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• W2033073750 abstract "Functional interactions between Fcγ-receptors (FcγR) and the β2 integrin Mac-1 (CD11b/CD18) have been described, but the molecular basis of this relationship remains unclear. Although the glycosylphosphatidylinositol-linked receptor FcγRIIIB of human neutrophils is constitutively associated with Mac-1, we found no evidence for direct physical association between Mac-1 and the FcγR of mouse macrophages, which are transmembrane proteins. Nevertheless, Mac-1 accumulated in the phagocytic cup following engagement of FcγR by IgG-opsonized particles. Blocking the CD18 chains of β2 integrins by using specific antibodies reduced Mac-1 accumulation in the cup. These antibodies or the addition of the recombinant CD11b I-domain inhibited the ingestion of IgG-opsonized particles. FcγR cross-linking stimulated cell adhesion to surfaces coated with Mac-1 ligands and in addition enabled macrophages to bind C3bi-opsonized particles, indicating that FcγR-derived signals induce activation of Mac-1. Measurements of fluorescence recovery after photobleaching revealed that whereas most (>80%) of Mac-1 is immobile in resting cells, stimulation of FcγR markedly increases the mobile fraction of the integrin. Activation of Mac-1 by FcγR required the activity of Src family tyrosine kinases, phosphatidylinositol 3-kinase and phospholipase C, with the release of diacylglycerol and stimulation of protein kinase C. Because elevated cytosolic Ca2+ was not required, we suggest that novel protein kinase C isoforms are involved in Mac-1 activation. These results suggest that FcγR stimulation promotes Mac-1 clustering into high avidity complexes in phagocytic cups by releasing the integrin from cytoskeletal constraints and enhancing its lateral diffusion. FcγR can enhance host defense by activating Mac-1 (and possibly other integrins), having a synergistic effect on pathogen engulfment and promoting the adherence of phagocytes at sites of infection. Functional interactions between Fcγ-receptors (FcγR) and the β2 integrin Mac-1 (CD11b/CD18) have been described, but the molecular basis of this relationship remains unclear. Although the glycosylphosphatidylinositol-linked receptor FcγRIIIB of human neutrophils is constitutively associated with Mac-1, we found no evidence for direct physical association between Mac-1 and the FcγR of mouse macrophages, which are transmembrane proteins. Nevertheless, Mac-1 accumulated in the phagocytic cup following engagement of FcγR by IgG-opsonized particles. Blocking the CD18 chains of β2 integrins by using specific antibodies reduced Mac-1 accumulation in the cup. These antibodies or the addition of the recombinant CD11b I-domain inhibited the ingestion of IgG-opsonized particles. FcγR cross-linking stimulated cell adhesion to surfaces coated with Mac-1 ligands and in addition enabled macrophages to bind C3bi-opsonized particles, indicating that FcγR-derived signals induce activation of Mac-1. Measurements of fluorescence recovery after photobleaching revealed that whereas most (>80%) of Mac-1 is immobile in resting cells, stimulation of FcγR markedly increases the mobile fraction of the integrin. Activation of Mac-1 by FcγR required the activity of Src family tyrosine kinases, phosphatidylinositol 3-kinase and phospholipase C, with the release of diacylglycerol and stimulation of protein kinase C. Because elevated cytosolic Ca2+ was not required, we suggest that novel protein kinase C isoforms are involved in Mac-1 activation. These results suggest that FcγR stimulation promotes Mac-1 clustering into high avidity complexes in phagocytic cups by releasing the integrin from cytoskeletal constraints and enhancing its lateral diffusion. FcγR can enhance host defense by activating Mac-1 (and possibly other integrins), having a synergistic effect on pathogen engulfment and promoting the adherence of phagocytes at sites of infection. Mac-1, a heterodimeric receptor primarily expressed in neutrophils and monocytes/macrophages, is composed of a specific α chain (CD11b) and the β2 chain (CD18) which is common to the other members of the β2 integrin family (1Ehlers M.R. Microbes Infect. 