Article |
Address correspondence to Isao Tachibana, Dept. of Molecular Medicine, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. Tel.: 81-6-6879-3833. Fax: 81-6-6879-3839. E-mail:itachi02{at}imed3.med.osaka-u.ac.jp
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Abstract |
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Key Words: integrins; osteoclasts; cell fusion; macrophages; multinucleated giant cells
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Introduction |
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Although their multifunctional characteristics and multipartnerships with other proteins have been increasingly reported, the definitive biological functions of tetraspanins still remain elusive. In this respect, studies of tetraspanin knockout mice have revealed the cellular functions for which a given tetraspanin is essential. One of these is a role of CD9 in gamete membrane fusion; CD9 knockout mice were infertile because CD9-null eggs were incapable of fusing with sperm (Miyado et al., 2000). Tetraspanins may play a more general role in cellcell fusion because additional reports have implicated tetraspanins in other fusion events. Antibodies against CD9 and CD81 inhibit the fusion of myoblasts, and CD9 transfection into myoblast-derived sarcoma cells enhances syncytium formation (Tachibana and Hemler, 1999). Anti-CD81 and -CD82 mAbs perturb the fusion of cells infected with human T cell leukemia virus type 1 (Fukudome et al., 1992). CD9 overexpression renders cells more susceptible to feline immunodeficiency virus and canine distemper virus, leading to elevated syncytium formation (Löffler et al., 1997; Willett et al., 1997). These results suggest that tetraspanins facilitate the fusion between gametes, myoblasts, and virus-infected cells. However, it is still unknown whether they play a similar role in cell fusion into other multinucleated cells, such as multinucleated giant cells (MGCs)* or osteoclasts.
Syncytia formed after the fusion of mononuclear phagocytes are called MGCs or osteoclasts. Multinucleation via cell fusion appears to endow monocytes/macrophages with the capacity to digest and resorb extracellular infectious agents, foreign materials, and other components that are too large to be internalized (Vignery, 2000). The presence of MGCs is a hallmark of granulomas, which are formed in inflammatory sites of tuberculosis, fungal infection, HIV infection, sarcoidosis, Crohn's disease, and tumors (Anderson, 2000; James, 2000). The physiological meanings of MGCs still remain unknown, but possible roles in the host defense against bacterial infection have been suggested; MGCs may limit the cell-to-cell spread of Mycobacterium tuberculosis (Byrd, 1998) and may have stronger candidacidal activity than macrophages (Enelow et al., 1992). Osteoclasts are formed by the fusion of mononuclear progenitors of the monocyte/macrophage lineage. These polykaryons are characterized by the presence of tartrate-resistant acid phosphatase (TRAP) activity and have a crucial role not only in physiological bone remodeling, but also in local bone disorders such as osteoporosis and bone tumors. However, the actual cut-off line that discriminates between osteoclasts and MGCs remains controversial (Vignery, 2000).
The mechanisms of the fusion of mononuclear phagocytes are not well understood, but previous papers have shown that several membrane proteins, such as CD44, CD47, CD98, macrophage fusion receptor, P2X7 receptor, ADAMs, and integrins, are involved (Vignery, 2000; Namba et al., 2001). In the present paper, we show that tetraspanins CD9 and CD81 play a preventive role in the fusion of mononuclear phagocytes.
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Results |
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Anti-CD9 and -CD81 mAbs promote the fusion of blood monocytes/alveolar macrophages
Anti-CD9 and -CD81 antibodies were previously shown to inhibit the fusion of sperm and egg (Takahashi et al., 2001), myoblasts (Tachibana and Hemler, 1999), or virus-infected cells (Fukudome et al., 1992; de Parseval et al., 1997). To examine the role of tetraspanins in monocyte fusion, we added anti-tetraspanin mAbs to the monocyte culture under fusogenic conditions containing Con A. Unexpectedly, anti-CD9 (BU16) and anti-CD81 (JS64) mAbs dramatically promoted monocyte fusion (Fig. 2). The fusion rates in the presence of BU16 and JS64 were elevated 3.5-fold and fourfold relative to those in control IgG cultures, respectively. Moreover, MGCs formed in the presence of these mAbs were larger in size than the control MGCs (Fig. 2 A). Another anti-CD9 mAb, MM2/57, also promoted the fusion, although its effect was weaker than those of BU16 and JS64. On the other hand, anti-CD14, -CD63, and -CD151 mAbs had little, if any, fusion-promoting effects. Anti-integrin ß1 and ß2 mAbs significantly inhibited the fusion (Fig. 2, A and B), consistent with previous reports (Most et al., 1990; Tabata et al., 1994). To examine the dose dependency, fusion rates in the presence of various concentrations of BU16, JS64, and isotype-matched IgG were determined (Fig. 2 C). The fusion-promoting effects of BU16 and JS64 were dose dependent, reached a plateau at 110 µg/ml, and decreased slightly at 20 µg/ml. Meanwhile, the isotype-matched control IgG had little effect on monocyte fusion even at 20 µg/ml. To exclude the possibility that the effects of BU16 and JS64 were mediated by Fc receptors, we generated divalent F(ab')2 fragments and examined their effects on monocyte fusion. As shown in Fig. 2 D, these F(ab')2 fragments exerted similar degrees of fusion-promoting effects as untreated mAbs. Finally, the coaddition of anti-CD9 and -CD81 mAbs appeared to have additive effects on the monocyte fusion (Fig. 2, A and D).
