Welsh L. Oncogene. 1994; 9: 651-660PubMed Google Scholar, 8Wennstrom S. Hawkins P. Cooke F. Hara K. Yonezawa K. Kasuga M. Jackson T. Claesson-Welsh L. Stephens L. Curr. Biol. 1994; 4: 385-393Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar). Addition of wortmannin, a fungal metabolite that inhibits PI 3-kinases in the nanomolar range, blocks PDGF receptor-induced membrane ruffling and actin assembly (8Wennstrom S. Hawkins P. Cooke F. Hara K. Yonezawa K. Kasuga M. Jackson T. Claesson-Welsh L. Stephens L. Curr. Biol. 1994; 4: 385-393Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar, 9Wymann M. Arcaro A. Biochem. J. 1994; 298: 517-520Crossref PubMed Scopus (125) Google Scholar). Similarly, addition of PI 3-kinase inhibitors abrogated membrane ruffling and actin polymerization in response to insulin (10Kotani K. Hara K. Kotani K. Yonezawa K. Kasuga M. Biochem. Biophys. Res. Commun. 1995; 208: 985-990Crossref PubMed Scopus (81) Google Scholar, 11Martin S.S. Haruta T. Morris A.J. Klippel A. Williams L.T. Olefsky J.M. J. Biol. Chem. 1996; 271: 17605-17608Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 12Walters R.J. Hawkins P. Cooke F.T. Eguinoa A. Stephens L.R. FEBS Lett. 1996; 392: 66-70Crossref PubMed Scopus (10) Google Scholar). Precisely how PI 3-kinases participate in actin assembly is not known, but pharmacological inhibition of PI 3-kinase inhibits GTP loading of Rac1 stimulated by PDGF, and addition of constitutively active forms of Rac1 induces membrane ruffling despite the presence of PI 3-kinase inhibitors (10Kotani K. Hara K. Kotani K. Yonezawa K. Kasuga M. Biochem. Biophys. Res. Commun. 1995; 208: 985-990Crossref PubMed Scopus (81) Google Scholar, 13Hawkins P.T. Eguinoa A. Qui R.-G. Stokoe D. Cooke F.T. Walters R. Wennstrom S. Claesson-Welsh L. Evans T. Symons M. Stephens L. Curr. Biol. 1995; 5: 393-403Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar). These data suggest that PI 3-kinase lies upstream of Rac1. In contrast, recent studies of epithelial cells spreading on collagen suggest that PI 3-kinase, which is required for motility, may lie downstream of Rho family GTPases (14Keely P.J. Westwick J.K. Whitehead I.P. Der C.J. Parise L.V. Nature. 1997; 390: 632-636Crossref PubMed Scopus (658) Google Scholar). The role of PI 3-kinase in actin assembly mediated by other types of receptors is less clear. For G protein-linked receptors, such as the thrombin receptor (15Kovacsovics T.J. Bachelot C. Toker A. Vlahos C.J. Duckworth B. Cantley L.C. Hartwig J.H. J. Biol. Chem. 1995; 270: 11358-11366Crossref PubMed Scopus (200) Google Scholar) and the chemotactic peptide receptor (16Arcaro A. Wymann M.P. Biochem. J. 1993; 296: 297-301Crossref PubMed Scopus (1068) Google Scholar, 17Vlahos C.J. Matter W.F. Brown R.F. Traynorkaplan A.E. Heyworth P.G. Prossnitz E.R. Ye R.D. Marder P. Schelm J.A. Rothfuss K.J. Serlin B.S. Simpson P.J. J. Immunol. 1995; 154: 2413-2422PubMed Google Scholar), inhibition of PI 3-kinase has been reported to have no effect on stimulus-induced actin polymerization. For immunoreceptor tyrosine activation motif-containing receptors, such as FcεRI, addition of wortmannin does not inhibit IgE-induced actin polymerization but does block the appearance of well-formed membrane ruffles in response to antigen (18Barker S.A. Caldwell K.K. Hall A. Martinez A.M. Pfeiffer J.R. Oliver J.M. Wilson B.S. Mol. Biol. Cell. 1995; 6: 1145-1158Crossref PubMed Scopus (124) Google Scholar). Several studies have demonstrated a role for PI 3-kinase in FcγR-mediated phagocytosis (19Ninomiya N. Hazeki K. Fukui Y. Seya T. Okada T. Hazeki O. Ui M. J. Biol. Chem. 1994; 269: 22732-22737Abstract Full Text PDF PubMed Google Scholar, 20Crowley M.T. Costello P.S. Fitzer-Attas C.J. Turner M. Meng F. Lowell C. Tybulewicz V.L.J. DeFranco A.L. J. Exp. Med. 1997; 186: 1027-1039Crossref PubMed Scopus (418) Google Scholar, 21Araki N. Johnson M.T. Swanson J.A. J. Cell Biol. 1996; 135: 1249-1260Crossref