2000; 2: 289-294Crossref PubMed Scopus (258) Google Scholar). As is the case for other integrins, Mac-1 (also known as CD11b/CD18, αMβ2, Mo-1, or CR3) activation is required for efficient binding to several ligands such as intercellular adhesion molecule 1, C3bi, or fibrinogen. Activation of the cells by specific agonists induces the receptor to undergo conformational changes, mobilization, and clustering by a process known as inside-out signaling (2Hughes P.E. Pfaff M. Trends Cell Biol. 1998; 8: 359-364Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar, 3van Kooyk Y. Figdor C.G. Curr. Opin. Cell Biol. 2000; 12: 542-547Crossref PubMed Scopus (294) Google Scholar). A variety of studies have demonstrated that Mac-1 participates in a number of important aspects of the innate immune response, including phagocyte adhesion, migration, and engulfment of complement-opsonized particles (1Ehlers M.R. Microbes Infect. 2000; 2: 289-294Crossref PubMed Scopus (258) Google Scholar, 4Coxon A. Rieu P. Barkalow F.J. Askari S. Sharpe A.H. von Andrian U.H. Arnaout M.A. Mayadas T.N. Immunity. 1996; 5: 653-666Abstract Full Text PDF PubMed Scopus (546) Google Scholar). Such functions are generally triggered by direct binding of ligands like intercellular adhesion molecule 1 and C3 complement fragment C3bi to Mac-1. In addition, Mac-1 interacts with and appears to serve as a signaling partner for glycosylphosphatidylinositollinked receptors such as urokinase-type plasminogen activator receptor and CD14 (5Todd III, R.F. Petty H.R. J. Lab. Clin. Med. 1997; 129: 492-498Abstract Full Text PDF PubMed Scopus (93) Google Scholar). Whereas Mac-1 can directly recognize components of the microbial wall (6Vetvicka V. Thornton B.P. Ross G.D. J. Clin. Investig. 1996; 98: 50-61Crossref PubMed Scopus (362) Google Scholar, 7Thornton B.P. Vetvicka V. Pitman M. Goldman R.C. Ross G.D. J. Immunol. 1996; 156: 1235-1246PubMed Google Scholar), phagocytosis via this receptor is most efficient when the target particles are coated with complement fragment C3bi, a process known as opsonization. Opsonic phagocytosis can also be mediated by Fcγ-receptors (FcγR), 1The abbreviations used are: FcγR, Fcγ-receptors; agg-IgG, aggregated human IgG; FCS, fetal calf serum; FRAP, fluorescence recovery after photobleaching; HBSS, Hank's buffered saline solution; HPMI, Hepes-buffered solution RPMI-1640; IgG-RBC or C3bi-RBC, IgG-or C3bi-opsonized red blood cells; mAb, monoclonal antibodies; MFI, mean fluorescence intensity; PI3K, phosphatidylinositol 3-kinase; FITC, fluorescein isothiocyanate; PMA, phorbol 12-myristate 13-acetate; Ab, antibody; BSA, bovine serum albumin; PE, phycoerythrin; PBS, phosphate-buffered saline; PKC, protein kinase C; TRITC, tetramethylrhodamine isothiocyanate; GST, glutathione S-transferase; GFP, green fluorescent protein; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester; DiC8, 1,2-dioctanoyl-sn-glycerol; PLC, phospholipase C. which recognize the constant region of IgG bound to antigenic particles (8Gessner J.E. Heiken H. Tamm A. Schmidt R.E. Ann. Hematol. 