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• W2086724321 abstract "Differing roles for [Ca2+]i transients in FcγR-mediated phagocytosis have been suggested based on the observations that antibody-opsonized erythrocyte phagocytosis by human neutrophils shows a [Ca2+]i dependence, while that by murine macrophages appears [Ca2+]i-independent. To explore whether this difference might reflect different receptor isoforms or different cell types, we studied the [Ca2+]i dependence of receptor-initiated phagocytosis by human FcγRIIa and a panel of FcγRIIa cytoplasmic domain mutants expressed in murine P388D1 cells and by human FcγR endogenously expressed on human neutrophils and monocytes. Wild-type and point mutants of huFcγRIIa stably transfected into murine P388D1 cells have different capacities to initiate a [Ca2+]i transient, which are closely correlated with quantitative phagocytosis (r = 0.94, p < 0.0001). Phagocytosis both by huFcγRIIa in P388D1 cells and by huFcγRIIa endogenously expressed on neutrophils and blood monocytes shows [Ca2+]i dependence. Phagocytosis of antibody-opsonized erythrocytes by neutrophils demonstrated greater susceptibility to [Ca2+]i quenching compared with FcγRIIa-specific internalization with E-IV.3, suggesting that the phagocytosis activating property of FcγRIIIb in neutrophils also engages a [Ca2+]i-dependent element. In contrast, phagocytosis by human FcγRIa, endogenously expressed on blood monocytes, is [Ca2+]i-independent. Despite the importance of a consensus tyrosine activation motif for both receptors, FcγRIa and FcγRIIa engage at least some distinct signaling elements to initiate phagocytosis. The recognition that both of the phagocytic receptors on murine macrophages and human FcγRIa associate with the FcεRI γ-chain, which contains a tyrosine activation motif distinct from that in the FcγRIIa cytoplasmic domain, suggests that [Ca2+]i-independent phagocytosis is a property associated with the utilization of γ-chains by FcγR. Differing roles for [Ca2+]i transients in FcγR-mediated phagocytosis have been suggested based on the observations that antibody-opsonized erythrocyte phagocytosis by human neutrophils shows a [Ca2+]i dependence, while that by murine macrophages appears [Ca2+]i-independent. To explore whether this difference might reflect different receptor isoforms or different cell types, we studied the [Ca2+]i dependence of receptor-initiated phagocytosis by human FcγRIIa and a panel of FcγRIIa cytoplasmic domain mutants expressed in murine P388D1 cells and by human FcγR endogenously expressed on human neutrophils and monocytes. Wild-type and point mutants of huFcγRIIa stably transfected into murine P388D1 cells have different capacities to initiate a [Ca2+]i transient, which are closely correlated with quantitative phagocytosis (r = 0.94, p < 0.0001). Phagocytosis both by huFcγRIIa in P388D1 cells and by huFcγRIIa endogenously expressed on neutrophils and blood monocytes shows [Ca2+]i dependence. Phagocytosis of antibody-opsonized erythrocytes by neutrophils demonstrated greater susceptibility to [Ca2+]i quenching compared with FcγRIIa-specific internalization with E-IV.3, suggesting that the phagocytosis activating property of FcγRIIIb in neutrophils also engages a [Ca2+]i-dependent element. In contrast, phagocytosis by human FcγRIa, endogenously expressed on blood monocytes, is [Ca2+]i-independent. Despite the importance of a consensus tyrosine activation motif for both receptors, FcγRIa and FcγRIIa engage at least some distinct signaling elements to initiate phagocytosis. The recognition that both of the phagocytic receptors on murine macrophages and human FcγRIa associate with the FcεRI γ-chain, which contains a tyrosine activation motif distinct from