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Anti-CD9 and -CD81 mAbs do not affect cell adhesion, aggregation, proliferation, or cytokine production
The effects of anti-CD9 and -CD81 mAbs may have been due to altered cell adhesion or aggregation, both of which are prerequisite for cellcell fusion. To examine these possibilities, we performed adhesion and aggregation assays. As shown in Fig. 3 A, anti-ß1 and -ß2 mAbs significantly inhibited monocyte adherence to the tissue culturetreated surfaces. This is consistent with a previous report showing that ß1 and ß2 integrins mediate adhesion to culture surfaces during macrophage fusion (McNally and Anderson, 2002). Meanwhile, anti-CD9 and -CD81 mAbs, and control IgG had no effect on the monocyte adherence. In aggregation assays, no mAb had a significant effect on the monocyte aggregation that was induced by 10 µg/ml Con A (unpublished data). However, when a lower concentration of Con A (5 µg/ml) was used, anti-ß2 integrin mAbs markedly inhibited the monocyte aggregation (Fig. 3 B). On the other hand, neither anti-ß1 integrin mAb, anti-CD9 and -CD81 mAbs, anti-CD14 mAb, nor control IgG affected the monocyte aggregation. We also examined whether anti-CD9 and -CD81 mAbs alter the proliferation of monocytes using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and observed that these mAbs had no effect on monocyte proliferation (unpublished data).
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Soluble GSTCD9 large extracellular loop protein inhibits monocyte fusion
There was a possibility that the results of the antibody-blocking experiments were caused by steric hindrance to molecules adjacent to the tetraspanins. Thus, to extend the observations made in the antibody experiments, we studied effects of a recombinant GST fusion protein that contained the large extracellular loop of human CD9 (GSTCD9; Shimizu et al., 2002). Various concentrations of GSTCD9 were added to the monocyte culture under fusogenic conditions containing Con A. As shown in Fig. 4, GSTCD9 inhibited monocyte fusion, and this inhibitory effect was dose-dependent in the range of 0.220 µg/ml. On the other hand, neither GST alone nor GSTmurine CD9 fusion protein (GSTmCD9) had significant fusion-inhibitory effect even at 20 µg/ml.
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Enhanced cell fusion by CD9- and CD81-null alveolar macrophages and bone marrow cells after in vitro or in vivo stimulation
To determine more definitively the roles of CD9 and CD81 in monocyte/macrophage fusion, we investigated MGC formation of CD9- and CD81-deficient murine alveolar macrophages. Alveolar macrophages were isolated by BAL from the lungs of wild-type, CD9-null, and CD81-null mice, but no MGCs were present in these mice (unpublished data). Equal numbers of collected macrophages were then induced to fuse into MGCs in vitro. Notably, after the stimulation, CD9 (-/-) and CD81 (-/-) alveolar macrophages formed threefold and fourfold more MGCs than wild-type macrophages, respectively (Fig. 7 A). To further examine if the deficiency of CD9 or CD81 alters MGC formation in vivo, we used a Propionibacterium acnesinduced lung inflammation model (Itakura et al., 2001). Wild-type and mutant mice were challenged by intratracheal injection of heat-killed P. acnes. After 7 d, lung paraffin sections were prepared and analyzed to detect MGCs. Although infiltration of inflammatory cells consisting of neutrophils, macrophages, and lymphocytes into the alveolar space was reported 3 d after P. acnes injection (Itakura et al., 2001), most of cells remaining at d 7 were macrophages. Remarkably, although few MGCs were present in wild-type mice, substantial numbers of MGCs were formed in the airspace of CD9- and CD81-null mice (Fig. 7 B, left). In a separate experiment to quantify the MGC formation, alveolar macrophages were separated by BAL after P. acnes injection, and the number of MGCs was determined (Fig. 7 B, right). The total numbers of cells isolated from wild-type and mutant mice were not significantly different (unpublished data), but nonetheless, sixfold and threefold more MGCs were detected in CD9- and CD81-null mice compared with wild-type mice, respectively.