PubMed Scopus (797) Google Scholar). In one, a role for this enzyme was suggested for the closure of phagosomes (21Araki N. Johnson M.T. Swanson J.A. J. Cell Biol. 1996; 135: 1249-1260Crossref PubMed Scopus (797) Google Scholar). Although quantitation of PI 3-kinase activity or F-actin was not performed, addition of wortmannin did not appear to inhibit the formation of "phagocytic cups," as determined by fluorescence micrographs of phalloidin-stained cells interacting with phagocytic targets (21Araki N. Johnson M.T. Swanson J.A. J. Cell Biol. 1996; 135: 1249-1260Crossref PubMed Scopus (797) Google Scholar). A study of FcεRI in mast cells suggests that stimulation of actin polymerization may not necessarily lead to membrane ruffles (18Barker S.A. Caldwell K.K. Hall A. Martinez A.M. Pfeiffer J.R. Oliver J.M. Wilson B.S. Mol. Biol. Cell. 1995; 6: 1145-1158Crossref PubMed Scopus (124) Google Scholar). Similarly, in DT40 lymphocytes expressing chimeric receptors encoding CD16 and the γ subunit of Fc receptors, addition of IgG-coated targets resulted in localized actin assembly and rudimentary plasma membrane protrusions, but phagocytosis did not occur (22Cox D. Chang P. Kurosaki T. Greenberg S. J. Biol. Chem. 1996; 271: 16597-16602Crossref PubMed Scopus (83) Google Scholar). This suggests that actin polymerization at the plasma membrane is not always coupled to pseudopod extension; distinct signals may be required for this function. Interestingly, a recent study suggested that pseudopod extension by FcγRI expressed in COS cells occurred in the absence of net actin assembly (23Lowry M.B. Duchemin A. Robinson J.M. Anderson C.L. J. Exp. Med. 1998; 187: 161-176Crossref PubMed Scopus (76) Google Scholar). Collectively, these studies suggest that actin assembly and pseudopod extension, two cellular events that normally coincide spatially and temporally, may be regulated by distinct signal transduction cascades. PI 3 kinases have been implicated in multiple aspects of membrane trafficking, including endocytosis, exocytosis, and membrane recycling (for review, see Ref. 24Shepherd P.R. Reaves B.J. Davidson H.W. Trends Cell Biol. 1996; 6: 92-97Abstract Full Text PDF PubMed Scopus (119) Google Scholar). During phagocytosis, significant amounts of plasma membrane are internalized in the form of phagocytic vacuoles. However, this is accompanied by no apparent decrease in membrane surface area (25Tapper H. Grinstein S. J. Immunol. 1997; 159: 409-418PubMed Google Scholar), suggesting that surface membrane is replenished from an intracellular source. To define the role of PI 3-kinase in phagocytosis, we used a variety of approaches to identify the stage in phagocytosis that was blocked during PI 3-kinase inhibition. These studies indicate that the block occurs during pseudopod extension, not during the very early phases (i.e. F-actin accumulation) or late phases (i.e. phagosomal closure) of ingestion and could be bypassed when requirements for pseudopod extension were minimized. The block in pseudopod extension coincided with a decrease in exocytic insertion of membrane, suggesting that PI 3-kinases are required for coordinating membrane insertion events and pseudopod extension. RAW LacR/FMLP.2 cells were derived from RAW 264.7 cells as described previously (3Cox D. Chang P. Zhang Q. Reddy P.G. Bokoch G.M. Greenberg S. J. Exp. Med. 1997; 186: 1487-1494Crossref PubMed Scopus (372) Google Scholar). Primary macrophages were isolated from the peritoneal cavities of C57Bl6 mice 3–5 days after the intraperitoneal injection of thioglycollate (thio-macrophages) as described previously (26Di Virgilio F. Meyer B.C. Greenberg S. Silverstein S.C. J. Cell Biol. 1988; 106: 657-666Crossref PubMed Scopus (157) Google Scholar). Cells were maintained in RPMI 1640 medium containing 10% fetal calf serum, 100 units/ml penicillin G, and 100 μg/ml streptomycin. Wortmannin and sulfhydral-modified BSA were from Calbiochem (La Jolla