1998; 76: 231-248Crossref PubMed Scopus (345) Google Scholar). Unlike the integrins, however, FcγR are capable of binding and responding to their ligands without priming, i.e. stimulation of the cells by other agonists. In vitro studies using single opsonins have demonstrated that engagement of either FcγR or Mac-1 suffices to initiate phagocytosis. However, interactions between the two systems have been suggested by various observations. Neutrophils from leukocyte adhesion deficiency syndrome patients, which have mutations in the gene encoding CD18 (9Etzioni A. Doerschuk C.M. Harlan J.M. Blood. 1999; 94: 3281-3288Crossref PubMed Google Scholar), have a reduced ability to ingest IgG-opsonized red blood cells (IgG-RBC) (10Gresham H.D. Graham I.L. Anderson D.C. Brown E.J. J. Clin. Investig. 1991; 88: 588-597Crossref PubMed Scopus (73) Google Scholar). Moreover, antibodies that block Mac-1 function depressed IgG-mediated phagocytosis without impairing the binding of FcγR to its ligands (11Graham I.L. Gresham H.D. Brown E.J. J. Immunol. 1989; 142: 2352-2358PubMed Google Scholar). Also, FcγR-mediated adhesion of neutrophils to immobilized immune complexes was inhibited by antibodies targeting either CD18 or CD11b (12Jones S.L. Knaus U.G. Bokoch G.M. Brown E.J. J. Biol. Chem. 1998; 273: 10556-10566Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 13Kusunoki T. Tsuruta S. Higashi H. Hosoi S. Hata D. Sugie K. Mayumi M. Mikawa H. J. Leukocyte Biol. 1994; 55: 735-742Crossref PubMed Scopus (34) Google Scholar), and Mac-1-deficient mice displayed impaired immune complex-mediated recruitment of neutrophils onto glomerular basement membrane in vivo or spreading on immune complexes in vitro (14Tang T. Rosenkranz A. Assmann K.J. Goodman M.J. Gutierrez-Ramos J.C. Carroll M.C. Cotran R.S. Mayadas T.N. J. Exp. Med. 1997; 186: 1853-1863Crossref PubMed Scopus (175) Google Scholar). These studies and that of Coxon et al. (15Coxon A. Cullere X. Knight S. Sethi S. Wakelin M.W. Stavrakis G. Luscinskas F.W. Mayadas T.N. Immunity. 2001; 14: 693-704Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar) suggest that Mac-1 is required for sustained leukocyte adhesion following FcγR engagement. The manner whereby FcγR and Mac-1 receptors interact remains largely obscure. One type of FcγR, the human FcγRIIIB, has been reported to exist in physical association with Mac-1. A carbohydrate-mediated interaction is thought to exist between these receptors, which undergo co-capping in human neutrophils (16Zhou M. Todd III, R.F. van de Winkel J.G. Petty H.R. J. Immunol. 1993; 150: 3030-3041PubMed Google Scholar, 17Poo H. Krauss J.C. Mayo-Bond L. Todd III, R.F. Petty H.R. J. Mol. Biol. 1995; 247: 597-603PubMed Google Scholar). However, association with FcγRIIIB is unlikely to account for all the observations reported above. The expression of FcγRIIIB is restricted to human neutrophils (18Ravetch J.V. Perussia B. J. Exp. Med. 1989; 170: 481-497Crossref PubMed Scopus (499) Google Scholar, 19Li M. Wirthmueller U. Ravetch J.V. J. Exp. Med. 1996; 183: 1259-1263Crossref PubMed Scopus (54) Google Scholar), yet cooperation between Mac-1 and FcγR has been described in cell types that lack this receptor isoform. Moreover, it is unclear how clustering and signaling would be initiated by the complex of quiescent Mac-1 and FcγRIIIB, which is a glycosylphosphatidylinositol-linked and therefore signaling-incompetent form of FcγR. In this study we sought to define whether the transmembrane forms of FcγR, such as those expressed in murine cells (which lack FcγRIIIB), undergo functional interactions with Mac-1. To this end we studied the possible role of Mac-1 in the phagocytosis of IgG-RBC in unprimed RAW264.7 mouse macrophages. In particular, we explored whether direct physical interactions exist between Mac-1 and FcγR in these cells, and we considered the possibility that FcγR engagement may lead to inside-out activation of Mac-1. Reagents—Dulbecco's modified Eagle's medium and fetal calf serum (FCS) were from Wisent Inc. BSA (IgG-free, low endotoxin), sheep RBC, and rabbit anti-sheep RBC IgG were obtained from ICN Biomedicals, and anti-sheep RBC IgM was from Accurate Chemicals. cDNA encoding the αMI-domain fused to GST was kindly provided by Dr. E. F. Plow of the Cleveland Clinic Foundation (Cleveland, OH). 