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CD9/CD81 double-null mice spontaneously develop MGCs in the lung and showed enhanced osteoclastogenesis in the bone
Because CD9 and CD81 appear closely related in view of amino acid sequence (the identity in the aligned human sequences is 31%; Boucheix and Rubinstein, 2001), one may compensate for the function of the other in CD9 or CD81 single-null mice. Therefore, we generated CD9/CD81 double-null mice by intercrossing CD9 (+/-)/CD81 (+/-) mice. CD9/CD81 double-null mice were viable, but smaller in size than wild-type mice and displayed osteopenic phenotype in association with angular kyphotic deformity of the spine (unpublished data). Fig. 8 shows hematoxylin- and eosin-stained lung sections and TRAP-stained bone sections from wild-type and double mutant mice. Notably, alveolar airspace enlargement with the infiltration of macrophages and a smaller number of lymphocytes into the space and septa was observed in CD9/CD81 double-null mice (Fig. 8, B and C). More importantly, although no MGCs were detected in wild-type (Fig. 8 A) or CD9 (+/-)/CD81 (+/-) mice (unpublished data), eosin-stained MGCs were spontaneously formed in the alveolar space of double mutant mice (Fig. 8, B and C). Most of these MGCs were large, round, eosin-stained cells containing 3 to
10 nuclei, and resembled those detected in CD9 or CD81 single-null mice that had been stimulated by intratracheal injection of P. acnes (Fig. 7 B). Few neutrophils or eosinophils were present, thus ruling out the occurrence of bacterial or parasitic infection. Findings from bones of CD9/CD81 double-null mice were also remarkable (Fig. 8 E). When compared with the wild-type (Fig. 8 D) and single-null littermates, TRAP-positive osteoclasts in tibiae increased 1.5-fold in number per millimeter bone surface (wild type, 1.78 ± 0.19/mm; CD9 (-/-) mice, 1.79 ± 0.70/mm; CD81 (-/-) mice, 1.67 ± 0.22/mm; CD9 (-/-)/CD81 (-/-) mice, 2.61 ± 0.29/mm; P < 0.005 versus wild type in the t test, n = 4). Dual-energy X-ray absorptiometry on femurs showed that bone-mineral density of double mutant mice were significantly reduced (wild-type, 25.1 ± 1.8 mg/cm2; CD9 (-/-) mice, 26.1 ± 0.4 mg/cm2; CD81 (-/-) mice, 25.5 ± 1.7 mg/cm2; CD9 (-/-)/CD81 (-/-) mice, 22.0 ± 1.5 mg/cm2; P < 0.05 versus the wild type).
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Discussion |
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All of anti-tetraspanin mAbs used in fusion assays of the present work were previously shown to inhibit cellular functions in vitro. In particular, the antihuman CD9 mAbs MM2/57 and BU16 were reported to inhibit virus-induced fusion (Löffler et al., 1997). The antimouse CD9 mAbs KMC8 and JF9 were reported to prevent spermegg fusion (Miyado et al., 2000; Takahashi et al., 2001). KMC8 and antimouse CD81 mAb 2F7 inhibited/delayed the fusion of the myoblast cell line C2C12 (Tachibana and Hemler, 1999). These anti-CD9 and -CD81 mAbs unexpectedly promoted the fusion of mononuclear phagocytes. It is unlikely that the effects were mediated by Fc portionFc receptor interactions, because isotype-matched control IgG had no effect on the fusion, and F(ab')2 fragments had similar effects to untreated mAbs. These results raise the possibility that CD9 and CD81 facilitate the fusion of virus-infected cells, spermegg, and myoblasts, while preventing the fusion of monocytes/macrophages via the same epitopes. A previous work used a soluble GSTCD81 extracellular loop fusion protein to demonstrate that this protein bound to neurons but not to astrocytes, and that it blocked neuron-induced astrocyte proliferative arrest, thus suggesting the presence of a ligand for CD81 on neurons (Kelic et al., 2001). In the present work, the GSTCD9 extracellular loop fusion protein inhibited monocyte fusion in a dose-dependent manner. This result further supports the involvement of CD9 protein in monocyte fusion, and may reflect a functional interaction between the CD9 extracellular loop and its putative ligand on an apposed cell surface.