CA). LY294002 was from Biomol (Plymouth Meeting, PA). IgG against sheep erythrocytes was from Diamedix (Miami, FL). A monoclonal antibody directed against the p85 subunit of phosphatidylinositol 3-kinase was from Transduction Laboratories (Lexington, KY). [32P]Orthophosphate and [γ-32P]ATP were from Dupont NEN. Aluminum-backed Silica Gel 60 thin layer chromatography plates were from EM Separations (Gibbstown, NJ). Rabbit serum against BSA was from Dako Corp. (Carpinteria, CA). Fluorescein isothiocyanate- and rhodamine-conjugated F(ab′)2 fragments of anti-rabbit IgG were from Jackson ImmunoResearch (West Grove, PA). Rhodamine-phalloidin, YO-PRO 1 iodide, and FM1-43 were from Molecular Probes (Eugene, OR). Carboxylate-modified latex beads of various sizes were from Bangs Laboratories (Fishers, IN). 5 × 106 sheep erythrocytes opsonized with rabbit IgG (EIgG) were added to adherent cells for 30 min as described previously (27Greenberg S. El Khoury J. Kaplan E. Silverstein S.C. J. Immunol. Methods. 1991; 139: 115-122Crossref PubMed Scopus (17) Google Scholar). For opsonization of latex beads, carboxylate-modified latex beads ranging in size from 1 to 6 μm were incubated with 4 mg/ml sulhydryl-modified BSA dissolved in 0.05 m 2-[N-morpholino]ethanesulfonic acid, pH 5.5, and 1 mg/ml 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, followed by washing and incubation in various dilutions of rabbit antiserum against BSA for 1 h at 25 °C. After washing three times in phosphate-buffered saline, aliquots of beads were removed for immunoblotting to determine the quantity of IgG deposited on the beads. The density of IgG/μm2 was determined based on the calculated surface areas of the different bead size. Batches were discarded if the density of IgG deposited varied >15% of the mean, which was 5000 molecules/μm2. For phagocytosis assays, cells were incubated with beads for 45 min at 37 °C. Excess beads were washed away and the extent of binding and ingestion of the beads was determined. To detect attached, but uningested beads, cells were incubated with fluorescein isothiocyanate-conjugated anti-rabbit IgG for 45 min at 4 °C, followed by fixation in 3.7% formaldehyde. Cells were subsequently permeabilized with 0.2% Triton X-100, and total cell-associated beads were stained with rhodamine-conjugated anti-rabbit IgG. Phagocytosis was quantified as the total number of beads per cell (i.e. rhodamine-stained) minus the number of bound but uningested beads (i.e. those that were accessible to staining with fluorescein isothiocyanate-conjugated anti-rabbit IgG). 5–10 high power fields were scored for attached and ingested particles, and phagocytosis assays were performed in duplicate. Adherent RAW LacR/FMLP.2 cells were grown to 90% confluence in 6-cm dishes and were washed and incubated in minimal essential medium minus phosphates (Life Technologies, Inc.) containing 1% BSA and 37 MBq/ml [32P]orthophosphate acid for 2 h at 37 °C. Cells were washed and incubated further with 8 × 107 EIgG for varying times followed by phospholipid extraction and thin layer chromatography as described in (17Vlahos C.J. Matter W.F. Brown R.F. Traynorkaplan A.E. Heyworth P.G. Prossnitz E.R. Ye R.D. Marder P. Schelm J.A. Rothfuss K.J. Serlin B.S. Simpson P.J. J. Immunol. 1995; 154: 2413-2422PubMed Google Scholar). Samples were run with a PIP3 standard obtained by incubation of purified phosphatidylinositol-4,5-bisphosphate with [γ-32P]ATP and anti-phosphatidylinositol 3-kinase immunoprecipitates derived from RAW LacR/FMLP.2 cells. Adherent RAW LacR/FMLP.2 cells on coverslips were incubated with 3 × 106 EIgG on ice for 30 min to allow for particle binding, and after washing with ice-cold buffer, cells were incubated for a further 5 min at 37 °C before fixation in 3.7% formaldehyde and staining with 0.33 μm rhodamine-phalloidin. Cells were imaged using a confocal scanning system (Bio-Rad MRC 600) as described previously (22Cox D. Chang