β1 and β2 Integrins Mediate Adhesion during Macrophage Fusion and Multinucleated Foreign Body Giant Cell Formation
2002; Elsevier BV; Volume: 160; Issue: 2 Linguagem: Inglês
10.1016/s0002-9440(10)64882-1
ISSN1525-2191
AutoresAmy K. McNally, James M. Anderson,
Tópico(s)T-cell and B-cell Immunology
ResumoAn in vitro system of interleukin (IL)-4-induced human monocyte-derived macrophage fusion was used to investigate the cell/substrate adhesive mechanisms that support multinucleated foreign body giant cell (FBGC) formation. Monocytes were cultured for 3 days and IL-4 was added to induce macrophage fusion and FBGC formation by day 7. Functionally defined anti-integrin antibodies demonstrated that initial monocyte adhesion is mediated by β2 integrins, whereas during the induction of macrophage fusion by IL-4, an additional dependence on β1 integrins is acquired. The combination of anti-β1 plus anti-β2 was most effective, reducing macrophage/FBGC adhesion to 10% of controls. Consistent with integrin-mediated signaling, the tyrosine kinase inhibitor genistein and the phosphatidylinositol-3-kinase inhibitors wortmannin and LY294002 also attenuated macrophage/FBGC adhesion. Confocal microscopic analysis revealed that β2 integrins are present on monocytes after initial adhesion and are strongly expressed on fusing macrophages, particularly in peripheral cell areas, and on FBGCs. In contrast, β1 integrins are not detected on monocytes but begin to appear during macrophage development and are strongly expressed on fusing macrophages and FBGCs. For the first time, these results demonstrate the IL-4-induced acquisition of cooperation between β1 and β2 integrins in the cell/substrate adhesive interactions that are required for multinucleated FBGC formation. An in vitro system of interleukin (IL)-4-induced human monocyte-derived macrophage fusion was used to investigate the cell/substrate adhesive mechanisms that support multinucleated foreign body giant cell (FBGC) formation. Monocytes were cultured for 3 days and IL-4 was added to induce macrophage fusion and FBGC formation by day 7. Functionally defined anti-integrin antibodies demonstrated that initial monocyte adhesion is mediated by β2 integrins, whereas during the induction of macrophage fusion by IL-4, an additional dependence on β1 integrins is acquired. The combination of anti-β1 plus anti-β2 was most effective, reducing macrophage/FBGC adhesion to 10% of controls. Consistent with integrin-mediated signaling, the tyrosine kinase inhibitor genistein and the phosphatidylinositol-3-kinase inhibitors wortmannin and LY294002 also attenuated macrophage/FBGC adhesion. Confocal microscopic analysis revealed that β2 integrins are present on monocytes after initial adhesion and are strongly expressed on fusing macrophages, particularly in peripheral cell areas, and on FBGCs. In contrast, β1 integrins are not detected on monocytes but begin to appear during macrophage development and are strongly expressed on fusing macrophages and FBGCs. For the first time, these results demonstrate the IL-4-induced acquisition of cooperation between β1 and β2 integrins in the cell/substrate adhesive interactions that are required for multinucleated FBGC formation. Multinucleated giant cells are formed from macrophage fusion1Murch AR Grounds MD Marshall CA Direct evidence that inflammatory multinucleate giant cells form by fusion.J Pathol. 1980; 137: 177-180Crossref Scopus (83) Google Scholar and are recognized as hallmark histiological features of chronic inflammatory responses to persistent microbial infections or nonphagocytosable foreign objects.2Chambers TJ Spector WG Inflammatory giant cells.Immunobiology. 1982; 161: 2283-2289Crossref Scopus (80) Google Scholar As such, the foreign body giant cell (FBGC) is a prominent cell type on retrieved biomedical materials and has been observed on a variety of prosthetic materials after extended periods of implantation, eg, 15 years.3Anderson JM Inflammatory response to implants.Am Soc Artif Intern Organs. 