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Keywords:

  • CD9;
  • CD63;
  • monocyte fusion;
  • multinucleated giant cell;
  • tetraspanin

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Members of the tetraspanin superfamily of proteins are implicated in a variety of complex cell processes including cell fusion. However, the contribution of individual tetraspanins to these processes has proved difficult to define. Here we report the use of recombinant extracellular regions of tetraspanins to investigate the role of specific members of this family in the fusion of monocytes to form multinucleated giant cells (MGC). In contrast to their positive requirement in sperm–egg fusion, previous studies using antibodies and knockout mice have indicated a negative regulatory role for tetraspanins CD9 and CD81 in this process. In an in vitro model of fusion using human monocytes, we have confirmed observations that antibodies to CD9 and CD81 enhance MGC formation; however, in contrast to previous investigations, we found that all members of a panel of antibodies to CD63 inhibited fusion. Moreover, recombinant proteins corresponding to the large extracellular domains (EC2s) of CD63 and CD9 inhibited MGC formation, whereas the EC2s of CD81 and CD151 had no effect. The potent inhibition of fusion and binding of labelled CD63 EC2 to monocytes under fusogenic conditions suggest a direct interaction with a membrane component required for fusion. Our findings indicate that the tetraspanins CD9, CD63 and CD81 are all involved in MGC formation, but play distinct roles.


Abbreviations:
B/B/N

BSS containing 0·2% BSA and 0·1% sodium azide

BSA

bovine serum albumin

BSS

balanced salt solution

Con A

concanavalin A

EC1

small extracellular region

EC2

large extracellular region

EC50

half-maximal (40%) effective concentration

FACS

fluorescence-activated cell sorter

FBGC

foreign body giant cell

FCS

fetal calf serum

FITC

fluorescein isothiocyanate

GST

glutathione S-transferase

HRP

horseradish peroxidase

IC50

half-maximal (50%) inhibitory concentration

LPS

lipopolysaccharide

MFI

median fluorescence intensity

MGC

multinucleated giant cell

PBS

phosphate-buffered saline

RFI

relative fluorescence intensity (relative to negative control)

SRB

sulforhodamine B

TEM

tetraspanin-enriched microdomains

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

The tetraspanins are a diverse family of membrane proteins with a wide tissue distribution in multicellular organisms.1,2 Thirty-three tetraspanin-encoding genes have been identified in humans, and roles for the proteins in cell adhesion, motility, signalling, virus susceptibility and fusion have been described.1–3 A key characteristic of tetraspanins is their propensity to interact with one another and with various other classes of transmembrane molecules (including immunoreceptors or co-receptors, growth factors or their receptors and integrins) as well as signalling/cytoskeletal proteins.1,4 They appear to act mainly as molecular organizers, regulating the formation of functional clusters of proteins in tetraspanin-enriched microdomains (TEM)1,3 or via the ‘tetraspanin web’.4 In addition to four membrane-spanning domains, tetraspanins have short cytoplasmic N and C termini and a small and a large extracellular domain (EC1 and EC2, respectively). Conserved sequences in the transmembrane region and conserved cysteine-containing motifs in the EC2 domain distinguish tetraspanins from other four-pass membrane proteins. Disulphide linkages formed by the four conserved cysteines (and up to four additional Cys residues) are important in the conservation of a subloop structure within the EC2.1 Apart from these residues, the EC2 domain shows greatest sequence diversity throughout the family and is thought to dictate many member-specific interactions and functions.2

Tetraspanins have been implicated in various cell-fusion processes. Most notably, CD9 has been shown to play a critical role in sperm–egg fusion because oocytes from CD9 knockout mice are unable to fuse with sperm, resulting in infertility.5 Fusion is restored by the expression of ectopic CD9, and the tetraspanin CD81 can compensate for the loss of CD9, suggesting similar roles for these tetraspanins. Monoclonal antibodies to the EC2 region of CD9, as well as recombinant proteins corresponding to CD9 EC2, inhibit fusion. CD9 and CD81 are also implicated in muscle cell fusion,6 and CD9, CD81 and CD82 have been linked with virus-induced syncitium formation.3 Recently a role for CD9 and CD81 in the formation of multinucleated giant cells (MGC) has been proposed.7

MGC formed by the fusion of monocytes/macrophages are a feature of granulomatous inflammation associated with chronic infections (e.g. in tuberculosis) or the response to foreign bodies.8 They are classified morphologically as Langhans’ type (formed mainly in response to interferon-γ, with a circular peripheral arrangement of nuclei) or foreign body giant cells (FBGCs; formed mainly in response to interleukin-4 or interleukin-13, with an irregular arrangement of nuclei).8 Osteoclasts, the cells involved in bone resorption, are also formed by fusion of cells of the monocyte lineage and may be considered to be giant cells. The role of MGCs in infection is poorly defined. Whilst it has been suggested that they may limit the spread of infection,9 recent evidence indicates that they may also contribute to inflammatory tissue damage through increased secretion of matrix metalloproteinase.10 MGCs are also present in chronic inflammatory conditions of uncertain aetiology (e.g. sarcoidosis, Crohn’s disease)11,12 and FBGC on medical implants are associated with degradation of biomaterials.8