2All rights, title, and interest in the cDNA encoding the α I-domain fused to GST are owned by The Cleveland Clinic Foundation. The purification of the recombinant proteins was performed as described (20Ustinov V.A. Plow E.F. J. Biol. Chem. 2002; 277: 18769-18776Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Hamster anti-mouse CD18 and rat anti-mouse CD11b-producing hybridoma, 2E6 and M1/70, respectively, were purchased from the American Type Culture Collection (ATCC), and purified rat IgG2b and rat anti-mouse CD16/CD32 (FcγRIII/II) were from Pharmingen. Rat anti-mouse CD11a mAb (M17/5.2) and anti-talin Ab were kind gifts from Drs. H. Ostergaard (Edmonton, Canada) and A. Kupfer (Denver, CO), respectively. F(ab′)2 fragments of anti-CD18 or anti-CD11a were prepared from the IgG fraction purified from hybridoma supernatants by using immobilized pepsin, according to the manufacturer's instructions (Pierce). FITC-conjugated anti-CD11b Ab (M1/70) and IgG2b isotype control and anti-hamster IgG were from Serotec and Caltag, respectively. Hamster IgG F(ab′)2 and Cy5-, Cy3-, or tritc-conjugated secondary Ab were from Jackson ImmunoResearch Laboratories. PP1, GF-109203X, and calphostin C were from Biomol. BAPTA-AM and Alexa 488- or rhodamine-conjugated phalloidin were from Molecular Probes. U73122 and 1,2-dioctanoyl-sn-glycerol (DiC8) were from Calbiochem. All other reagents were from Sigma. Aggregated human IgG (agg-IgG) was prepared by heating 10 mg/ml human IgG to 63 °C for 20 min followed by spinning at 14,000 rpm for 10 min. The freshly prepared supernatant was used at 250 μg/ml final concentration. Cell Culture and Transfections—RAW264.7 macrophages were cultured in Dulbecco's modified Eagle's medium with 10% heat-inactivated FCS and transferred onto acid-washed and poly-l-lysine (1 μg/ml)-coated glass coverslips 1 day before the assays. For immobilized immune complex binding assays, RAW264.7 cells grown on glass coverslips were lifted by treating with PBS containing 2 mm EDTA for 15 min at 4 °C. Cells were washed with PBS and allowed to recover at room temperature for 5 h in Hepes-buffered RPMI 1640 (HPMI). To study Fc receptor mobility, cells on poly-l-lysine-coated coverslips were transiently transfected with the FcγRI-γ-γ-GFP cDNA (21Kim M.K. Pan X.Q. Huang Z.Y. Hunter S. Hwang P.H. Indik Z.K. Schreiber A.D. Clin. Immunol. 2001; 98: 125-132Crossref PubMed Scopus (24) Google Scholar), a gift from Dr. A. D. Schreiber (Philadelphia), using FuGENE 6 (Roche Applied Science), according to the manufacturer's instructions, and used within 24 h of transfection. Phagocytosis and Binding Determinations—Sheep RBC were opsonized with IgG as described (22Botelho R.J. Teruel M. Dierckman R. Anderson R. Wells A. York J.D. Meyer T. Grinstein S. J. Cell Biol. 2000; 151: 1353-1368Crossref PubMed Scopus (428) Google Scholar). C3bi opsonization was performed by first incubating RBC with sub-agglutinating concentrations of IgM (1:10) in PBS with 0.5 mm CaCl2 and MgCl2 for 1 h at room temperature. Excess IgM was then washed off, and RBC were incubated with C5-deficient serum (1:6) for 20 min at 37 °C with frequent mixing. C3bi-RBC were then washed and used immediately for binding assays. To induce IgG-mediated phagocytosis, RAW264.7 cells were exposed to IgG-opsonized RBC (∼5 RBC/macrophage) in HPMI with 1% heat-inactivated FCS at 37 °C for 30 min. Cells were subjected to hypoosmolar shock by adding H2O for 20 s followed by three washes with PBS prior to fixation with 4% paraformaldehyde overnight or with methanol at -20 °C