1988; 11: 101-107Crossref Scopus (481) Google Scholar A role for these giant cells in biomaterial degradation involving oxidative mechanisms has been indicated by the discovery of material surface cracking on retrieved polyurethane directly, and only underneath adherent FBGCs4Zhao Q Topham NS Anderson JM Hiltner A Lodoen G Payet R Foreign body giant cells and polyurethane biostability: in vivo correlation of cell adhesion and surface cracking.J Biomed Mater Res. 1991; 25: 177-183Crossref PubMed Scopus (293) Google Scholar and on regions of failed pacemaker leads with high concentrations of inflammatory cells.5Wiggins MJ Wilkoff B Anderson JM Hiltner A Biodegradation of polyether polyurethane inner insulation in bipolar pacemaker leads.J Biomed Mater Res. 2001; 58: 302-307Crossref PubMed Scopus (46) Google Scholar Biomaterial-associated FBGC formation requires the extravasation of blood monocytes and their subsequent adhesion to and macrophage development and fusion on implanted material surfaces.6Anderson JM Multinucleated giant cells.Curr Opin Hematol. 2000; 7: 40-47Crossref PubMed Scopus (287) Google Scholar Our previous research implicated β2 integrins (also known as the CD18 family of leukocyte-specific integrins) in initial monocyte adhesion to four different chemically modified polystyrene culture surfaces.7McNally AK Anderson JM Complement C3 participation in monocyte adhesion to different surfaces.Proc Natl Acad Sci USA. 1994; 91: 10119-10123Crossref PubMed Scopus (163) Google Scholar However, the cell/substrate adhesive interactions that promote macrophage development and support optimal FBGC formation have not been elucidated. Toward this end, and ultimately toward a fundamental mechanistic understanding of the macrophage fusion process, we developed an in vitro system of human monocyte-derived macrophage fusion and FBGC formation using interleukin (IL)-4 or IL-13 as the fusion-inducing cytokine.8McNally AK Anderson JM Interleukin-4 induces foreign body giant cells from human monocytes/macrophages. Differential lymphokine regulation of macrophage fusion leads to morphological variants of multinucleated giant cells.Am J Pathol. 1995; 147: 1487-1499PubMed Google Scholar, 9DeFife KM Jenney CR McNally AK Colton E Anderson JM Interelukin-13 induces monocyte/macrophage fusion and macrophage mannose receptor expression.J Immunol. 1997; 158: 3385-3390PubMed Google Scholar This culture system requires the presence of autologous serum and a sufficient density of adherent macrophages; it does not generate significant macrophage fusion or FBGC formation in the absence of IL-4 or IL-13. The importance of IL-4 in FBGC formation on biomaterials in vivo was confirmed by antibody inhibition,10Kao WJ McNally AK Hiltner A Anderson JM Role for interleukin-4 in foreign body giant cell formation on a poly(etherurethane urea) in vivo.J Biomed Mater Res. 1995; 29: 1267-1275Crossref PubMed Scopus (131) Google Scholar and a role for IL-4-induced mannose receptor activity11Stein M Keshav S Harris N Gordon S Interleukin-4 potently enhances murine macrophage mannose receptor activity: a marker of alternative immunologic macrophage activation.J Exp Med. 1992; 176: 287-292Crossref PubMed Scopus (1430) Google Scholar in the molecular mechanism of macrophage fusion was proposed.12McNally AK Anderson JM Interleukin-4-induced macrophage fusion is prevented by inhibitors of mannose receptor activity.Am J Pathol. 1996; 149: 975-985PubMed Google Scholar Subsequently, we discovered that IL-4-induced macrophage fusion occurs considerably more readily and efficiently on culture surfaces to which the arginine-glycine-aspartate (RGD) integrin recognition sequence has been covalently attached than it does on standard cell culture polystyrene.13Anderson JM DeFife K McNally A Collier T Jenney C Monocyte, macrophage and foreign body giant cell interactions with molecularly engineered surfaces.J Mater Sci Mater Med. 1999; 10: 579-588Crossref PubMed Google Scholar Immobilized RGD does not increase degrees of initial monocyte adhesion, and this more optimal macrophage fusion also requires the presence of as yet unidentified adsorbed serum components. Therefore, the immobilized RGD peptide is not, by itself, a sufficient substrate for FBGC formation. Nevertheless, this finding indicates an important role for integrin-mediated adhesion in IL-4-induced macrophage development and/or fusion. Integrins comprise a large group of heterodimeric transmembrane molecules that mediate both cell-extracellular matrix and cell-cell interactions.14Gonzalez-Amaro R Sanchez-Madrid F Cell adhesion molecules: selectins and integrins.Crit Rev Immunol. 