Monocytes/macrophages can be induced to form MGC in various ways in vitro, in response to cytokines, conditioned medium, phorbol esters and the indirect activity of lectins.8 Takeda et al. found that antibodies to tetraspanins CD9 and CD81, but not an antibody to CD63, enhanced concanavalin A (Con A)-induced fusion of human monocytes and mouse alveolar macrophages in vitro. In addition, macrophages from CD9 and CD81 null mice showed enhanced formation of MGC in vitro, and CD9/CD81 double-knockout mice showed spontaneous formation of MGC. This led the authors to speculate that CD9 and CD81 act together to regulate monocyte/macrophage fusion in a negative manner.7

It has generally been difficult to dissect out the activities of individual tetraspanins within complex biological processes. The absence of a dramatic phenotypic abnormality in most tetraspanin knockout animals suggests overlapping functions. Interpretation of studies using cross-linking antibodies is complicated by the association of individual tetraspanins with other tetraspanins (and other proteins) in the context of TEM. Also, antibodies with different epitope specificities and affinities may have quite different effects. Here we report a complementary approach, using antibodies and a range of recombinant tetraspanin EC2 domains to investigate the roles of these membrane proteins in MGC formation. Soluble forms of tetraspanin EC2 regions have been shown to have biological activity in a number of systems, including sperm–egg fusion,5,13 hepatitis C virus binding,14,15 leucocyte transmigration16 and human immunodeficiency virus (HIV) infection of macrophages.17 We demonstrate for the first time that CD63 plays a direct positive role in monocyte fusion, leading to MGC formation. Our results also indicate direct involvement of CD9, but concur with previous suggestions that it acts as a negative regulator for monocyte fusion.7 CD81 may also normally contribute to preventing MGC formation, but the lack of effect with the EC2 region argues that the involvement of this tetraspanin is indirect.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Antibodies

Hybridoma cells secreting mouse anti-CD63 IgG1 (H5C618) were obtained from the Developmental Studies Hybridoma Bank, University of Iowa, IA, and antibody was purified from culture supernatant using protein G–Sepharose (Amersham-Pharmacia, Amersham, UK). Fab fragments were prepared by digestion with papain for 2 hr and purified on protein A–Sepharose (Amersham-Pharmacia). Purity of univalent Fab fragments was determined by Coomassie Blue staining of sodium dodecyl sulphate–polyacrylamide (SDS-PAGE) gels and western blotting using anti-mouse IgG conjugated to horseradish peroxidase (HRP) (Sigma, Poole, UK); no intact IgG or F(ab′)2 was detected. Anti-CD63 IgM (LP9)19 was purified in-house from hybridoma culture supernatant using Thiosorb M (Millipore, Watford, UK). Anti-CD63 IgG1 antibodies 16·1 and 16·520 were kind gifts from Keith Skubitz (Department of Medicine, University of Minnesota, MN). Anti-CD151 IgG1 14A2 was a kind gift from Leonie Ashman (School of Biomedical Sciences, University of Newcastle, Australia) and anti-CD81 IgG1 (clone 1D6) and anti-CD52–fluorescein isothiocyanate (FITC) conjugate was from Serotec (Oxford, UK). Hybridoma cells secreting anti-CD9 IgG1 (602-29)21 were kindly provided by Peter Andrews (Department of Biomedical Sciences, University of Sheffield, Sheffield, UK). Anti-CD9 IgG1 Alma-1 was a kind gift from Francois Lanza (INSERM U.311, Strasbourg, France).

Production of glutathione S-transferase fusion proteins

The production and characterization of the tetraspanin EC2 glutathione S-transferase (GST) fusion proteins has been described in detail previously.13 Briefly, tetraspanin EC2 regions that had been cloned into the pGEX-KG vector were expressed in BL21 Escherichia  coli and cultured at 37° for 4 hr after induction with isopropyl thio-β-d-galactoside (IPTG). Cells were pelleted and lysed with Novagen Bugbuster (VWR International, Lutterworth, UK) in the presence of a protease inhibitor cocktail. Recombinant protein was purified in a single step by affinity chromatography on glutathione beads (Amersham-Pharmacia). Protein purity was analysed by Coomassie staining of SDS-PAGE gels and conformation of the EC2s was assessed using conformation-sensitive antibodies in western blotting and by assessing the depletion of GST-tagged material following immunoprecipitation with anti-tetraspanin IgG (Figs S1–S3). To remove GST, glutathione Sepharose beads (Amersham-Pharmacia) were saturated with GST–CD63 and treated with 10 units of thrombin protease (Sigma) per mg of EC2 for 4 hr at 21°. Thrombin was removed by incubation with p-aminobenzamidine agarose beads. Free GST or GST–CD63 EC2 could not be detected by silver staining of SDS-PAGE gels after treatment. His6-EC2 tetraspanins were kindly provided by Christopher Liu and Richard Hynes (Howard Hughes Medical Institute, Massachusetts Institute of Technology, MA). As described previously, correct conformers were purified and analysed by reverse-phase high-performance liquid chromatography (HPLC) connected to an LCQ electrospray mass spectrophotometer (Finnigan-MAT, San Jose, CA) and folding was also verified using conformation-sensitive antibodies.22

Fluorescent labelling of GST fusion proteins

For imaging studies, GST and GST–tetraspanin EC2 proteins, produced as described above, were labelled with FITC using conventional methods23 and with Alexafluor 647 (Cambridge Bioscience, Cambridge, UK) according to the manufacturer’s instructions. The molar dye : protein ratios were as follows: GST–FITC, 4·8:1; GST–CD9 EC2–FITC 4·2:1; GST–CD63 EC2–FITC, 4·8:1; GST–Alexafluor 647, 3·5:1; and GST–CD63 EC2–Alexafluor 647, 1·7:1.