for 10 min. For IgG-RBC binding assays, cells were exposed to IgG-opsonized RBC under the same conditions as for phagocytosis except that incubation was for 5 min at 4 °C followed by 15 min at room temperature. Cells were washed extensively with PBS prior to fixation with methanol. For C3bi-RBC binding assays, RAW264.7 cells were serum-starved for 2 h and then incubated with or without 100 nm PMA or 100 μm of DiC8 for 20 min prior to the addition of C3bi-RBC. Where indicated, the cells were pretreated with 10 μm PP1, 100 μm LY294002 (30 min), 10 μm GF-10923X (30 min), 10 μm BAPTA-AM (30 min), 10 μm U73122, or 500 nm calphostin C (15 min at 37 °C followed by 15 min under light) in serum-free medium prior to addition of C3bi-RBC. C3bi-RBC were incubated with the cells for 20 min before vigorous washing and fixing with methanol. Where specified, the cells were stimulated with agg-IgG. Blocking Experiments—Cells were washed and incubated for 10 min at room temperature with HPMI containing 5% FCS to block nonspecific binding sites. The cells were next treated for 15 min at room temperature with or without (control) 5 μg/ml anti-CD18 (2E6), anti-CD11a (M17/5.2), or non-immune hamster IgG (all in F(ab′)2 fragments) with 1% FCS. Next, IgG opsonized RBC were added to the incubation mixture, and binding or phagocytosis experiments were conducted in the presence of the blocking Ab, as described above. To detect CD18 staining, the secondary Ab (TRITC anti-hamster IgG) was preabsorbed with IgG-RBC to eliminate cross-reactivity with opsonized RBC, by incubation for 1 h. The accumulation of CD18 in the cup versus membrane under normal and blocking conditions was determined from confocal images by determining the pixel density in the cup and in inactive plasma membrane regions by using NIH/Scion Image software. For blocking experiments using the recombinant proteins GST-αMI domain or GST, IgG-RBC were preincubated for 5 min with or without 10 μm recombinant protein in HPMI containing 0.2% BSA, and this suspension was then added to cells adherent to poly-l-lysine-coated coverslips. Phagocytosis was quantified as described above. Immunostaining and Flow Cytometry—For internalization experiments cells were preincubated with 1 μg/ml F(ab′)2 fragments of anti-CD18 (2E6), of non-immune hamster IgG, or of FITC-conjugated anti-CD11b Ab (M1/70), as described above, prior to washing and incubation with or without agg-IgG for 30 min at 37 or 4 °C, as specified. For surface staining at 4 °C, 0.1% azide was added to the incubation buffers. Cells were washed in PBS, fixed, and incubated with secondary Ab to detect human IgG and CD18. For flow cytometric analysis cells were scraped from tissue culture flasks, washed, and incubated with agg-IgG in Dulbecco's modified Eagle's medium for 30 min at 37 °C (or, in the case of controls, at 4 °C in the presence of azide) and washed twice with ice-cold Hanks' buffered saline solution (HBSS) containing 2.5% FCS and 0.1% azide prior to blocking with 5% FCS for 10 min and staining at 4 °C for 30 min with FITC-labeled anti-human-IgG, 2E6/FITC anti-hamster, FITC anti-CD11b, or the corresponding control Ab. Live cells excluding propidium iodide were analyzed for surface staining of human IgG or integrin chains with FACSCalibur and CellQuest software (BD Biosciences). To determine the number of integrin molecules per cell, the phycoerythrin (PE) fluorescence quantitation kit (Quantibrite™ PE, BD Biosciences) was used according to the manufacturer's instructions with saturating amounts of PE-anti-CD18 and -anti-CD11b mAb (Caltag Laboratories). The amounts of PE/Ab were determined by generating a standard curve with unconjugated R-PE (Molecular Probes), and the number of Ab molecules per cell were calculated. To assess affinity changes