With regard to genuine tetraspanins (Boucheix and Rubinstein, 2001), mice lacking CD9 (Miyado et al., 2000), CD37 (Knobeloch et al., 2000), CD81 (Maecker and Levy, 1997; Miyazaki et al., 1997), and Tssc6 (Tarrant et al., 2002) have been produced by gene targeting. CD9-null mice are infertile due to defective fusion capacity of their eggs. CD81-null mice are deficient in T celldependent IgG production and in Th2 cytokine secretion (Maecker et al., 1997). Recently, CD81-null mice were also reported to have reduced reproductive capacity after repeated backcrosses (Deng et al., 2000). Moreover, exogenously overexpressed CD81 rescued the fusibility of CD9-null eggs (Kaji et al., 2002). Thus, CD9 and CD81 may share an essential role in gamete fusion processes. Meanwhile, no abnormal findings in other cell fusion events and no morphological abnormalities have been reported in tetraspanin knockout mice. However, given that several different tetraspanins form complexes with each other, the loss of a particular tetraspanin may be compensated by other tetraspanins. We presumed that single tetraspanin knockout mice under particular conditions or multiple tetraspanin knockout mice may reveal novel tetraspanin functions. In fact, in the present work, in response to in vitro and in vivo stimulation, CD9- and CD81-null macrophages and bone marrow cells displayed enhanced fusion capacity. Furthermore, CD9/CD81 double-null mice spontaneously developed MGCs in the lung and showed increased osteoclastogenesis in the bone. It is tempting to presume that osteopenic phenotype of CD9/CD81 double-null mice is due to enhanced osteoclastogenesis. However, bone mass is maintained under a balance between bone resorption and formation. In fact, CD9 is present on osteoblast progenitors, and this CD9 molecule is also likely to be involved (Hayashi et al., 2000). Thus, detailed morphometric analysis and studies to evaluate the activities of osteoclasts and osteoblasts are obviously required to elucidate mechanisms in the osteopenic phenotype; such studies are currently in progress. Collectively, our data obtained using mAb and knockout mouse experiments suggest that CD9 and CD81 function to inhibit the fusion of mononuclear phagocytes, and that these tetraspanins may be able to compensate for each other.
Because the roles of CD9 and CD81 in monocyte/macrophage fusion proposed based on the present paper are contradictory to the previous hypothesis that these tetraspanins facilitate cellcell fusion, the mechanisms of the tetraspanin contribution to cellcell fusion now appear to be complex. One possible reason for this may be that the functions of tetraspanins are dependent on the cell lineage. Notably, although lectins such as Con A and phytohemagglutinin induce the fusion of macrophages, these lectins paradoxically inhibit the fusion of virus-infected cells, myoblasts, and gametes (Chambers, 1977). This evidence may indicate a fundamental distinction between macrophage and nonmacrophage cell fusion.
Integrins are well known to form complexes with tetraspanins, and tetraspanins may modulate the adhesive functions of integrins during cellcell fusion (Boucheix and Rubinstein, 2001). In fact, it was shown in previous papers that the fusion of blood monocytes involves ß1 and ß2 integrins (Most et al., 1990; Tabata et al., 1994). In the present work, both anti-ß1 and -ß2 integrin mAbs inhibited the fusion of monocytes, probably due to blocking of monocyte adherence and aggregation, respectively. However, these adhesive and aggregative functions are not likely to be modified by tetraspanins because neither anti-CD9 nor anti-CD81 mAb altered monocyte adhesion or aggregation. We also observed that ß1 and ß2 integrins were complexed with CD9 and CD81 in freshly isolated blood monocytes, and this complex formation was up-regulated under normal culture conditions. Complexes between CD9 and CD81 were also increased; these up-regulations may at least partly reflect the elevated levels of total CD9 and CD81 proteins, but are consistent with the previous report that CD9 and ß1 integrins assemble into a molecular complex during maturation of monocytes (Kurita-Taniguchi et al., 2001). However, under fusogenic conditions, the complex formation of tetraspanin (CD9 or CD81)integrin (ß1 or ß2) was down-regulated, whereas tetraspanintetraspanin (CD9CD81) complex formation occurred normally. A mild detergent (Brij99) was used in coimmunoprecipitation experiments, indicating that the molecular complexes in cell lysates could be part of raft-like membrane microdomains (Claas et al., 2001). Possibly, the different regulation of tetraspaninintegrin and tetraspanintetraspanin complexes might reflect differential localization of these complexes into distinct microdomains. Further studies will be needed to clarify the contribution of tetraspaninintegrin complexes to monocyte/macrophage fusion.