1999; 19: 389-429PubMed Google Scholar These adhesion molecules are now well known as important mediators of signal transduction between the extracellular and intracellular environments.15Schwartz MA Shaller MD Ginsberg MH Integrins: emerging paradigms of signal transduction.Annu Rev Cell Dev Biol. 1995; 11: 549-559Crossref PubMed Scopus (1474) Google Scholar, 16Giancotti FG Ruoslahti E Integrin signaling.Science. 1999; 285: 1028-1032Crossref PubMed Scopus (3859) Google Scholar Several of the known integrins recognize the RGD sequence in their ligands,17Ruoslahti E RGD and other recognition sequences for integrins.Annu Rev Cell Dev Biol. 1996; 12: 697-715Crossref PubMed Scopus (2614) Google Scholar with an interaction between fibronectin and α5/β1 as a classic example.18Pytela R Pierschbacher MD Ruoslahti E Identification and isolation of a 140 kilodalton cell surface glycoprotein with properties of a fibronectin receptor.Cell. 1985; 40: 191-198Abstract Full Text PDF PubMed Scopus (690) Google Scholar Monocytes/macrophages are believed to express three integrins in the β1 family, namely, the fibronectin receptors α4/β1 and α5/β1 and the laminin receptor α6/β1. In addition, there are four members of the leukocyte-specific β2 integrin family with αL, αM, αX, or αD in association with β2. αL/β2 and αD/β2 primarily appear to mediate intercellular adhesion. αX/β2 and, particularly, αM/β2 are capable of interactions with a diversity of ligands and mediate cell-particle or cell-substrate interactions.19Berton G Lowell CA Integrin signaling in neutrophils and macrophages.Cell Signal. 1999; 11: 621-635Crossref PubMed Scopus (180) Google Scholar These ligands include fragments of complement C3, fibrinogen, Factor X, and high-molecular weight kininogen, which might be predicted to adsorb from blood onto material surfaces during surgical implantation of a biomedical device.20Anderson JM Ziats NP Bonfield TL McNally AK Topham NS Human blood protein and cell interactions with cardiovascular materials.in: Akutsu T Koyanagi H Artificial Heart. vol 3. Springer-Verlag, New York1991: 45-55Google Scholar αV/β3, which is known primarily as a vitronectin receptor, can also interact with certain other RGD-containing extracellular matrix proteins.19Berton G Lowell CA Integrin signaling in neutrophils and macrophages.Cell Signal. 1999; 11: 621-635Crossref PubMed Scopus (180) Google Scholar Both the β1 and β2 integrin families have been implicated in monocyte/macrophage adhesion to endothelium and extravasation to sites of inflammation.21Luscinskas FW Kansas GS Ding H Pizcueta P Schleiffenbaum BE Tedder TF Gimbrone MA Monocyte rolling, arrest and spreading on IL-4-activated vascular endothelium under flow is mediated via sequential action of L-selectin, beta 1-integrins, and beta 2-integrins.J Cell Biol. 1994; 125: 1417-1427Crossref PubMed Scopus (353) Google Scholar αM/β2 is an important phagocytic receptor that may also mediate frustrated phagocytosis of a nonphagocytosable substance such as a biomaterial.22Henson PM Henson JE Fittschen C Kimani G Bratton DL Riches DWH Phagocytic cells: degranulation and secretion.in: Gallin JI Goldstein IM Snyderman R Inflammation: Basic Principles and Clinical Correlates. Raven, New York1988: 363-390Google Scholar In early work, IL-4 was shown to increase expression of β2 integrins on monocytes23Te Velde AA Klomp JPG Yard BA de Vries JE Figdor CG Modulation of phenotypic and functional properties of human peripheral blood monocytes by IL-4.J Immunol. 1988; 140: 1548-1554PubMed Google Scholar and, more recently, to increase β1 expression on and tyrosine phosphorylation in fibroblasts.24Doucet C Brouty-Boye D Pottin-Clemenceau C Jasmin C Canonica GW Azzarone B IL-4 and IL-13 specifically increase adhesion molecule and inflammatory cytokine expression in human lung fibroblasts.Int Immunol. 