that in the FcγRIIa cytoplasmic domain, suggests that [Ca2+]i-independent phagocytosis is a property associated with the utilization of γ-chains by FcγR. Receptors for the Fc region of IgG (FcγR) 1The abbreviations used are: FcγRreceptors for the Fc region of IgGEAbovine erythrocytes opsonized with the IgG fraction of rabbit antibovine erythrocyte polyclonal antiseraBAPTA1,2-bis(2-aminophenoxy)ethane-N,N,N‘,N‘-tetraacetic acidmAbmonoclonal antibodyGAMpolyclonal F(ab′)2 goat anti-mouse IgGPBSphosphate-buffered salinePIphagocytic index (number of erythrocytes phagocytosed per 100 phagocytes)EBbiotinylated bovine erythrocytesEBAavidin coated EBFMLPformylmethionylleucylphenylalanineE-IV.3 and E-22erythrocytes coated with the anti-FcγRII mAb IV.3 Fab fragments or the FcγRI mAb 22 F(ab′)2 fragments through a biotin-avidin bridge. provide a link between antibody-antigen complexes and cellular-based effector functions and are critical in the regulation of the inflammatory response(1Hulett M.D. Hogarth P.M. Adv. Immunol. 1994; 57: 1-127Crossref PubMed Scopus (430) Google Scholar, 2Ravetch J.V. Cell. 1994; 78: 553-560Abstract Full Text PDF PubMed Scopus (339) Google Scholar). Significant structural diversity between the three gene families encoding FcγR is observed(1Hulett M.D. Hogarth P.M. Adv. Immunol. 1994; 57: 1-127Crossref PubMed Scopus (430) Google Scholar, 2Ravetch J.V. Cell. 1994; 78: 553-560Abstract Full Text PDF PubMed Scopus (339) Google Scholar, 3Ravetch J.V. Kinet J.P. Annu. Rev. Immunol. 1991; 9: 457-492Crossref PubMed Scopus (1282) Google Scholar, 4Kimberly R.P. Salmon J.E. Edberg J.C. Arthritis Rheum. 1995; 38: 306-314Crossref PubMed Scopus (127) Google Scholar, 5van de Winkel J.G.J. Capel P.J.A. Immunol. Today. 1993; 14: 215-221Abstract Full Text PDF PubMed Scopus (631) Google Scholar). Nonetheless, FcγR share certain intracellular signaling pathways. The common themes in FcγR signaling pathways involve the activation of protein tyrosine kinases followed by a transient rise in intracellular Ca2+ levels. The [Ca2+]i increase is essential for many cellular functions and is required for the phagocyte FcγR-induced oxidative burst(6Clapham D.E. Cell. 1995; 80: 259-268Abstract Full Text PDF PubMed Scopus (2268) Google Scholar, 7Macintyre E.A. Abdul-Gaffer R. O'Flynn K. Pilkington G.R. Farace F. Morgan J. Linch D.C. J. Immunol. 1988; 141: 4333-4343PubMed Google Scholar). receptors for the Fc region of IgG bovine erythrocytes opsonized with the IgG fraction of rabbit antibovine erythrocyte polyclonal antisera 1,2-bis(2-aminophenoxy)ethane-N,N,N‘,N‘-tetraacetic acid monoclonal antibody polyclonal F(ab′)2 goat anti-mouse IgG phosphate-buffered saline phagocytic index (number of erythrocytes phagocytosed per 100 phagocytes) biotinylated bovine erythrocytes avidin coated EB formylmethionylleucylphenylalanine erythrocytes coated with the anti-FcγRII mAb IV.3 Fab fragments or the FcγRI mAb 22 F(ab′)2 fragments through a biotin-avidin bridge. Many lines of evidence in both human and murine systems indicate that tyrosine phosphorylation events are critical for phagocyte FcγR functions, including phagocytosis(8Greenberg S. Chang P. Silverstein S.C. J. Exp. Med. 1993; 177: 529-534Crossref PubMed Scopus (168) Google Scholar, 9Greenberg S. Trends Cell Biol. 1995; 5: 93-99Abstract Full Text PDF PubMed Scopus (167) Google Scholar, 10Edberg J.C. Kimberly R.P. J. Immunol. 