Other proteins involved in the fusion of mononuclear phagocytes, such as ADAMs (Namba et al., 2001), CD44, CD47 (Vignery, 2000), and CD98 (Tabata et al., 1994), may be affected by CD9 and CD81. An interaction between CD9 and ADAMs was suggested in the fusion between sperm and eggs (Zhu and Evans, 2002). Both CD44 and CD47 were reported to associate with CD9 (Jones et al., 1996; Longhurst et al., 1999). CD98 associates with ß1 integrins and is important in integrin activation (Fenczik et al., 2001). Tetraspanins may indirectly influence CD98 function through their participation in integrin complexes. Roles of these proteins in tetraspanin-regulated monocyte/macrophage fusion remains unknown, but the fact that CD9 associates with multiple monocyte/macrophage fusion-related proteins further supports the involvement of tetraspanins in this particular type of fusion.
In conclusion, the present paper has demonstrated a novel fusion-regulatory function of tetraspanins. While facilitating the fusion of gametes, myoblasts, and virus-infected cells, CD9 and CD81 are essential to prevent the fusion of mononuclear phagocytes. Given that multinucleation endows cells with more resorptive capacity for extracellular components such as bone or infectious agents (Vignery, 2000), functional alterations of tetraspanins may contribute to the progression of osteoporosis, infection, and granulomatous diseases.
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Materials and methods |
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Isolation of blood monocytes, alveolar macrophages, and bone marrow cells
Peripheral blood mononuclear cells were isolated from heparinized whole blood by Ficoll-Hypaque density gradient centrifugation. Mononuclear cells were collected and cultured on MSP plates (Japan Immunoresearch). The adherent monocytes were detached and suspended in RPMI 1640 medium containing 10% heat-inactivated FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin (culture medium). Human alveolar macrophages were collected by BAL from patients in Osaka University Hospital (Osaka, Japan) with informed consent. Murine alveolar macrophages were obtained by BAL from 68-wk-old ddY male mice. Lungs of mice were subjected to lavage six times with 1.0 ml saline. Collected cells consisting mostly of macrophages were suspended in DME containing 5% human serum. Murine bone marrow cells were obtained from tibiae of 812-wk-old C57BL/6 male mice and suspended in MEM containing 10% FCS.
Immunoblotting
Cells were lysed in lysis buffer containing 1% Brij99, 25 mM Hepes, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 2 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin. Samples containing equal protein concentrations were electrophoresed on SDS-PAGE and transferred to Immobilon-P membranes (Millipore). The membranes were probed with mAbs diluted at 1 µg/ml, followed by incubation with peroxidase-conjugated goat antimouse IgG (H+L; Bio-Rad Laboratories) diluted 1:2,000. Immunoreactive bands were visualized with Renaissance® Chemiluminescent Reagent (NEN Life Science Products).
Immunoprecipitation
Lysates containing equal amounts of protein were incubated with anti-integrin and -tetraspanin mAbs. Immune complexes were collected with protein A-Sepharose (Sigma-Aldrich), separated by SDS-PAGE under nonreducing conditions, and transferred to an Immobilon-P membrane. Immunoblotting was performed with biotinylated mAbs followed by peroxidase-conjugated streptavidin (Zymed Laboratories).