of β2 integrins, cells were incubated with an activation epitope-specific mAb (mAb24, a gift from Dr. N. Hogg, London, UK). Cells were treated at 37 °C for 15 min using mAb24 or mouse IgG1 (isotype control) in HBSS with 10 mm Hepes and 1% FCS with or without agg-IgG. Parallel incubations were performed in Ca2+,Mg2+-free HBSS, containing 5 mm MgCl2, 1 mm EGTA, and 1% FCS. Alexa 488-conjugated F(ab′)2 fragments of anti-mouse IgG were used as secondary Ab. Fluorescence Recovery after Photobleaching (FRAP)—The mobility of integrins and Fc receptors was estimated using FRAP. Cells layered on poly-l-lysine were either transfected with FcγRI-γ-γ-GFP cDNA or exposed to 1 μg/ml FITC anti-CD11b Ab for 10 min at room temperature. The cells were washed, and the dorsal surface of flat lamellae was imaged immediately. Spots of ∼2 μm in diameter were photobleached using the full power of the 488 nm laser line of the Zeiss LSM 510 confocal microscope, resulting in a 70–90% reduction of the fluorescence intensity. A similar spot from the area that remained unbleached was selected to serve as a control. Sequential images were acquired after photobleaching with a decreased laser intensity to minimize further photobleaching for a period up to 120 s. The data were exported and analyzed in Microcal Origin 6.0 software. Starting values were set as 100% recovery, and photobleached values were normalized to the unbleached regions at all time points to correct for any bleaching incurred during measurement or for changes in focal plane. The percent recovery (mobile fraction) of the measured population of labeled CD11b Ab or FcγR-GFP was determined as the ratio of the final fluorescence to the pre-bleach fluorescence. The half-time to recovery (t ½) was also quantified. Images of the cells were taken before and after photobleaching and compared to verify that no gross morphological changes or drifting had occurred in the regions of interest. Immobilized Immune Complex Binding Assays—BSA and BSA/anti-BSA (immune complex)-coated coverslips were prepared as described (14Tang T. Rosenkranz A. Assmann K.J. Goodman M.J. Gutierrez-Ramos J.C. Carroll M.C. Cotran R.S. Mayadas T.N. J. Exp. Med. 1997; 186: 1853-1863Crossref PubMed Scopus (175) Google Scholar). Briefly, acid-washed coverslips were coated with poly-l-lysine (100 μg/ml) and treated with 2.5% glutaraldehyde for 15 min. Coverslips were washed and coated with BSA (1 mg/ml) for 30 min and then blocked with 0.1 m glycine for 2 h. To create immobilized BSA/anti-BSA immune complexes, BSA-coated coverslips were incubated with 40 μgof rabbit anti-BSA IgG in PBS for 1 h. RAW264.7 cells suspended in HPMI were plated onto BSA or BSA/anti-BSA-coated coverslips and incubated for 8 min at 37 °C. Cells were fixed with 4% paraformaldehyde and stained with phalloidin and/or primary Ab to integrins or talin, followed by secondary Ab. Immunofluorescence and Confocal Microscopy—Following treatment, RAW264.7 cells were washed with PBS and fixed with 4% paraformaldehyde in PBS overnight at 4 °C or for 20 min at room temperature. Immunostaining was performed by permeabilization with 0.1% Triton X-100 in PBS containing 100 mm glycine for 20 min before blocking for 1 h with 5% FCS in PBS. Staining with anti-integrins Ab and phalloidin was for 1 h at room temperature in PBS containing 1% FCS. Following washing, samples were incubated for 1 h at room temperature with appropriate secondary antibodies. Samples were analyzed by differential interference contrast and fluorescence confocal microscopy using a Zeiss LSM 510 microscope with a 100× oil immersion objective. FITC, Cy5, and Cy3 channels were examined using the conventional laser excitation lines and filter sets. Statistical Analysis—All data were expressed as mean values ± S.E. Student's