1994; 152: 5826-5835PubMed Google Scholar). In addition, in many systems examined, tyrosine kinase activity is required for the receptor-induced rise in [Ca2+]i (presumably through tyrosine phosphorylation of phospholipase Cγ1 and generation of inositol 1,4,5-trisphosphate). However, the role of [Ca2+]i in Fcγ receptor-mediated phagocytosis has been controversial. For example, work in murine macrophage cell lines suggests that transients in [Ca2+]i are not essential for phagocytosis of antibody-opsonized erythrocytes (EA) (11McNeil P.L. Swanson J.A. Wright S.D. Silverstein S.C. Taylor L.D. J. Cell Biol. 1986; 102: 1586-1592Crossref PubMed Scopus (47) Google Scholar, 12DiVirgilio F. Meyer B.C. Greenberg S. Silverstein S.C. J. Cell Biol. 1988; 106: 657-666Crossref PubMed Scopus (146) Google Scholar, 13Greenberg S. El Koury J. DiVirgilio F. Kaplan E.M. Silverstein S.C. J. Cell Biol. 1991; 113: 757-767Crossref PubMed Scopus (141) Google Scholar). In contrast, phagocytosis of EA by human neutrophils is significantly impaired by chelation of intracellular calcium and abrogation of [Ca2+]i transients(14Lew D.P. Andersson T. Hed J. DiVirgilio F. Pozzan T. Stendahl O. Nature. 1985; 315: 509-511Crossref PubMed Scopus (139) Google Scholar, 15Rosales C. Brown E.J. J. Immunol. 1991; 146: 3937-3944PubMed Google Scholar). The ability of [Ca2+]i-depleted neutrophils to mediate phagocytosis initiated by other cell surface receptors suggests that the [Ca2+]i-dependent EA phagocytosis by human neutrophils may reflect a particular property of the Fcγ receptors on these cells(16Della Bianca V. Grzeskowiak M. Rossi F. J. Immunol. 1990; 144: 1411-1417PubMed Google Scholar). Indeed, each of the studies of the [Ca2+]i-dependence of Fcγ receptor mediated phagocytosis has used EA probes that engage all available Fcγ receptor types and has not systematically distinguished between different cell types or cells derived from different species. Recent data indicate that important species differences do exist for Fcγ receptors. For example, human FcγRIIA and FcγRIIIB, expressed on neutrophils, do not have murine homologues(1Hulett M.D. Hogarth P.M. Adv. Immunol. 1994; 57: 1-127Crossref PubMed Scopus (430) Google Scholar, 2Ravetch J.V. Cell. 1994; 78: 553-560Abstract Full Text PDF PubMed Scopus (339) Google Scholar, 3Ravetch J.V. Kinet J.P. Annu. Rev. Immunol. 1991; 9: 457-492Crossref PubMed Scopus (1282) Google Scholar). Human FcγRIIA stably transfected into the murine macrophage cell line P388D1 mediates receptor-specific phagocytosis but in a [Ca2+]i-dependent fashion(17Odin J.A. Edberg J.C. Painter C.J. Kimberly R.P. Unkeless J.C. Science. 1991; 254: 1785-1788Crossref PubMed Scopus (135) Google Scholar). In contrast, murine FcγRII (the IIb isoform) does not have a tyrosine activation motif nor does it trigger a [Ca2+] transient or protein tyrosine phosphorylation(18Alber G. Kent U.M. Metzger H. J. Immunol. 1992; 149: 2428-2436PubMed Google Scholar). Murine FcγRII is also unable to mediate phagocytosis in macrophages in either a [Ca2+]i-dependent or independent fashion(19Takai T. Li M. Sylvestre D. Clynes R. Ravetch J.V. Cell. 1994; 76: 519-529Abstract Full Text PDF PubMed Scopus (825) Google Scholar). These observations, coupled with the recent data of Stendahl and co-workers (20Stendahl O. Krause K.-H. Krischer J. Jerstrom P. Theler J.-M. Clark R.A. Carpentier J.-L. Lew D.P. Science. 1994; 265: 1439-1441Crossref PubMed Scopus (127) Google Scholar) that [Ca2+]istorage organelles accumulate at contact sites during phagocytosis in human neutrophils prompted us to reexamine the question of the [Ca2+]i dependence of Fcγ receptor-mediated phagocytosis by human Fcγ receptors, endogenously expressed by human cells and stably transfected into the P388D1 murine macrophage cell line. Our data indicate that human FcγRIIa uses [Ca2+]i-dependent elements to mediate receptor-specific phagocytosis and that huFcγRIIa point mutants with a varying ability to initiate a [Ca2+]i transient show a closely corresponding variation in quantitative phagocytosis. Human FcγRIIa, endogenously expressed on neutrophils and blood monocytes, also shows partial [Ca2+]i dependence for phagocytosis. In contrast, phagocytosis by human FcγRIa, endogenously expressed on blood monocytes, is [Ca2+]i-independent. Despite the importance of tyrosine phosphorylation for phagocytosis and the use of a consensus tyrosine activation motif by both receptors(1Hulett M.D. Hogarth P.M. Adv. Immunol. 