1988; 10: 1421-1433Crossref Scopus (174) Google Scholar In neutrophils, the crosslinking of β2 integrins results in tyrosine kinase-dependent increases in β1 integrin expression.25Werr J Eriksson AA Hedqvist P Lindbom L Engagement of beta2 integrins induces surface expression of beta1 integrin receptors in human neutrophils.J Leukoc Biol. 2000; 68: 553-560PubMed Google Scholar Likewise, it has been proposed that integrin ligation and clustering in phagocytic cells activates cytoplasmic tyrosine kinases, that in turn leads to the activation of downstream signaling pathways involving phosphatidylinositol-3-kinase, phospholipases C and D, PKC, and others. These collective and highly regulated events control cytoskeletal rearrangements, focal contact formation, cell mobility, cell survival, and the synthesis of inflammatory mediators by phagocytic cells.19Berton G Lowell CA Integrin signaling in neutrophils and macrophages.Cell Signal. 1999; 11: 621-635Crossref PubMed Scopus (180) Google Scholar The present study was designed to investigate the nature of cell/substrate interactions that support optimal IL-4-induced macrophage fusion and FBGC formation. Specifically, we questioned integrin involvement in these adhesive interactions with an experimental approach that included anti-integrin antibody and pharmacological inhibition of adhesion, comparison of initial monocyte adhesion to macrophage/FBGC adhesion, and evaluation of integrin expression in monocytes, fusing macrophages, and FBGCs by fluorescence confocal scanning laser microscopy. RGD-modified (Pronectin F-coated; Smartplastic) 96-well culture plates were from ICN, Irvine, CA. Lab Tek II 8-well chamber slides for confocal microscopy were from Nalge Nunc. Macrophage-serum-free medium (SFM) was from Life Technologies, Inc., Grand Island, NY. Human recombinant IL-4 (R & D Systems, Minneapolis, MN) was reconstituted in 0.5% bovine serum albumin (low endotoxin; Sigma Chemical Co., St. Louis, MO) according to the manufacturer's instructions and stored in aliquots at −80°C. Anti-integrin antibodies (Table 1) were acquired as preservative-free or were dialyzed against phosphate-buffered saline (PBS) to remove preservative before functional studies. Additional reagents were obtained as follows: purified plasma fibronectin and its proteolytic fragments, Chemicon, Temecula, CA; nonspecific purified control IgGs, Chemicon; RNase A, Life Technologies, Inc.; Cy-5-conjugated goat anti-mouse IgG, Jackson Immuno Research, West Grove, PA; rhodamine-phalloidin and YO-YO-1, Molecular Probes, Eugene, OR; cell signaling reagents, Biomol (Plymouth Meeting, PA); Gel/Mount, Biomeda (Foster City, CA); all other reagents, Sigma.Table 1Anti-Integrin AntibodiesCloneSpeciesIsotypeSourceAnti-function-blocking β1JB1AMouseIgG1Chemicon6S6MouseUnidentifiedChemicon β2YFC118.3RatIgG2bChemiconMHM23MouseIgG1DAKO β3B3AMouseIgG1Chemicon25E11MouseIgG2aChemicon αM60.1MouseIgG1P. Beatty, University of UtahAnti-detecting β14B7RMouseIgG1Santa Cruz Biotechnology β2P4H9-A11MouseIgG3Chemicon β3BB10MouseIgG1Chemicon Open table in a new tab Human monocytes were isolated by a nonadherent method as previously described8McNally AK Anderson JM Interleukin-4 induces foreign body giant cells from human monocytes/macrophages. Differential lymphokine regulation of macrophage fusion leads to morphological variants of multinucleated giant cells.Am J Pathol. 1995; 147: 1487-1499PubMed Google Scholar and added to polypropylene culture tubes in an ice bath at a concentration of 2 × 105 cells per tube in 0.1 ml of macrophage-SFM supplemented with 20% autologous serum. Various anti-integrin antibodies or nonspecific isotype-matched control IgGs, each at a concentration of 50 μg/ml, were added and the incubation was continued on ice with gentle shaking for 1 hour to allow antibodies to interact with monocytes. The cells were then transferred to 96-well culture plates and incubated at 37°C for 1 hour. Nonadherent cells were removed with two washes of 0.2 ml each of prewarmed (37°C) PBS containing calcium and magnesium ions (PBS++). Adherent cells were fixed in methanol for 1 minute, air-dried, and stained with May-Grunwald/Giemsa. For each sample, adhesion was evaluated by averaging the number of nuclei in two low-power fields (∼200 cells) and expressing the result as a percentage of untreated control monocytes. Results represent the mean ± SEM of data from three different monocyte donors. Macrophage fusion was induced with IL-4 using a modification of our previously described culture system8McNally AK Anderson JM Interleukin-4 induces foreign body giant cells from human monocytes/macrophages. Differential lymphokine regulation of macrophage fusion leads to morphological variants of multinucleated giant cells.Am J Pathol. 