two-tailed t tests were performed to assess the significance of differences using InStat software (GraphPad, San Diego). CD18 Is Required for Optimal IgG-mediated Phagocytosis of RBC—We initially confirmed the expression and quantified the density of CD18 and CD11b on the surface of RAW264.7 cells by flow cytometry (see “Experimental Procedures”). An average of ∼3 × 105 CD18 molecules/cell of CD18 and 1.6 × 105 CD11b molecules/cell was estimated, which is similar to values reported for monocyte-derived macrophages (23Ross G.D. Reed W. Dalzell J.G. Becker S.E. Hogg N. J. Leukocyte Biol. 1992; 51: 109-117Crossref PubMed Scopus (88) Google Scholar). These data indicate that in RAW264.7 cells nearly half of the CD18 chains were associated with CD11b chains to form the Mac-1 integrin receptor. To assess whether a functional interaction exists between β2 integrins and transmembrane FcγR, we examined the effect of blocking monoclonal antibodies (mAb) against CD18 (24Metlay J.P. Witmer-Pack M.D. Agger R. Crowley M.T. Lawless D. Steinman R.M. J. Exp. Med. 1990; 171: 1753-1771Crossref PubMed Scopus (504) Google Scholar) on the phagocytosis of IgG-RBC by RAW264.7 cells. Fig. 1 shows that pretreatment with F(ab′)2 fragments of the blocking mAb 2E6 impaired phagocytosis significantly. The phagocytic index dropped from 287 ± 31 in the controls (n = 9) to 132 ± 4 in antibody-treated cells (n = 4; p ≤ 0.001). This effect was specific, because a comparable concentration of non-immune F(ab′)2 fragments produced no significant inhibition of phagocytosis (Fig. 1). Inhibition of phagocytosis by anti-CD18 was not due to interference with the binding of the opsonins to the FcγR, because measurements of the number of IgG-RBC associated with the phagocytes revealed no difference between control and antibody-treated cells (Fig. 1, solid bars). Although we had no available F(ab′)2 fragments of blocking mAb against the α chain of Mac-1, we speculated that this receptor may have been responsible, at least in part, for the CD18-dependent component of phagocytosis, based on its abundance and on the data of Graham et al. (11Graham I.L. Gresham H.D. Brown E.J. J. Immunol. 1989; 142: 2352-2358PubMed Google Scholar), who found that Mac-1 is the major β2 integrin contributing to Fc-mediated phagocytosis in primary human monocytes and neutrophils. A unique domain of about 200 amino acids of the extracellular moiety of CD11b, known as the I-domain, has been implicated in the binding of Mac-1 to several ligands (20Ustinov V.A. Plow E.F. J. Biol. Chem. 2002; 277: 18769-18776Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). To verify whether Mac-1 is required for optimal phagocytosis of IgGRBC, we tested the effect of a recombinant GST fusion protein of the I-domain of CD11b (GST-αMI-domain) on phagocytosis. Parallel control experiments were performed by using the GST protein alone. Fig. 1 shows that in the presence of the GST-αMI-domain fusion protein, the phagocytic index was reduced by 55% (to 128 ± 27) as compared with untreated controls, whereas preincubation in the presence of GST alone had no effect on phagocytosis. These results confirm that CD11b/CD18 is implicated in FcγR-induced phagocytosis, at least in part through the I-domain of CD11b. In addition to CD11b/CD18, we found that RAW264.7 cells express also CD11a/CD18 (LFA-1). We therefore examined the possible role of this β2 integrin during IgG-induced phagocytosis. F(ab′)2 fragments of blocking mAb against the α chain of LFA-1 did not alter phagocytosis of IgG-RBC (Fig. 1). These results imply that not all β2 integrin family members participate in IgG-mediated phagocytosis. We therefore focused on CD18 and CD11b during the rest of this study. Accumulation of CD18 and CD11b at the Phagocytic Cup—To