Monocyte fusion assay

Peripheral blood monocytes were derived from peripheral whole blood of healthy, anonymized volunteers by Ficoll–Hypaque density centrifugation, as described previously.24 The study was approved by the South Sheffield Research Ethics Committee (protocol number SSREC/02/299). Mononuclear cells were seeded at 5 × 105 cells/chamber in 0·5 ml of RPMI-1640 containing 10% fetal calf serum (FCS) in an eight-chambered slide (Lab-Tek®; Nunc, Fisher Scientific, Loughborough, UK). After overnight culture, adherent cells were cultured in RPMI containing 10% (v/v) FCS in the presence or absence of 10 μg/ml of concanavalin A (Con A; Sigma) for 72 hr at 37°. Antibodies to tetraspanin, and recombinant tetraspanin EC2 proteins, were added at the stated concentrations at the same time as Con A. The cells were washed with phosphate-buffered saline (PBS), fixed and permeabilized with acetone (5 min at room temperature), rehydrated with PBS then labelled with FITC–anti-CD52 and the nuclei were counterstained with propidium iodide. Fusion rates were determined by counting the number of nuclei in fused cells (three or more nuclei per cell) and unfused cells in six randomly chosen fields using a Nikon Eclipse E400 immunofluorescence microscope (Nikon, Tokyo, Japan). In some cases, the numbers of nuclei per MGC in six random fields was recorded.

Measurement of cell number and cell adhesion

Mononuclear cells, prepared as described above, were resuspended in medium and cultured at 5 × 105 cells/200 μl in 96-well tissue culture plates in the absence or presence of Con A (10 μg/ml) and in the presence or absence of GST alone or tetraspanin GST–EC2 (20 μg/ml). After 72 hr, cell number was assessed using the sulforhodamine B (SRB) assay.25 The effects on cell adhesion were assessed in parallel by removing non-adherent cells by washing [three times with 200 μl of balanced salt solution (BSS)] prior to carrying out the SRB assay.

Cell aggregation assay

Adherent monocytes, prepared as described above, were seeded into a 96-well tissue culture plate at 2 × 105 cells/100 μl of medium and cultured for 12 hr with Con A (5 μg/ml) and in the presence or absence of GST–EC2 or His6-EC2 tetraspanins (20 μg/ml). The numbers of aggregates containing 4–10 and 10–20 cells were counted in six randomly chosen fields using light microscopy.

Analysis of binding of GST fusion proteins to monocytes

Adherent monocytes were cultured in the presence or absence of Con A (10 μg/ml) for 2 hr. Cells were harvested and incubated with fluorescent GST proteins (6 μg/ml) diluted in RPMI-1640 containing 10% (v/v) FCS for 45 min at 37° or on ice. (For dose–response curves, monocytes were incubated with various concentrations of labelled GST proteins on ice.) In some cases, cells were incubated with labelled GST proteins in the presence of excess (100×) unlabelled protein. Cells were washed with cold BSS containing 0·2% bovine serum albumin (BSA) and 0·1% azide (B/B/N) prior to fixation with 2% paraformaldehyde and analysis using a fluorescence-activated cell sorter (FACS) (FACSort; Beckton Dickinson, Cowley, UK). For fluorescence microscopy, monocytes cultured in the presence or absence of Con A and prepared as for FACS analysis (described above) were deposited on slides by cytocentrifugation (Shandon, Runcorn, UK). After fixation in acetone and rehydration with PBS, cells were incubated with FITC-labelled GST or with GST–CD63 EC2 for 1 hr, washed and stained as described above.

Cell-surface expression of tetraspanins on fusing monocytes

Adherent monocytes cultured in the presence or absence of 10 μg/ml of Con A were harvested using Cell Dissociation Solution (Sigma) and washed in B/B/N. Approximately 2 × 105 cells were incubated with anti-CD9 (602·29), anti-CD63 (H5C6), anti-CD81 (1D6) or IgG1 isotype control (10 μg/ml) immunoglobulins on ice for 45 min. The cells were washed twice with ice-cold B/B/N and incubated for 30 min with FITC-labelled anti-mouse immunoglobulin (Sigma), washed again and then analysed by flow cytometry, as described above.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Effects of anti-CD9 and anti-CD63 immunoglobulins on MGC formation