In vitro fusion assay of monocytes, macrophages, and bone marrow cells
For human blood monocyte/alveolar macrophage fusion experiments, 2 x 105 cells were suspended in 100 µl culture medium, plated in a 96-well tissue culture plate (Falcon), and induced to fuse into MGCs by the addition of 10 µg/ml Con A for 3 d. To estimate the degree of cell fusion in the absence or presence of 10 µg/ml mAb or recombinant GSTCD9 protein, nuclei were visualized using Wright stain. Fusion rates were determined by calculating the percentages of the number of nuclei within MGCs (three or more nuclei per cell) per total number of nuclei in six independent fields. Between 400 and 600 nuclei were counted in each field. For murine alveolar macrophage fusion, 3 x 105 macrophages were suspended in 50 µl DME containing 5% human serum, plated onto a 96-well plate, and then induced to fuse into MGCs by the addition for 3 d of 10 nM 1,25(OH)2D3 and 50 µl culture supernatant that was obtained from cultures of Con Astimulated murine spleen cells. The number of MGCs per well in triplicate cultures was determined. For murine bone marrow cells, 2 x 106 cells were suspended in 500 µl
MEM containing 10% FCS, plated onto a 24-well plate, and then induced to fuse by the addition for 7 d of 50 ng/ml sRANKL and 20 ng/ml M-CSF. Cells were then fixed and stained for TRAP (Li et al., 2002). The number of TRAP-positive multinucleated cells (three or more nuclei per cell) per well in triplicate cultures was determined.
In vivo fusion assay of murine alveolar macrophages
300 µg heat-killed and sonicated P. acnes (ATCC 11828; American Type Culture Collection) was suspended in 100 µl PBS and administered intratracheally to CD9 (-/-), CD81 (-/-), and wild-type mice under anesthesia as described previously (Itakura et al., 2001). After 7 d, the mice were killed and lung paraffin sections were prepared to observe MGC formation in the lung. In separate experiments, alveolar macrophages were isolated by BAL as described above, and the number of MGCs per lung was determined.
Cell adhesion assay
200,000 monocytes were suspended in 100 µl RPMI 1640 containing 10 µg/ml Con A and plated onto the wells of a 96-well tissue culture plate (Falcon). Cells were then allowed to adhere to the plate for 12 h in the absence or presence of 10 µg/ml mAbs. After nonadherent cells were removed by rinsing, the remaining adherent cells were evaluated in triplicate cultures using an MTT assay.
Cell aggregation assay
200,000 monocytes were cultured in 100 µl RPMI 1640 with 5 µg/ml Con A in the absence or presence of 10 µg/ml mAbs for 12 h in wells of a 96-well nontissue culture-treated plate (Linbro). The number of cell aggregates (>4 cells/aggregate) was determined in six independent fields.
Immunofluorescence
Murine alveolar macrophages were cultured in Lab-Tek® glass chamber slides (Nunc), fixed in 3% PFA, and then permeabilized with 0.5% Triton X-100. Nonspecific recognition of Fc receptors was blocked with 20% goat serum. The permeabilized cells were stained with 1 µg/ml control mAb (R3595) or antimouse CD9 mAb (KMC8) and incubated with FITC-conjugated antirat immunoglobulin at a 1:50 dilution and 0.5 µg/ml propidium iodide (Sigma-Aldrich). Immunofluorescence images were obtained using an inverted immunofluorescence microscope (Axioplan 2; Carl Zeiss MicroImaging, Inc.).
Mice
The generation of CD9 (-/-) mice and CD81 (-/-) mice was described previously (Miyazaki et al., 1997; Miyado et al., 2000). These mice were backcrossed more than five generations into the C57BL/6 background. CD9 (-/-)/CD81 (-/-) mice were produced by intercrossing CD9 (+/-)/CD81 (+/-) mice. The genotyping of all breeding pairs was confirmed by PCR analysis. The mice were maintained in a barrier facility, and all animal procedures were performed in accordance with the Osaka University (Osaka, Japan) guidelines on Animal Care. 711-wk-old mice matched for age and sex were used in all experiments.
Histological analysis of lung and bone sections
1 ml 10% buffered formaldehyde was instilled into the lung of each mouse via an intratracheal cannula, and then the whole lung was excised and fixed in 10% buffered formaldehyde. The fixed lung was embedded in paraffin, sectioned sagittally, and stained with hematoxylin and eosin. Tibiae were fixed with ethanol, and the undecalcified bones were embedded in glycolmethacrylate. 3-µm longitudinal sections from the proximal parts were stained for TRAP. The number of TRAP-positive osteoclasts was determined as described previously (Liu et al., 2001).
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Acknowledgments |
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Submitted: 3 December 2002
Revised: 5 May 2003
Accepted: 5 May 2003
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References |
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