1994; 57: 1-127Crossref PubMed Scopus (430) Google Scholar, 2Ravetch J.V. Cell. 1994; 78: 553-560Abstract Full Text PDF PubMed Scopus (339) Google Scholar, 3Ravetch J.V. Kinet J.P. Annu. Rev. Immunol. 1991; 9: 457-492Crossref PubMed Scopus (1282) Google Scholar, 4Kimberly R.P. Salmon J.E. Edberg J.C. Arthritis Rheum. 1995; 38: 306-314Crossref PubMed Scopus (127) Google Scholar, 5van de Winkel J.G.J. Capel P.J.A. Immunol. Today. 1993; 14: 215-221Abstract Full Text PDF PubMed Scopus (631) Google Scholar, 21Ernst L.K. Duchemin A.M. Anderson C.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6023-6027Crossref PubMed Scopus (155) Google Scholar, 22Scholl P.R. Geha R.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8847-8850Crossref PubMed Scopus (96) Google Scholar, 23Greenberg S. Chang P. Silverstein S.C. J. Biol. Chem. 1994; 269: 3897-3902Abstract Full Text PDF PubMed Google Scholar, 24Durden D.L. Liu Y.B. Blood. 1994; 84: 2102-2108Crossref PubMed Google Scholar, 25Keiner P.A. Rankin B.M. Burkhardt A.L. Schieven G.L. Gilliland L.K. Rowley R.B. Bolen J.B. Ledbetter J.A. J. Biol. Chem. 1993; 268: 24442-24448Abstract Full Text PDF PubMed Google Scholar, 26Agarwal A. Salem P. Robbins K.C. J. Biol. Chem. 1993; 268: 15900-15905Abstract Full Text PDF PubMed Google Scholar, 27Huang M.M. Indik Z. Brass L.F. Hoxie J.A. Schreiber A.D. Brugge J.S. J. Biol. Chem. 1992; 267: 5467-5473Abstract Full Text PDF PubMed Google Scholar, 28Shen Z. Lin C.-T. Unkeless J.C. J. Immunol. 1994; 152: 3017-3023PubMed Google Scholar, 29Ghazizadeh S. Bolen J.B. Fleit H.B. Biochem. J. 1995; 305: 669-674Crossref PubMed Scopus (60) Google Scholar, 30Hunter S. Kamoun M. Schreiber A.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10232-10236Crossref PubMed Scopus (30) Google Scholar), they engage at least some distinct signaling elements to initiate phagocytosis. The recognition that the human FcγRIa associates with the FcεRI γ-chain, which contains the tyrosine activation motif (21Ernst L.K. Duchemin A.M. Anderson C.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6023-6027Crossref PubMed Scopus (155) Google Scholar, 22Scholl P.R. Geha R.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8847-8850Crossref PubMed Scopus (96) Google Scholar) as do both of the phagocytic receptors on murine macrophages(23Greenberg S. Chang P. Silverstein S.C. J. Biol. Chem. 1994; 269: 3897-3902Abstract Full Text PDF PubMed Google Scholar), suggests that [Ca2+]i-independent phagocytosis is a property associated with FcγR utilizing γ-chains. Human FcγRIIa, while engaging many elements in common with human FcγRIa such as p72syk and phospholipase Cγ1(25Keiner P.A. Rankin B.M. Burkhardt A.L. Schieven G.L. Gilliland L.K. Rowley R.B. Bolen J.B. Ledbetter J.A. J. Biol. Chem. 1993; 268: 24442-24448Abstract Full Text PDF PubMed Google Scholar, 26Agarwal A. Salem P. Robbins K.C. J. Biol. Chem. 1993; 268: 15900-15905Abstract Full Text PDF PubMed Google Scholar, 27Huang M.M. Indik Z. Brass L.F. Hoxie J.A. Schreiber A.D. Brugge J.S. J. Biol. Chem. 1992; 267: 5467-5473Abstract Full Text PDF PubMed Google Scholar, 28Shen Z. Lin C.-T. Unkeless J.C. J. Immunol. 1994; 152: 3017-3023PubMed Google Scholar, 29Ghazizadeh S. Bolen J.B. Fleit H.B. Biochem. J. 1995; 305: 669-674Crossref PubMed Scopus (60) Google Scholar, 30Hunter S. Kamoun M. Schreiber A.