As reported previously, Con A induced peripheral blood monocytes to undergo fusion to form MGC (Figs 1a,b and 2a,b), which were predominantly of the Langhans’ type7,26 (Fig. 1b). No fusion was observed for monocytes cultured in the absence of Con A (Fig. 1a). Treatment with anti-tetraspanin immunoglobulins had a range of effects on MGC formation: anti-CD63 immunoglobulins such as H5C6 inhibited MGC formation, even as monomeric Fab fragments (Fig. 2c,d), whereas anti-CD81 immunoglobulin 1D6 and anti-CD9 immunoglobulin 602-29 enhanced MGC formation (Fig. 2e,f). These effects were quantitatively analyzed as fusion index, which measures the percentage of nuclei in MGC, and also in some cases as the mean number of nuclei per MGC (Fig. 3). All anti-CD63 immunoglobulins tested, including an IgM (LP9), significantly decreased MGC formation by both of these measures (Fig. 3a,b). By contrast, the monoclonal anti-CD9 Alma-1 had a slight, but significant, enhancing effect on fusion index (Fig. 3a). Although an effect on fusion index was not apparent for 602-29, this anti-CD9 immunoglobulin significantly enhanced MGC size, as shown quantitatively in Fig. 3b. Similarly, the anti-CD81 immunoglobulin, 1D6, significantly enhanced MGC formation, whereas an antibody to the tetraspanin CD151 (14A2), had no effect on MGC formation (Fig. 3a,b). No effects were observed with isotype control antibody (data not shown).

image

Figure 1.  Concanavalin A (Con A) induces giant cell formation in human monocytes. Adherent human blood monocytes were incubated in medium alone (a) or with 10 μg/ml of Con A (b) for 72 hr. After fixation, cells were stained with fluorescein isothiocyanate (FITC)-labelled anti-CD52 (green) and the nuclei were counterstained with propidium iodide (red). Scale bar, 100 μm.

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image

Figure 2.  Effects of anti-tetraspanin immunoglobulins on concanavalin A (Con A)-induced monocyte fusion. Adherent human monocytes were incubated for 72 hr in medium alone (a), or in medium with Con A (10 μg/ml; b), Con A and anti-tetraspanin immunoglobulins (20 μg/ml): anti-CD63 immunoglobulin, H5C6 (c), anti-CD63 H5C6 Fab fragments (d), anti-CD81, (e) or anti-CD9, 602·29 (f). (In initial experiments, concentrations of up to 100 μg/ml of immunoglobulin were used, but no increased effects were observed. All antibodies exhibited saturating binding at 20 μg/ml.) After fixation, cells were stained with fluorescein isothiocyanate (FITC)-labelled anti-CD52 and the nuclei were counterstained with propidium iodide. Scale bar, 100 μm.

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image

Figure 3.  Effects of anti-tetraspanin immunoglobulins on concanavalin A (Con A)-induced fusion index and giant cell size. Adherent human monocytes were incubated for 72 hr in medium alone, in medium with Con A (10 μg/ml) or in medium with Con A and anti-tetraspanin immunoglobulins (20 μg/ml). Fusion index (a) was determined as the percentage of nuclei within multinucleated giant cells (MGC; three or more nuclei per cell) as a ratio to the total number of nuclei. To assess the mean number of nuclei within MGC (b), nuclei within all MGC in a slide chamber were counted. The results shown are for two to six independent experiments (each using monocytes from different donors) performed in duplicate, shown as means ± standard error (SE). The significance of difference [determined by a Kruskal–Wallis analysis of variance (anova)] is between antibody-treated samples and the Con A alone control, unless otherwise indicated. ***P < 0·001, **P < 0·01. NS, not significant.

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Analysis of cell-surface expression by FACS showed that after 2 hr of stimulation with Con A, a small increase in CD9, CD63 and CD81 was observed on the fusing population of cells (Fig. S2a,b). After 72 hr, the levels of CD9 and CD81 on this population were decreased whereas the levels of CD63 remained similar (Fig. S2d). By contrast, for monocytes cultured for 72 hr in the absence of Con A, there was a substantial increase in the cell-surface levels of CD9 and CD81, whereas the cell-surface expression of CD63 remained low (Fig. S2c). These findings on tetraspanin expression are generally consistent with those reported previously.7

Effects of GST–EC2 tetraspanins on MGC formation

As mentioned previously, soluble forms of tetraspanin EC2 regions are functionally active in a number of cell processes.5,13–17 We therefore investigated a range of recombinant human tetraspanin EC2 domains produced as GST fusion proteins for their effects on MGC formation. GST alone had no effect on cell fusion (Fig. 4a), whereas recombinant GST–CD63 and GST–CD9 EC2s clearly inhibited MGC formation (Fig. 4b,c). In a quantitative analysis, the tetraspanin EC2 protein GST–CD81 had no significant effect on MGC formation (Fig. 4d). Mouse CD9 EC2, which has 77% identity with human CD9 EC2, could not inhibit MGC formation as a GST fusion protein (Fig. 4d). Similarly, a CD9 EC2 containing a C153A mutation had only a small (albeit significant) negative effect on MGC formation (Fig. 4d). This mutant was not recognized by conformation-specific antibodies and was not expected to contain a correctly folded subloop in its EC2 region. GST–CD63 and GST–CD9 EC2s also had significant effects on the size of the MGC (Fig. 4e).