P. Kurosaki T. Greenberg S. J. Biol. Chem. 1996; 271: 16597-16602Crossref PubMed Scopus (83) Google Scholar). Total cellular F-actin was quantitated as described (22Cox D. Chang P. Kurosaki T. Greenberg S. J. Biol. Chem. 1996; 271: 16597-16602Crossref PubMed Scopus (83) Google Scholar) with the following modifications: RAW LacR/FMLP.2 cells were plated at 2 × 104 cells/well in 96 well tissue culture plates and treated with vehicle (dimethyl sulfoxide), 100 nm wortmannin, or 100 μmLY294002 at 37 °C for 30 min. 2 × 106 EIgG were added at 37 °C for 5 min, and cells were stained for F-actin with rhodamine-phalloidin as described above. Rhodamine fluorescence (excitation, 540 nm; emission, 590 nm) was measured using a fluorescence plate reader (CytoFluor II; Millipore) and normalized to cell number by dividing by a subsequent measurement of fluorescence (excitation, 485 nm; emission, 530 nm) after addition of 5 μm YO-PRO. Experiments were performed in triplicate. Thio-macrophages were pretreated with vehicle (dimethyl sulfoxide), 100 nmwortmannin, or 100 μm LY294002 for 30 min at 37 °C and then applied to 13-mm2 round coverslips previously coated with 1 mg/ml human IgG. Cells were allowed to spread for varying intervals at 37 °C followed by fixation and staining for F-actin (to facilitate delineation of cell margins). The cell diameter in close contact with the coverslip was measured, and the apparent adherent surface area was calculated. The adherent surface areas of ∼30 cells/coverslip were measured, and experiments were performed in duplicate. Thio-macrophages were preincubated with various concentrations of FM1-43 for 3 min at 25 °C and added to 96-well plates that had been previously coated with 1 mg/ml human IgG. Using a fluorescence plate reader (CytoFluor II), fluorescence (excitation, 485 nm; emission, 530 nm) was monitored in the continual presence of dye for varying time intervals at 25 °C. Cells and dye alone gave negligible fluorescence readings. FM1-43 fluorescence values were normalized to cell number after a subsequent fluorescence measurement (excitation, 360; emission, 460) after addition of 2 μm 4,6diamidino-2-phenylindole. PI 3 kinase has been shown to be activated by most receptors capable of mediating actin polymerization. To test the role of this family of enzymes in phagocytosis, we determined the effects of two structurally unrelated PI 3-kinase inhibitors, wortmannin and LY294002, on FcγR-mediated phagocytosis. Addition of either wortmannin or LY294002 produced a concentration-dependent inhibition of phagocytosis with an IC50 of ∼4.5 nm and 3 μm, respectively (Fig. 1). These results are similar to those reported for phagocytosis in guinea pig neutrophils (using wortmannin) and bone marrow-derived macrophages (19Ninomiya N. Hazeki K. Fukui Y. Seya T. Okada T. Hazeki O. Ui M. J. Biol. Chem. 1994; 269: 22732-22737Abstract Full Text PDF PubMed Google Scholar, 21Araki N. Johnson M.T. Swanson J.A. J. Cell Biol. 1996; 135: 1249-1260Crossref PubMed Scopus (797) Google Scholar). The IC50 values in this study were similar to those reported for inhibition of PI 3-kinases, but not for a PI 4-kinase (28Nakanishi S. Catt K.J. Balla T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5317-5321Crossref PubMed Scopus (313) Google Scholar) and myosin light chain kinase (29Nakanishi S. Kakita S. Takahashi I. Kawahara K. Tsukuda E. Sano T. Yamada K. Yoshida M. Kase H. Matsuda Y. Hashimoto Y. Nonomura Y. J. Biol. Chem. 1992; 267: 2157-2163Abstract Full Text PDF PubMed Google Scholar). Because mTOR is another target of wortmannin (30Brunn G.J. Williams J. Sabers C. Wiederrecht G. Lawrence J.C. Abraham R.T. EMBO J. 1996; 15: 5256-5267Crossref PubMed Scopus (631) Google Scholar), we tested the effects of rapamycin on phagocytosis; however, 10 μm rapamycin had no effect on phagocytosis (not shown). These results suggest that the inhibitory effects of wortmannin and LY294002 on FcγR-mediated phagocytosis