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10232-10236Crossref PubMed Scopus (30) Google Scholar), must also engage [Ca2+]i-dependent elements. NHS-LC-biotin, sulfo-NHS-biotin, and streptavidin were obtained from Pierce. BAPTA-AM and Indo-1/AM were from Molecular Probes (Eugene, OR). A 10 mM stock of BAPTA-AM in dimethyl sulfoxide was prepared and stored at −20°C. Genistein was from Life Technologies, Inc. and was stored as a 20 mg/ml stock in dimethyl sulfoxide at −20°C. Anti-FcγR mAbs 32.2, 22 (FcγRI, CD64) and IV.3 (FcγRII, CD32) Fab or F(ab′)2 fragments were obtained from Medarex (Annandale, NJ); IgM anti-H-2Dd was obtained from Pharmingen (San Diego, CA). Fab and F(ab′)2 fragments were free of intact IgG as detected by silver stain analysis after SDS-polyacrylamide gel electrophoresis and size exclusion high performance liquid chromatography. Unconjugated F(ab′)2 goat anti-mouse IgG (GAM) for mAb cross-linking and phycoerythrin- and fluorescein isothiocyanate-conjugated GAM for immunofluorescence flow cytometry studies were obtained from Jackson Immunoresearch (West Grove, PA) or Boehringer Mannheim. Fetal calf serum and RPMI 1640 were obtained from Life Technologies, Inc. All other reagents were from Sigma. Mutant FcγRIIA cDNAs were made by oligonucleotide primer-directed site-specific mutagenesis of a human FcγRIIA cDNA generously provided by J. Kochan (Hoffman-La Roche, Nutley, NJ)(31Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4558) Google Scholar). Mutants, confirmed by sequencing (Sequenase 2.0, U. S. Biochemical Corp.), were subcloned into pcEXV-3 (32Miller J. Malek T.R. Leonard W.J. Greene W.C. Shevach E.M. Germain R.N. J. Immunol. 1985; 134: 4212-4217PubMed Google Scholar) and transfected by CaPO4 precipitation in the presence of 25-100 μM chloroquine into the murine macrophage cell line, P388D1. Stable transfectants were screened and selected by flow cytometry, and assessment of receptor expression was demonstrated between 1.1 and 2.5 × 106 receptors/cell. Fresh anti-coagulated human peripheral blood was separated by centrifugation through a discontinuous two-step Ficoll-Hypaque gradient (33Edberg J.C. Redecha P.B. Salmon J.E. Kimberly R.P. J. Immunol. 1989; 143: 1642-1649PubMed Google Scholar). Mixed mononuclear cells were isolated from the upper interface and washed with Hanks' balanced salt solution. Neutrophils were isolated from the lower interface and washed with Hanks' balanced salt solution. Contaminating erythrocytes in the neutrophil harvest were lysed with hypotonic saline (0.2% NaCl) for 20 s followed by 1.6% NaCl and a final wash with Hanks' balanced salt solution. After final washes, cells were resuspended to 5 × 106 cells/ml in PBS prior to immunofluorescent staining or in RPMI containing 10% fetal calf serum prior to phagocytosis. Treatment of cells with BAPTA (to quench intracellular Ca2+ levels) or genistein (to block protein tyrosine kinase activity) was performed as described previously(10Edberg J.C. Kimberly R.P. J. Immunol. 1994; 152: 5826-5835PubMed Google Scholar, 17Odin J.A. Edberg J.C. Painter C.J. Kimberly R.P. Unkeless J.C. Science. 1991; 254: 1785-1788Crossref PubMed Scopus (135) Google Scholar, 34Kimberly R.P. Ahlstrom J.W. Click M.E. Edberg J.C. J. Exp. Med. 1990; 171: 1239-1255Crossref PubMed Scopus (94) Google Scholar). Briefly, cells were incubated with BAPTA-AM (1-100 μM) in buffer without free Ca2+ for 30 min at room temperature (neutrophils and monocytes) or 37°C (P388D1 transfectants) followed by one wash. Buffer containing Ca2+ was then added and handled as described below. For genistein treatment, cells were preincubated with 100 μg/ml genistein for 30 min, and then the genistein was maintained at the same concentration through the phagocytic assay. Controls included loading cells with the BAPTA-AM and genistein solvent (dimethyl sulfoxide) at appropriate concentrations for the same period of time. As an alternative to BAPTA-AM treatment for quenching [Ca2+]i, cells were allowed to drain their intracellular Ca2+ stores as described by Rosales et al.