image

Figure 4.  Effects of recombinant tetraspanin second extracellular domains (EC2) on concanavalin A (Con A)-induced monocyte fusion. Adherent human monocytes were incubated for 72 hr with Con A (10 μg/ml) in the presence or absence of recombinant tetraspanin EC2 [glutathione S-transferase (GST)–-EC2] or GST alone (20 μg/ml). Panel a shows Con A-stimulated monocytes labelled with propidium iodide and anti-CD52 immunoglobulin; panels b and c show the effects of EC2s GST–CD63 and GST–CD9, respectively, on Con A-induced fusion. Panels d and e show fusion index and the mean number of nuclei, respectively, within multinucleated giant cells (MGC). Panel f compares the effect of GST–CD9 and GST–CD81 alone, or in combination (each at a final concentration of 20 μg/ml) on fusion index. Results are for three to six independent experiments (with different donors) performed in duplicate and shown as means ± standard error (SE); the significance of difference [determined by a Kruskal–Wallis analysis of variance (anova)] is between samples treated with recombinant protein and Con A alone, unless otherwise indicated. ***P < 0·001, **P < 0·01. NS, not significant.

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As anti-CD81 immunoglobulins enhance MGC formation, the lack of an effect with GST–CD81 was unexpected. To investigate the possibility that although inactive alone, it might co-operate with GST–CD9, we co-incubated GST–CD9 and GST–CD81 in the monocyte fusion assay. Interestingly, GST–CD81 significantly abrogated the inhibition of MGC formation by GST–CD9 (Fig. 4f).

Inhibition of MGC formation is not dependent on the presence of GST

Although GST alone had no effect on MGC formation, we considered that in a fusion protein it might affect tetraspanin EC2 activity. To investigate this, we cleaved GST–CD63 EC2 and removed the free GST by affinity chromatography. Cleaved CD63 EC2 actually showed a slightly higher activity than intact GST–CD63 EC2 [Fig. 5a; half-maximal (50%) inhibitory concentration (IC50) values of 1·1 and 35 nm, respectively]. To confirm that inhibition was not the result of residual GST, highly purified His6-tagged CD9 EC2 and CD63 EC2 were also tested and both proteins showed significant inhibition of MGC formation, similar to that of GST–CD63 EC2 (Fig. 5b). As GST and all of the GST–tetraspanin EC2 proteins had been produced under identical conditions in E. coli, it was considered unlikely that inhibition of MGC formation might be caused by the presence of contaminating LPS. However, because LPS has been reported to inhibit MGC formation,26 this was investigated. At lipopolysaccharide (LPS) concentrations ranging from 1 ng/ml to 2 μg/ml, no inhibition of Con A-induced MGC formation was observed (data not shown). Indeed, at high concentrations of LPS, an enhancement of Con A-induced giant cell formation was apparent (data not shown).

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Figure 5.  Effects of N-terminal tags on the inhibition of fusion by recombinant tetraspanin second extracellular domains (EC2). Adherent human monocytes were incubated for 72 hr with concanavalin A (Con A) (10 μg/ml) and fusion index was measured in the presence or absence of: (a) increasing concentrations of EC2 GST–CD63 or cleaved EC2 CD63 (generated from the former by cleavage and removal of GST) or (b) His6-tagged EC2 proteins (20 μg/ml). Results are the means of at least three separate experiments ± standard error of the mean (SEM). NS, not significant.

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Effects of tetraspanin EC2 region on cell number, monocyte adhesion and aggregation

For MGC formation to occur, monocytes must be adherent and able to reach a critical density through cell aggregation.27 Inhibition of MGC formation by CD9 or CD63 EC2s might therefore be caused by effects on cell viability, proliferation, adhesion or aggregation. A colorometric test was used to determine total and adherent numbers of monocytes after 72 hr of culture under normal or fusogenic conditions and in the presence or absence of recombinant CD9 and CD63 EC2s. No significant effects on total numbers of monocytes cultured under any of these different conditions was observed (data not shown), indicating that the EC2 proteins do not have cytotoxic or antiproliferative effects. When any non-adherent cells were removed by vigorous washing, again no significant inhibitory effects of the EC2 proteins on adhesion were found. Microscopic observations have also confirmed that these proteins do not inhibit adhesion of monocytes cultured under standard conditions on glass chamber slides (data not shown). The effect of GST–EC2 and His6-tagged tetraspanins on monocyte aggregation induced by a low dose of Con A (5 μg/ml; suboptimal for MGC formation) was also examined, but no inhibition of aggregation was observed with recombinant CD63 or CD9 EC2 proteins (Fig. 6).

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Figure 6.  Effect of recombinant tetraspanin second extracellular domains (EC2) on the aggregation of monocytes. Aggregation of adherent human monocytes was assessed in the presence of a dose of concanavalin A (Con A) (5 μg/ml) that was suboptimal for fusion. The number of aggregates of between 4–10 and 10–20 monocytes was counted from six randomly chosen fields in each slide chamber. The results are the means ± standard deviation (SD) of a single experiment performed in triplicate. GST, glutathione S-transferase.