investigate the relationship between FcγR and Mac-1, we first studied the distribution of CD18 and CD11b during phagocytosis of IgG-RBC. Fig. 2, A–D, shows that both CD18 and CD11b accumulate in phagocytic cups, identified by the presence of adherent IgG-RBC and, more importantly, by the enrichment in F-actin. Fig. 2E displays a typical case of CD18 accumulation in the cup and Fig. 2F illustrates the line-scanning method used to quantify the enrichment in CD18 and CD11b at the cup, in comparison to its density in the contralateral membrane. In 45 determinations, the density of CD18 at the cup consistently exceeded the levels found in other regions of the plasmalemma (62 ± 2% over the overall plasmalemma level). These results are consistent with the phagosomal localization of Mac-1 reported previously (25Morrissette N.S. Gold E.S. Guo J. Hamerman J.A. Ozinsky A. Bedian V. Aderem A.A. J. Cell Sci. 1999; 112: 4705-4713Crossref PubMed Google Scholar) in lipopolysaccharideprimed murine peritoneal macrophages during phagocytosis of IgG-zymosan. FcγR and CD11b Chains Do Not Interact Constitutively—Because FcγR are known to accumulate at the phagocytic cup, the concomitant accumulation of Mac-1 suggests that the two types of receptors may interact physically. To define whether the two receptor types are constitutively associated, we analyzed whether internalization of FcγR induced by soluble IgG complexes drives also the internalization of Mac-1. When added at 4 °C, agg-IgG binds to the surface of RAW264.7 cells, which can be readily verified by immunofluorescence (Fig. 3, A and G) or flow cytometry (cf. Fig. 3G , inset) by using an anti-human secondary antibody. At 37 °C, the receptor clustering triggered by agg-IgG induces endocytosis of the FcγR, which can be visualized by immunofluorescence (Fig. 3, D and J) and quantified by flow cytometry using antibodies to human IgG (cf. Fig. 3J , inset) or FcγR (not shown; see Ref. 26Booth J.W. Kim M.K. Jankowski A. Schreiber A.D. Grinstein S. EMBO J. 2002; 21: 251-258Crossref PubMed Scopus (76) Google Scholar for example). Note that whereas a sizable fraction of FcγR becomes internalized under these conditions, the density of Mac-1 at the cell surface remained unaltered. This was apparent by microscopy (cf. Fig. 3, B and H with E and K, respectively) but more accurately established by flow cytometry (cf. Fig. 3, C and I with F and L, respectively). Similar results were obtained when internalization was induced by cross-linking FcγR by using sequentially mouse IgG and anti-mouse IgG (not shown). These data strongly suggest that Mac-1 and FcγR are not constitutively associated. Nevertheless, it is conceivable that constitutively associated receptors may undergo dissociation upon endocytosis and that Mac-1 recycles rapidly to the surface. We therefore used an alternative approach to evaluate the interaction of the receptors, which did not require receptor cross-linking. This was accomplished by comparing the lateral mobility of Mac-1 and of FcγR by using fluorescence recovery after photobleaching (FRAP). Receptors on the dorsal membrane, near the edge of the cells, were chosen for these measurements to minimize the curvature and thereby ensure that the area under study was within the confocal plane. As depicted in Fig. 4, A–F, and quantified" @default.
• W2033073750 created "2016-06-24" @default.
• W2033073750 creator A5038691946 @default.
• W2033073750 creator A5063705444 @default.
• W2033073750 creator A5090229237 @default.
• W2033073750 date "2003-11-01" @default.
• W2033073750 modified "2023-10-12" @default.
• W2033073750 title "Fcγ-receptors Induce Mac-1 (CD11b/CD18) Mobilization and Accumulation in the Phagocytic Cup for Optimal Phagocytosis" @default.
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