were attributable to inhibition of one or more members of the PI 3-kinase family of enzymes. A previous study demonstrated that wortmannin inhibits PLA2 in vivo but not in vitro (31Cross M.J. Stewart A. Hodgkin M.N. Kerr D.J. Wakelam M.J.O. J. Biol. Chem. 1995; 270: 25352-25355Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar), suggesting that this class of phospholipases may lie downstream of a PI 3-kinase. In addition, FcγR-mediated phagocytosis is accompanied by enhanced activation of one or more isoforms of PLA2, and inhibition of PLA2activity leads to decreased phagocytosis (32Lennartz M.R. Brown E.J. J. Immunol. 1991; 147: 621-626PubMed Google Scholar). To determine whether the inhibition of phagocytosis by wortmannin is mediated via inhibition of PLA2, we preincubated cells with either 100 nmwortmannin or 10 μm bromophenacyl bromide (an irreversible inhibitor of PLA2) followed by the absence or presence of 2 μm exogenous arachidonate to attempt to bypass PLA2 blockade (32Lennartz M.R. Brown E.J. J. Immunol. 1991; 147: 621-626PubMed Google Scholar). Both wortmannin and bromophenacyl bromide inhibited phagocytosis of EIgG. Addition of exogenous arachidonate restored phagocytosis by 38 ± 3% in cells incubated with bromophenacyl bromide but did not do so in cells treated with wortmannin. This indicates that the decrease in phagocytosis in cells treated with wortmannin was not attributable to inhibition of PLA2 activity. To test whether ligation of Fcγ receptors results in the activation of PI 3 kinase(s)in vivo, [32P]orthophosphate-labeled macrophages were challenged with EIgG, phospholipids were extracted, and the phosphoinositide content was analyzed by thin layer chromatography. There was a rapid increase in the accumulation of PIP3 after FcγR ligation, which peaked at 15 s and declined by 1–2 min (Fig.2). Immunoprecipitation of the 85-kDa subunit of PI 3-kinase did not reveal enhanced phosphotyrosine content of this subunit (not shown). The increase in the accumulation of PIP3 that occurred during phagocytosis is consistent with an increase in the activity of one or more isoforms of PI 3-kinase. PI 3 kinase activity has been previously shown to be necessary for actin assembly mediated by PDGF or insulin (12Walters R.J. Hawkins P. Cooke F.T. Eguinoa A. Stephens L.R. FEBS Lett. 1996; 392: 66-70Crossref PubMed Scopus (10) Google Scholar, 33Heldman A.W. Kandzari D.E. Tucker R.W. Crawford L.E. Fearon E.R. Koblan K.S. Goldschmidt-Clermont P.J. Circ. Res. 1996; 78: 312-321Crossref PubMed Scopus (13) Google Scholar). Like receptor tyrosine kinases, ligation of Fcγ receptors results in activation of several tyrosine kinases, such as Syk, and tyrosine kinase activity is required for actin assembly mediated by this class of immunoreceptor tyrosine activation motif-bearing receptors (2Greenberg S. Chang P. Silverstein S.C. J. Exp. Med. 1993; 177: 529-534Crossref PubMed Scopus (171) Google Scholar). To determine whether the inhibition of phagocytosis by wortmannin or LY294002 correlated with an inhibition of FcγR-directed actin polymerization, we stained RAW 264.7 cells undergoing phagocytosis for F-actin using rhodamine-phalloidin. F-actin-rich phagocytic cups were visible in control cells treated with vehicle alone (Fig.3, A and B), similar to those seen previously (27Greenberg S. El Khoury J. Kaplan E. Silverstein S.C. J. Immunol. Methods. 