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Specific binding of CD63 EC2 to Con A-stimulated monocytes

We next postulated that the effect of the recombinant CD9 and CD63 EC2s on MGC formation might be caused by specific interactions with membrane receptors involved in cell fusion. The recombinant proteins were directly labelled with fluorescent dyes, which did not affect their capacity to inhibit MGC formation (data not shown). Preliminary investigations indicated little specific binding of CD63 or CD9 EC2 proteins to unstimulated monocytes. We surmised that the tetraspanins might interact with molecules up-regulated early in the fusion process. Binding of labelled tetraspanin EC2 proteins to monocytes stimulated by 2 hr of treatment with Con A was examined by flow cytometry. As shown in Fig. 7a,b, after 2 hr of stimulation with Con A a population (R2) of large (aggregated/fusing) monocytes was observed. Fluorescence microscopy after staining with anti-CD52 indicated that these included fused monocytes, typically with < 10 nuclei/cell (Fig. 8c). No specific binding of FITC-labelled GST–CD63 or GST–CD9 EC2s to unstimulated monocytes was observed (Figs 7c,e and 8b), or to contaminating lymphocytes (R3) and platelets (R4) present in the preparations (Fig. 7a–f). However, following stimulation with Con A, staining of the R2 population of fusing monocytes with FITC–GST–CD63, and to a lesser extent with FITC–GST–CD9 EC2 was observed (Fig. 7c–f). In both cases, staining was brighter than with the similarly labelled GST protein alone (Fig. 7d,e; Fig. 8a,c). Staining of Con A-stimulated monocytes with the FITC-labelled tetraspanin EC2 proteins was observed at 4° (Fig. 7c,d) and at 37° (Fig. 7e,f), indicating binding to a cell-surface component, rather than just uptake. As some binding of labelled GST alone to Con A-stimulated monocytes was observed, attempts were made to verify the specificity of labelled GST–CD63 EC2 binding in the presence of excess, unlabelled proteins (Fig. 8d). GST–CD63 EC2 staining of Con A-stimulated monocytes was inhibited by unlabelled GST–CD63 EC2, His6-CD63 EC2 and free CD63 EC2. No inhibition of binding was obtained in the presence of excess unlabelled GST or, interestingly, in the presence of GST–CD9 EC2 proteins, suggesting that CD63 EC2 and CD9 EC2 have different binding partners.

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Figure 7.  Binding of recombinant tetraspanin second extracellular domains (EC2) to fusing monocytes. Adherent human monocytes were treated with concanavalin A (Con A) (10 μg/ml) for 2 hr. The degree of fusion was assessed by flow cytometry of dissociated monocytes. The top panels show the scatter profiles of cells incubated with medium alone (a) or with Con A (b). Cell populations were delineated on the basis of granularity and size: R1 and R2 are positive for the monocyte marker, CD14; R3 and R4 correspond to contaminating lymphocytes and platelets (CD52+, CD14), respectively. The lower panels show the binding of fluorescein isothiocyanate (FITC)-labelled glutathione S-transferase (GST)–CD63, GST–CD9 EC2s and GST to cells in each region at either 4° (c,d) or 37° (e,f), following 2 hr of incubation with medium alone (c,e) or with medium containing Con A (d,f).

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Figure 8.  Binding of recombinant tetraspanin second extracellular domains (EC2) to fusing monocytes. Adherent human monocytes were treated with medium alone or with medium containing concanavalin A (Con A) (10 μg/ml) for 2 hr, then labelled with fluorescein isothiocyanate (FITC)-labelled recombinant tetraspanin EC2 or glutathione S-transferase (GST) control for 1 hr at 4°. (a) Fusing monocytes incubated with FITC–GST; (b) control monocytes and (c) fusing monocytes incubated with FITC–GST–CD63 EC2. (d) Fusing monocytes were pre-incubated with the unlabelled EC2 proteins or with GST alone (100 μg/ml) for 15 min at 4° then incubated with Alexafluor 647–GST–CD63 EC2 (6 μg/ml) as above. (e) Dose–response analysis of binding of FITC–GST–CD63 EC2 or FITC–GST. The inset graph shows the dose–response at concentrations up to 10 μg/ml, and the main graph shows the dose–response at concentrations up to 50 μg/ml. Experiments were performed and analysed by fluorescence-activated cell sorting, as described in the Fig. 7 legend. The results are the means ± standard error (SE) of three experiments performed in duplicate. ***P < 0·001, **P < 0·01. Bar, 10 μm. MFI, median fluorescence intensity; RFI, relative fluorescence intensity (relative to negative control).

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To investigate in more detail the nature of the binding of GST–CD63 to fusing monocytes, dose–response titrations were performed (Fig. 8e). As previously described, FITC–GST–CD63 shows binding to Con A-stimulated monocytes, but not to unstimulated monocytes. Interestingly, the binding appears biphasic. At lower doses of GST–CD63–FITC, the binding appears to be high affinity [half-maximal (40%) effective concentration (EC50) = 9 × 10−9 m] and saturates at ∼27 μm (10 μg/ml). However, if higher concentrations of GST–CD63–FITC (up to 1·35 μm) are included in the analysis, a lower-affinity binding (EC50 = 92 μm) that does not saturate is observed. The lower-affinity binding appears specific, in that it is not observed with similarly labelled GST–FITC and may be caused by intercalation of the labelled EC2 protein into TEM on the monocyte membranes.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Tetraspanin proteins are molecular organizers, forming functional networks (TEM) that link membrane proteins with cytoplasmic signalling and cytoskeletal elements. TEM are proposed to participate in complex cellular functions, such as motility, cell–cell contacts and membrane fusion.1–3 However, the presence of multiple tetraspanins in TEM has hindered the assignation of specific roles to individual tetraspanins. In this report we have used recombinant tetraspanin extracellular domains (EC2) to define the function of tetraspanins in a complex biological event – the formation of MGC from monocytes. Previous work using antibodies and knockout mice suggested that the tetraspanins CD9 and CD81 both act as negative regulators of MGC formation, whereas the tetraspanin CD63 appeared uninvolved.7 Here, we confirm that antibodies to CD9 and CD81 affect MGC formation, but our investigations using EC2 domains indicate that these proteins have distinct roles. In addition, we present strong evidence that CD63 is important in this process.