1995; 147: 1487-1499PubMed Google Scholar as described below. This protocol generates a mixture of fusing macrophages and FBGCs within 7 days and promotes relatively high fusion rates, eg, 67 ± 3% of nuclei within multinucleated cells of >2 nuclei per cell, n = 5 monocyte donors. Monocytes were added to 96-well RGD-modified culture plates at 2 × 105 cells per well in 0.1 ml of macrophage-SFM supplemented with 20% autologous serum. After 1.5 hours at 37°C, nonadherent cells and unadsorbed serum proteins were removed by aspirating the medium and washing with 0.2 ml of prewarmed PBS++. The remaining adherent monocytes were recovered with 0.2 ml of prewarmed, unsupplemented macrophage-SFM and incubated in a humidified atmosphere of 5% CO2 and 95% air at 37°C. On day 3, the medium was replaced with 0.2 ml of prewarmed macrophage-SFM. At this time, increasing concentrations of inhibitors, 50 μg/ml of functionally defined anti-integrin monoclonal antibodies (mAbs) (Table 1), or the same concentrations and combinations of control IgGs as detailed in the figure legends were added. These were immediately followed by the addition of 15 ng/ml of IL-4 to induce macrophage fusion. The cultures were then incubated as above until day 7, when they were washed twice with prewarmed PBS++ to remove nonadherent cells and fixed with methanol. Macrophage/FBGC adhesion was determined as above on May-Grunwald/Giemsa-stained cells. To generate samples for confocal microscopy, monocytes were plated onto eight-well glass chamber slides at 5 × 105 cells per well in 0.25 ml of macrophage-SFM supplemented with 20% autologous serum. After 1.5 hours, nonadherent cells were removed by washing as above with 0.5 ml of prewarmed PBS++. Adherent cells were recovered with 0.5 ml per well of macrophage-SFM and incubated as above. On day 3, macrophage fusion was induced with IL-4 as above except that 10% heat-treated (1 hour × 56°C) autologous serum was used to supplement macrophage-SFM. On day 7, the cultures were washed with prewarmed PBS++ and fixed and permeabilized for 2 minutes with acetone at −20°C. Some cultures were also fixed after 1.5 hours (day 0) and on day 3 for comparison with day 7 cultures. Samples were stored at 4°C before immunofluorescent staining for integrins (day 0 and day 3 samples) or integrins, F-actin, and nuclei (day 7 samples) as outlined below. The diluent and washing buffer for all procedures was PBS++. Day 7 samples were first treated with RNase A (1 μg/ml) for 1.5 hours at 37°C and washed three times for 5 minutes. Nonspecific sites were blocked with 10% goat serum for 1 hour at 37°C. Primary detecting antibodies (Table 1) or isotype-matched control IgGs were diluted to 20 μg/ml in 5% goat serum and applied for 1 hour at 37°C. After four 5-minute washes at room temperature, Cy-5-conjugated goat anti-mouse IgG diluted 1:100 (7.5 μg/ml) (day 0 and day 3 samples) or a mixture of Cy-5-conjugated goat anti-mouse IgG, rhodamine-phalloidin diluted 1:100 (2 U/ml), and YO-YO-1 diluted 1:10,000 (0.1 μmol/L) was added for 1 hour at room temperature. The samples were then washed three times for 10 minutes each and mounted under glass coverslips using Gel/Mount. Samples were viewed by confocal scanning laser microscopy (MRC-600; Bio-Rad, Richmond, CA) with settings adjusted to blacken any residual background fluorescence from the corresponding nonspecific control antibodies. A classic feature of integrin-mediated adhesion is a dependence on divalent cations.26Ruoslahti E Pierschbacher MD New perspectives in cell adhesion: RGD and integrins.Science. 