(35Rosales C. Brown E.J. J. Biol. Chem. 1992; 267: 1443-1448Abstract Full Text PDF PubMed Google Scholar). Levels of intracellular Ca2+ were directly determined in all cases as described below. Intracellular [Ca2+]i was determined in Indo-1/AM-loaded cells using an SLM 8000 fluorimeter and the simultaneous 405/490 nm fluorescence emission ratio as described previously(17Odin J.A. Edberg J.C. Painter C.J. Kimberly R.P. Unkeless J.C. Science. 1991; 254: 1785-1788Crossref PubMed Scopus (135) Google Scholar, 34Kimberly R.P. Ahlstrom J.W. Click M.E. Edberg J.C. J. Exp. Med. 1990; 171: 1239-1255Crossref PubMed Scopus (94) Google Scholar). Briefly, suspensions of cells at 107/ml in Ca2+- and Mg2+-free phosphate-buffered saline, pH 7.4, were incubated with 5 μM Indo-1/AM at 37°C for 15 min and washed in PBS. Cells preparations to be opsonized with receptor-specific mAb Fab were resuspended in Ca2+-and Mg2+-free PBS at 107 cells/ml, incubated with saturating concentrations of mAb IV.3 Fab (0.5 μg/ml) or mAb 22 F(ab′)2 (2 μg/ml) at 37°C for 5 min, and washed in PBS. All cell preparations were resuspended in 1.1 mM Ca2+, 1.6 mM Mg2+ PBS at 37°C for 5 min and then immediately transferred to a continuously stirred cell cuvette maintained at 37°C in the SLM 8000. With excitation at 355 nm, the simultaneous fluorescence emission at 405 and 490 nm was measured, integrated, and recorded each second. After establishing a base line for 60 s, goat anti-mouse F(ab′)2 was added (35 μg/ml final concentration), and data acquisition was continued for an additional 3.5 min. Each sample was individually calibrated by lysing cells in 1% Triton X-100 to determine the maximal emission ratio and by adding EDTA (20 mM final concentration) to determine the minimal ratio. The Indo-1 fluorescence emission ratio was converted to [Ca2+]i by the method of Grynkiewicz et al.(36Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). Biotinylated bovine erythrocytes (EB) and biotinylated anti-FcγR were prepared as described previously(37Edberg J.C. Kimberly R.P. J. Immunol. Methods. 1992; 148: 179-187Crossref PubMed Scopus (27) Google Scholar). Briefly, erythrocytes (at 1 × 109 cells/ml in 0.1 M carbonate buffer (pH 8.6)) were incubated with 250 μg/ml of sulfo-NHS-biotin for 20 min at 4°C with mixing. EB (1 × 109 E/ml) were coated with an equal volume of streptavidin (250 μg/ml) for 30 min at 4°C with mixing. The streptavidin-coated EB (EBA) were then washed and resuspended to 1 × 109 erythrocytes/ml for immediate use. mAb were biotinylated with NHS-LC-biotin in 0.1 M carbonate buffer (pH 8.6). Typically, 50 μg/ml NHS-LC-biotin was used to biotinylate mAb (1 mg/ml) for 60 min at room temperature with occasional mixing. The free biotin was removed by extensive dialysis against PBS (pH 7.4). Small volume dialysis (ranging from 50-100 μl for the mAb) was performed in a dialysis chamber (Pierce). mAb-conjugated erythrocytes were prepared by incubating EBA with dilutions of biotinylated anti-FcγR mAb(37Edberg J.C. Kimberly R.P. J. Immunol. Methods. 1992; 148: 179-187Crossref PubMed Scopus (27) Google Scholar). mAb-coated EBA were resuspended in RPMI 1640-fetal calf serum, an aliquot was removed for analysis by indirect immunofluorescence, and the remaining cells were used immediately for the phagocytosis or attachment assays. EA were prepared by incubating bovine erythrocytes with a 1:4 dilution of the maximal subagglutinating titer of rabbit anti-bovine erythrocyte IgG as described previously(38Salmon J.E. Edberg J.C. Kimberly R.P. J. Clin. Invest. 