Antibodies that bind to CD9 and CD81 increase the size of MGC; in contrast, anti-CD63 immunoglobulins strongly inhibit MGC formation. However, the soluble recombinant EC2 proteins of both CD9 and CD63 were potent inhibitors of monocyte fusion, whereas CD81 EC2 had no effect, despite our demonstration of its biological activity in other systems.13,15,17 The effects of the EC2 proteins are clearly specific, as no inhibition was observed with recombinant EC2s of CD81 or with murine CD9 EC2, which shows 77% sequence identity to human CD9 EC2. Our results for CD9 are consistent with the observations made by Takeda et al.7 This group also found that both CD9 and CD81 were down-regulated under fusogenic conditions; in addition, alveolar and bone-marrow-derived macrophages from CD9−/− and CD81−/− mice showed enhanced MGC formation, and CD9/CD81 double-knockout mice spontaneously developed MGCs in the lung and showed enhanced osteoclastogenesis in bone. This led to the suggestion that both CD9 and CD81 act as negative regulators of MGC formation. Our data for CD9 is consistent with this, but the lack of a direct effect with CD81 EC2 suggests an indirect role for this tetraspanin (e.g. anti-CD81 antibodies may modulate the activity of CD9 within the context of TEM). There is certainly good evidence that CD9 and CD81 associate in TEM.1 Interestingly, when CD9 and CD81 EC2s were used together, the effect of CD9 EC2 on MGC formation was significantly abrogated. This also suggests that whilst CD81 is not directly involved in monocyte fusion, it can modulate the activity of CD9 in this process.

The inhibitory effects of various recombinant CD63 EC2 proteins and four different anti-CD63 immunoglobulins strongly suggest a positive role for this tetraspanin in MGC formation. Takeda et al.7 noted that CD63 expression was increased under fusogenic conditions but did not observe inhibition of fusion with anti-CD63 immunoglobulin. This lack of an effect may be caused by differences between assay systems. For example, Takeda et al. used the anti-CD63 immunoglobulin under conditions that gave control fusion rates of only 20%. By contrast, in the inhibition of fusion (with GST–CD9 EC2), they achieved fusion rates of ∼60% although the effects of antibodies under these conditions were not examined. It is likely that inhibition of fusion is more clearly observed with higher fusion rates, as in the experiments described here.

Unlike CD9, there have been no reports to date of a role for CD63 in cell–cell fusion. However, antibodies to CD63 specifically inhibit HIV infection of macrophages and it has been suggested that CD63 may be involved in virus–cell fusion.28 We have also previously shown that CD63 EC2 inhibits HIV infection of macrophages, probably by blocking viral uptake but possibly also by interfering with trafficking or fusion within intracellular vesicles.17 The molecule has well-documented roles in membrane trafficking,29 and cell-surface CD63 is rapidly internalized on antibody binding.30 Inhibition of MGC formation by anti-CD63 immunoglobulins might therefore be caused by increased internalization of a required cell-surface component, either CD63 itself or an interacting molecule. It is possible that the recombinant proteins might induce internalization of membrane CD63 and associated molecules in some way, although our previous data suggest that monocyte cell-surface levels of CD63 are not lowered by incubation with recombinant CD63 EC2.17

Whilst our results indicate positive and negative regulatory roles for CD63 and CD9 in MGC formation, respectively, it is not yet clear how the EC2 proteins affect this process at the molecular level. The inhibitory effects of CD9 and CD63 EC2s suggest direct interactions in cis or in trans with molecules on the monocyte surface, and their specific binding to Con A-stimulated monocytes is consistent with this. Dose–response curves show biphasic binding of CD63 EC2 to Con A-stimulated monocytes, with an EC50 for the higher-affinity component in the nanomolar range, similar to that required for inhibition of MGC formation. Therefore, one possibility is that the EC2 proteins are competing with cell-surface tetraspanins for specific binding to a partner molecule directly involved in fusion. There are few reported ligands for tetraspanins. Direct interactions of CD9 with the transmembrane immunoglobulin gene superfamily members EWI-F and EWI-2 have been shown,1 but these proteins have no known role in fusion. CD9 has been reported to associate with CD44,31–33 with CD4734 and with CD98,35 all of which have postulated involvement in MGC formation.27,36,37 However, these molecular interactions have only been demonstrated by co-immunoprecipitation in mild detergents, indicating that they are indirect and probably occur within TEM.1 Similarly, both CD9 and CD63 also associate indirectly with β1 and β2 integrins in various cell types,38 but whilst antibodies to β1 and β2 integrins inhibit MGC formation7,37,39 this is thought to be caused by effects on monocyte adhesion40 and/or aggregation37 rather than by fusion itself. The interaction of tetraspanins with other molecules recently implicated in monocyte fusion, such as the purinergic receptor41 or DC-STAMP,42 has not to our knowledge been investigated.