1987; 238: 491-497Crossref PubMed Scopus (3908) Google Scholar Therefore, we first asked whether the divalent cation chelator ethylenediaminetetraacetic acid (EDTA) or the more calcium-specific chelator ethyleneglycoltetraacetic acid (EGTA) would inhibit adhesion in our culture system. As is depicted in Figure 1, both reagents completely or nearly completely inhibited both initial monocyte and macrophage/FBGC adhesion. If EDTA or EGTA was removed, adhesion was restored to control levels (not shown). Further, to address the participation of selected nonintegrin receptors, we tested reagents that have been demonstrated to inhibit various other types of monocyte/macrophage interactions for their abilities to inhibit initial monocyte adhesion or macrophage/FBGC adhesion (Figure 1). At least three different concentrations of each reagent at or higher than their reported effective concentrations were evaluated; data that are shown represent the highest concentration of each reagent tested. Heparin, which interferes with interactions mediated by the low-density lipoprotein receptor-related protein/α2-macroglobulin receptor,27Chappell DA Fry GL Waknitz MA Muhonen LE Pladet MW Iverius PH Strickland DK Lipoprotein lipase induces catabolism of normal triglyceride-rich lipoproteins via the low density lipoprotein receptor-related protein/a2-macroglobulin receptor in vitro. A process facilitated by cell-surface proteoglycans.J Biol Chem. 1993; 268: 14168-14175Abstract Full Text PDF PubMed Google Scholar did not reduce either initial monocyte or macrophage/FBGC adhesion. Dextran sulfate or bacterial lipopolysaccharide, which are polyanionic anions that bind to macrophage scavenger receptors,28Krieger M Acton S Ashkenas J Pearson A Penman M Resnick D Molecular flypaper, host defense.J Biol Chem. 1993; 268: 4569-4572Abstract Full Text PDF PubMed Google Scholar also did not decrease adhesion. Chondroitin sulfates are ligands for CD44, an integral membrane protein that mediates cell-cell and cell-extracellular matrix interactions29Naor D Sionov VR Ish-Shalom D CD44: structure, function, and association with the malignant process.Adv Cancer Res. 1997; 71: 241-319Crossref PubMed Google Scholar and that also has been reported to participate in macrophage fusion.30Sterling H Saginario C Vignery A CD44 occupancy prevents macrophage multinucleation.J Cell Biol. 1998; 143: 837-847Crossref PubMed Scopus (94) Google Scholar However, neither chondroitin sulfate A or B reduced monocyte or macrophage/FBGC adhesion relative to control cells. We then asked whether soluble whole fibronectin, a 120-kd fibronectin fragment containing the RGD integrin recognition sequence, or a 40-kd fibronectin fragment without this sequence would compete with the receptors mediating initial monocyte or macrophage/FBGC adhesion. Results from these experiments are shown in Figure 2, in which it can be seen that neither of these proteins or fragments affected initial monocyte adhesion at three different concentrations. However, the 120-kd RGD-containing fibronectin fragment reduced adhesion in cultures of fusing macrophages/FBGCs in a concentration-dependent manner by ∼60% at 0.25 mg/ml. The 40-kd fragment was slightly inhibitory at this concentration, whereas whole plasma fibronectin or albumin again had no apparent effect. These results suggest that initial monocyte adhesion is mediated by interactions other than with substrate-adsorbed fibronectin or the RGD sequence. However, during macrophage development and the IL-4-induced onset of macrophage fusion, it seems that cell/substrate interactions are altered and may include adhesion to relevant regions of adsorbed fibronectin, other RGD-containing proteins, and/or immobilized RGD. To address the identities of integrins that support monocyte or macrophage/FBGC adhesion, we used functionally defined anti-integrin antibodies (Table 1). As is depicted in Figure 3A, neither anti-β1 (clone JB1a), anti-β3 (clone B3A), or these two antibodies in combination inhibited initial monocyte adhesion to culture surfaces. However, anti-β2 (clone YFC118.3) consistently reduced monocyte adhesion alone or in combination with anti-β1 and/or anti-β3 to ∼30% of control adhesion. In addition, the anti-β2 antibody MHM23 completely inhibited monocyte adhesion, whereas another anti-β1, clone 6S6, had no apparent effect (not shown). Isotype-matched control IgGs were not inhibitory. In Figure 3B, it can be seen that upon the IL-4 induction of macrophage fusion and FBGC formation, additional adhesive interactions operate to mediate the attachment of these cells to culture surfaces. At the day 7 time point, anti-β1 alone reduced macrophage/FBGC adhesion to 40% of untreated controls. Again, anti-β2 was a strong inhibitor and reduced adhesion to ∼30% of untreated controls. The combination of anti-β1 and anti-β2 was most effective and essentially abrogated adhesion by its reduction