1990; 85: 1287-1295Crossref PubMed Scopus (205) Google Scholar). For quantitation of mAb-coated EBA or EA phagocytosis by fresh human cells, erythrocytes were mixed with 100 μl of fresh neutrophils (5 × 106 cells/ml) or fresh mononuclear cells (5 × 106 monocytes/ml, determined by myeloperoxidase staining) at a ratio of 25:1 (mAb-coated EBA) or 50:1 (EA) (37Edberg J.C. Kimberly R.P. J. Immunol. Methods. 1992; 148: 179-187Crossref PubMed Scopus (27) Google Scholar). The cell mixture was pelleted for 5 min at room temperature at 44 × g and then incubated at 37°C for 20 min (neutrophils) or 1 h (mononuclear cells). After the nonphagocytosed erythrocytes were lysed with hypotonic saline (0.2% NaCl for 20 s followed by the addition of an equal volume of 1.6% NaCl), phagocytosis was quantitated by light microscopy. The data are expressed as phagocytic index (PI, the number of ingested particles/100 neutrophils or monocytes). Phagocytosis by transfected P388D1 cells was determined in an adherent assay system. P388D1 cells (5 × 105 cells/ml) were allowed to adhere to round glass coverslips in culture dishes overnight at 37°C. Coverslips were then transferred to clean culture dishes and EA- or mAb-coated EBA were added (50 μl at 5 × 107 erythrocytes/ml) were added and incubated for 1 h at 37°C. Noninternalized erythrocytes were lysed by brief immersion of the coverslip in dH2O followed by immersion in buffer. Phagocytosis was quantitated by light microscopy and expressed as phagocytic index as described above. Heat-treated and serum-treated zymosan were prepared as described previously(38Salmon J.E. Edberg J.C. Kimberly R.P. J. Clin. Invest. 1990; 85: 1287-1295Crossref PubMed Scopus (205) Google Scholar). Briefly, heat-treated zymosan were prepared by boiling 10 mg of zymosan for 10 min. Serum-treated zymosan were prepared by incubating 2 mg of zymosan with 2 ml of normal human serum for 30 min at 37°C. Following washing, both heat-treated zymosan and serum-treated zymosan were resuspended to 2.5 × 108/ml. For phagocytosis, heat-treated zymosan or serum-treated zymosan were mixed with neutrophils (5:1, zymosan/neutrophil ratio), pelleted, and incubated for 20 min at 37°C. Phagocytosis was assessed by light microscopy. Determination of FcγRIIIb alleles, NA1 and NA2, was performed by quantitative flow cytometry with mAbs CLB-FcR-gran 1, CLB-gran 11, and GRM1(33Edberg J.C. Redecha P.B. Salmon J.E. Kimberly R.P. J. Immunol. 1989; 143: 1642-1649PubMed Google Scholar, 38Salmon J.E. Edberg J.C. Kimberly R.P. J. Clin. Invest. 1990; 85: 1287-1295Crossref PubMed Scopus (205) Google Scholar). The assignment of NA type was confirmed by leukoagglutination as described previously (38Salmon J.E. Edberg J.C. Kimberly R.P. J. Clin. Invest. 1990; 85: 1287-1295Crossref PubMed Scopus (205) Google Scholar) and by immunoprecipitation of selected donors(33Edberg J.C. Redecha P.B. Salmon J.E. Kimberly R.P. J. Immunol. 1989; 143: 1642-1649PubMed Google Scholar). Phenotyping of donors for the LR-HR alleles of FcγRIIa was performed by quantitative flow cytometry using mAbs 41.H16 and IV.3 as described previously(39Gosselin E.J. Brown M.F. Anderson C.L. Zipf T.F. Guyre P.M. J. Immunol. 1990; 144: 1817-1822PubMed Google Scholar, 40Salmon J.E. Edberg J.C. Brogle N.L. Kimberly R.P. J. Clin. Invest. 1992; 89: 1274-1281Crossref PubMed Scopus (251) Google Scholar). Phagocytosis data are displayed as the mean ± S.D. Ca2+ data are representative experiments. Differences in phagocytosis between phagocytic probes were compared with a Student's t test and differences between probes over a range of BAPTA concentrations (see Fig. 4B) was determined using two-way analysis of variance. Several recent observations including information on species differences in Fcγ receptor isoforms and function (1Hulett M.D. Hogarth P.M. Adv. Immunol. 