1991; 139: 115-122Crossref PubMed Scopus (17) Google Scholar). In contrast to results obtained using growth factors, addition of wortmannin (Fig. 3, C andD) and LY294002 (not shown) had no obvious effect on the accumulation of F-actin beneath attached particles. However, careful observation of the cortical cytoplasm beneath attached cells revealed that, in nearly all cases, F-actin-rich pseudopods did not extend beyond ∼50–70% of the circumferences of the particles (Fig. 3,C and D). This was apparent only for particles attached to the sides of the macrophages, because the degree of pseudopod extension around particles that were attached to the dorsal surfaces of macrophages was difficult to appreciate. To determine whether the inhibition of PI 3 kinase in RAW 264.7 cells resulted in a failure to polymerize a sufficient quantity of actin necessary for complete pseudopod extension, we quantitated F-actin content in cells challenged with EIgG in the presence or absence of wortmannin or LY294002. In vehicle-treated cells there was an increase in the total amount of F-actin present after the addition of EIgG (Fig.3 E), which was blocked by the presence of 2 μmcytochalasin D (data not shown). The presence of either wortmannin or LY294002 did not inhibit increases in F-actin content after addition of EIgG and, in fact, led to slightly enhanced accumulations of F-actin (Fig. 3 E; p = 0.08 and 0.04, respectively). These data indicate that inhibition of PI 3-kinase did not impair actin assembly in response to FcγR ligation. The apparent block in maximal pseudopod extension in the presence of wortmannin (Fig. 3, C and D), with a concomitant preservation of FcγR-directed actin polymerization (Fig. 3 E), suggested that pathways leading to cytoskeletal assembly were not the target of PI 3-kinase inhibition. Because PI 3 kinase(s) have been implicated in the regulation of various membrane-trafficking events (for review, see Ref. 24Shepherd P.R. Reaves B.J. Davidson H.W. Trends Cell Biol. 1996; 6: 92-97Abstract Full Text PDF PubMed Scopus (119) Google Scholar), we considered the possibility that inhibition of this pathway produced a limitation in the membrane available for participation in pseudopod extension. In principle, the source of this membrane can be from the plasma membrane itself (e.g. from areas adjacent to those directly participating in particle ingestion) or from an intracellular membrane source (Fig. 4 A). To distinguish these possibilities, we first assessed the effect of reducing the total number of particles added to the macrophages on sensitivity to PI 3-kinase inhibition. However, reduction in the number of particles did not lead to a restoration of phagocytosis in the presence of wortmannin (Fig. 4 B), indicating that availability of plasma membrane was not a limiting factor for phagocytosis during PI 3-kinase inhibition. To determine whether inadequate pseudopod extension in the presence of PI 3-kinase inhibitors was attributable to a failure of the recruitment of membrane derived from an intracellular source, we challenged macrophages with phagocytic particles of varying sizes. We determined whether the phagocytic blockade by PI 3-kinase inhibitors was relieved by reducing particle size and, hence, the magnitude of pseudopod extension required for complete particle engulfment. By screening a variety of latex particles derivatized by several proteins, we found that covalent modification of carboxylated polystyrene beads with sulfhydryl-modified BSA afforded the lowest extent of nonspecific binding to macrophages. We chose a subclone of RAW 264.7 cells to study, RAW LacR/FMLPR.2, because this clone among several others demonstrated the least tendency to bind latex particles nonspecifically. Finally, we chose to opsonize the BSA-derivatized particles with an equivalent surface density of IgG, because this would allow for changes in particle size without altering ligand density and, hence, efficiency of FcγR engagement. Quantitative immunoblotting was performed to determine IgG opsonin density, and batches of beads of varying sizes were prepared that varied by no more than 15% in opsonin density. IgG-opsonized latex beads ranging from 1 to 6 μm in diameter were ingested by RAW LacR/FMLP.2 cells. Ingestion of these beads resembled phagocytosis of EIgG, because cytochalasin D inhibited phagocytosis by >90% regardless of the bead size. However, inhibition by cytochalasin D was slightly mor
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