to 10% of untreated controls. Interestingly, a mAb to αM, which did not affect the initial adherence of monocytes, also strongly inhibited macrophage/FBGC adhesion. Neither anti-β3, clone B3A, another anti-β3, clone 25E11 (not shown), or isotype-matched control IgGs were inhibitory. To ask whether anti-β1 or anti-β2 affected macrophage fusion as well as macrophage/FBGC adhesion, we examined the remaining adherent cells after antibody treatments. After inhibition of adhesion by anti-β1, the 40% remaining cells were clearly observed with multiple pseudopodial extensions and to be fusion competent. These cells had undergone degrees of fusion (67 ± 4%, n = 3 experiments) that were comparable to untreated control cultures (67 ± 3%, n = 5 experiments). In the same experiments after anti-β2 treatment, the majority of the 30% remaining adherent cells were mononuclear macrophages with few pseudopods (22 ± 10% fusion, n = 3 experiments). Integrin-mediated phagocytic cell signaling pathways have been demonstrated to involve tyrosine and phosphatidylinositol-3-kinases and phosphatidylinositide turnover via PLC activation.19Berton G Lowell CA Integrin signaling in neutrophils and macrophages.Cell Signal. 1999; 11: 621-635Crossref PubMed Scopus (180) Google Scholar Therefore, we asked whether macrophage/FBGC adhesion would be disrupted by the inclusion of various inhibitors of these pathways. Genistein, a widely used inhibitor of tyrosine phosphorylation, reduced adhesion in a concentration-dependent manner (Figure 4A). In addition, increasing doses of the phosphatidylinositol-3-kinase inhibitors LY294002 (Figure 4B) or wortmannin (Figure 4C) completely abrogated macrophage/FBGC adhesion, whereas the PKG and PKA inhibitor H-8, at concentrations of up to 10 μmol/L, did not (not shown). These collective results extend our antibody inhibition experiments and are consistent with integrin-mediated signaling during the induction of macrophage fusion by IL-4. To evaluate the expression of integrins during monocyte-to-macrophage development and in fusing macrophages/FBGCs, we used fluorescence confocal scanning laser microscopy and anti-integrin detecting antibodies (Table 1) applied to permeabilized cells at various time points in the 7-day culture period. Rhodamine phalloidin and the nucleic acid stain YO-YO-1 were also used to visualize F-actin and nuclei, respectively, in fusing macrophages/FBGCs. Representative results are presented in Figure 5 for monocytes after initial adhesion (1.5 hours) and after a period of monocyte-to-macrophage development (day 3). β1 integrins were not detected on adherent monocytes, but are apparently induced during macrophage development and were observable on day 3 of the culture period. In contrast, β2 integrins were readily detectable on freshly adherent monocytes and were even more strongly expressed with macrophage development on day 3. β3 integrins were undetectable or very weakly expressed at either time point. After the IL-4 induction of macrophage fusion and FBGC formation, the expression of β1 integrins was much more striking (Figure 6A). In fusing macrophages, β1 integrins were observed throughout the cytoplasm. In multinucleated FBGCs, β1 integrins were observed to be most concentrated in central cytoplasmic areas. They were also detectable throughout the cell and in the extreme periphery just outside of the peripheral rings of punctate actin that are characteristic of these giant cells.31DeFife KM Jenney CR Colton E Anderson JM Cytoskeletal and adhesive structural polarizations accompany IL-13-induced human macrophage fusion.J Histochem Cytochem. 1999; 47: 65-74Crossref PubMed Scopus (74) Google Scholar β2 integrins were also strongly expressed on fusing macrophages, particularly in peripheral cell areas, and were frequently co-localized with F-actin (yellow fluorescence, Figure 6B). Similarly to β1, upon FBGC formation, β2 integrins were observed to be most concentrated in central cytoplasmic regions together with the nuclei. Further, β2 was also expressed directly outside of FBGC punctate actin rings, appearing to distinctly outline these multinucleated giant cells at the cell/substrate interface. Finally, as was the case for monocytes and day 3 macrophages, β3 integrins were essentially undetectable
Referência(s)