In attempts to identify potential partners for CD9 and CD63 that are involved in monocyte fusion, we performed ‘pull-downs’ using our GST–EC2 proteins, but to date have not identified proteins that specifically associate with the EC2s on monocytes under fusogenic conditions (data not shown). However, recent work on the binding of our tetraspanin EC2 proteins to endothelial cells suggests a mechanism for their action, involving intercalation into existing TEM (O. Barreiro, M. Zamai, Yanez-Mo, E. Tejera, P. Lopez-Romero, P.N. Monk, E. Gratton, V. Caiola and F. Sanchez-Madrid, submitted). In this case, pull-down experiments may not identify any ‘specific’ binding partners. A second possibility therefore is that the recombinant EC2 proteins act by binding in this way to monocytes and disrupt the architecture of TEM that are crucial to fusion. The low-affinity binding of CD63 EC2–FITC to Con A-simulated monocytes observed at high doses of CD63 EC2–FITC suggests that this may be occurring. It is possible that there may be high- and low-affinity components to intercalation, such that low concentrations of tetraspanin EC2s are sufficient to disrupt TEM function, but further low-affinity interactions with TEM are observed at higher EC2 concentrations. The existence of specific membrane domains involved in mononuclear phagocyte fusion has previously been speculated.27 It is interesting to note that despite the well-established role of CD9 in sperm–egg fusion and the inhibition of this process by CD9 EC2, no specific binding partner for CD9 has yet been identified on oocytes or sperm.43

Soluble forms of other membrane proteins thought to be directly involved in cell–cell fusion (e.g. CD44, CD47, CD200) also inhibit mononuclear phagocyte fusion44,45 at similar concentrations to the EC2 proteins reported here. CD47 interacts with CD20045 [also known as the macrophage fusion receptor (or MFR)] but a cell-surface ligand for CD44 has not been reported.

In summary, we have used recombinant EC2 domains to dissect the role of tetraspanins in MGC formation. We have shown that the EC2s of CD9 and CD63 can specifically inhibit this process, either by interacting directly with as-yet-unidentified partner molecules or by disrupting components of TEM, whose correct configurations are required for fusion. MGC form in vivo in response to chronic inflammation11 or the presence of foreign bodies, such as surgical implants, where they may contribute to their degradation.8 It will be of interest to determine if the EC2 domains also affect the development of multinucleated osteoclasts from monocyte precursors. Agents that inhibit the formation of MGC, derived from CD9 or CD63 EC2 proteins, might therefore have various therapeutic applications in the treatment of chronic inflammatory disorders.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

V. P. and F. M. would like to acknowledge financial support from the Ford Foundation International Fellowship Program and the Humane Research Trust, respectively.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Fig. S1  Tetraspanin recombinant protein analyses using Western blotting. Fifteen microgram of the tetraspanin proteins were run on 12.5% SDS-PAGE gels with (a) or without (b) b-mercaptoethanol prior to blotting. Membranes were blocked in 5% w/v milk powder in PBS-0.01% Tween 20. In a, blots were probed with anti-GST-HRP (Sigma, UK) diluted1/3000 in blocking buffer. In b, blots were probed with specific mAbs: Alma-1 (hCD9), H5C6 (CD63), 1.3.3.2.2 (CD81) (Santa Cruz, US) each at 1/1000 dilution in blocking buffer followed by 1/3000 dilution of anti-mouse IgG-HRP (Sigma, UK) diluted in blocking buffer.

Fig. S2  Assessment of quality of recombinant EC2 proteins by immunoprecipitation and Western blotting. Recombinant proteins (6 mg/ml) were incubated with 150 mg/ml isotype control (a) or conformation-sensitive anti-tetraspanin antibody (b) (Alma-1 for CD9: 1.3.3.22 for CD81) for 1 hr followed by protein-G-Sepharose for 1 hr on ice. The supernates were run on non-reducing gels and subjected to Western blotting with (A) the relevant anti-tetraspanin antibody followed by anti-mouse-HRP or (B) anti-GST-HRP antibody (Sigma) as described in Fig. 1.1 above. There is a considerable reduction in the intensity of the upper bands (GST-CD9-EC2 and GST-CD81-EC2) in the anti-GST blot following immunoprecipitation using the conformation sensitive antibodies (B). In the case of CD81, not all of correctly folded protein has been removed by immunoprecipitation (A).

Fig. S3  Assessment of quality of recombinant EC2 proteins by ELISA. Following immunoprecipitation of GST-EC2 proteins as described above, supernates were also titrated in ELISA. After coating overnight, ELISA plates (Nunc) were probed with anti-GST-HRP or the relevant anti-tetraspanin antibody (Alma-1 for CD9, 1.3.3.22 for CD81) followed by anti-mouse IgG-HRP. Plates were developed using TMB substrate (Sigma) and read at 450 nm. A reduction in GST-reactivity is observed following immunoprecipitation with the relevant anti-tetraspanin antibody for both GST-EC2-CD81 and GST-EC2-CD9.

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