1994; 57: 1-127Crossref PubMed Scopus (430) Google Scholar, 2Ravetch J.V. Cell. 1994; 78: 553-560Abstract Full Text PDF PubMed Scopus (339) Google Scholar, 17Odin J.A. Edberg J.C. Painter C.J. Kimberly R.P. Unkeless J.C. Science. 1991; 254: 1785-1788Crossref PubMed Scopus (135) Google Scholar, 19Takai T. Li M. Sylvestre D. Clynes R. Ravetch J.V. Cell. 1994; 76: 519-529Abstract Full Text PDF PubMed Scopus (825) Google Scholar) have prompted a reconsideration of the studies supporting [Ca2+]i-dependent and [Ca2+]i-independent Fcγ receptor-mediated phagocytosis. For example, studies by Stendahl et al.(20Stendahl O. Krause K.-H. Krischer J. Jerstrom P. Theler J.-M. Clark R.A. Carpentier J.-L. Lew D.P. Science. 1994; 265: 1439-1441Crossref PubMed Scopus (127) Google Scholar) have shown that there is an accumulation of [Ca2+]i storage organelles during phagocytosis in human neutrophils. These observations suggest the possibility that the Fcγ receptors expressed in human neutrophils are functionally distinct from murine Fcγ receptors in engaging [Ca2+]i-dependent elements for phagocytosis. Indeed, initial studies of human FcγRIIA truncation mutants stably transfected into P388D1 cells have shown that all truncations unable to initiate a [Ca2+]i transient are unable to mediate receptor-specific phagocytosis(17Odin J.A. Edberg J.C. Painter C.J. Kimberly R.P. Unkeless J.C. Science. 1991; 254: 1785-1788Crossref PubMed Scopus (135) Google Scholar). The evidence for an essential role for [Ca2+]i in FcγRIIa phagocytosis is strengthened by the ability of BAPTA, a chelator of [Ca2+]i, to block phagocytosis by FcγRIIa wild-type receptor in P388D1 cells(17Odin J.A. Edberg J.C. Painter C.J. Kimberly R.P. Unkeless J.C. Science. 1991; 254: 1785-1788Crossref PubMed Scopus (135) Google Scholar). Accordingly, we have examined these relationships in a series of FcγRIIa transfectants with point mutations in the region of the cytoplasmic domain containing the YXXL tyrosine activation motif. Mutations in this region (Fig. 1) can lead to altered binding and activation of p72syk, which in turn phosphorylates phospholipase Cγ-1 leading to the generation of inositol 1,4,5-trisphosphate and [Ca2+]i transients(41Gauen L.K. Zhu Y. Letourneur F. Hu Q. Bolen J.B. Matis L.A. Klausner R.D. Shaw A.A. Mol. Cell. Biol. 1994; 14: 3729-3741Crossref PubMed Scopus (128) Google Scholar). Studies of transfected FcγR mutants indicate that receptor-mediated phagocytosis is also altered (42Mitchell M.A. Huang M.M. Chien P. Indik Z.K. Pan X.P. Schreiber A.D. Blood. 1994; 84: 1753-1759Crossref PubMed Google Scholar, 43Kolanus W. Romeo C. Seed B. EMBO J. 1992; 11: 4861-4868Crossref PubMed Scopus (38) Google Scholar Fifteen mutants were constructed (Fig. 1), and stable transfectants expressing between 1.1 and 2.6 × 106 receptors/cell were selected. The [Ca2+] transient observed after cross-linking each mutant receptor was measured and ranged from no response for several mutants of tyrosine residues within the tyrosine activation motif to a flux of approximately 500 nM (Fig. 2). The measured [Ca2+]i transients were abrogated by pretreatment of cells with BAPTA, were unaffected by 10 mM EGTA extracellularly, and therefore were due to mobilization of [Ca2+]i from intracellular stores. Among the 15 cell lines expressing different mutant FcγRIIa, there was no significant relationship between quantitative receptor expression measured" @default.
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• W2086724321 title "The Ca2+ Dependence of Human Fcγ Receptor-initiated Phagocytosis" @default.
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