1 INSERM U448, Faculty of Medicine, 8 rue du Général Sarrail, 94010 Créteil Cedex, France
2 Department of Neuroscience, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA
3 Institute for Cancer Research and Treatment, University of Torino School of Medicine, 10060 Candiolo, Torino, Italy
4 INSERM U433, Faculty of Medicine R. Laennec, rue Guillaume Paradin, 69372 Lyon Cedex 08, France
Correspondence to: L. Boumsell; E-mail: boumsell{at}im3.inserm.fr
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Abstract |
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Keywords: cell migration, semaphorin receptors
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Introduction |
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A role of sCD100 in the processes regulating human cell migration had also been established. We demonstrated that sCD100 inhibits the migration of cells from the monocytic and B-cell lineages (7). However, the identity of the receptors mediating this inhibitory effect remained to be elucidated. In the neural system, cell-surface receptors for repulsive semaphorins have been identified as members of two transmembrane receptor families, namely the neuropilins and the plexins (810). However, ß1-integrins have recently been identified as receptors for class 7 (SEMA7A/CD108) protective semaphorins (11). For class 3 secreted semaphorins, full signaling required the formation of plexinneuropilin 1/2 complexes, where neuropilin 1/2 acts as a binding subunit, and plexin plays the role of the downstream transducing molecule (12). Furthermore, direct binding of semaphorins to plexins has been demonstrated. Thus, plexin B1 can directly bind CD100/SEMA4D (13), while plexin C1 (VESPR/CD232) was found to interact with SEMA7A/CD108 on monocytes (14). More recently, other types of molecules have been identified as immune receptors for murine semaphorins. Thus, the C-type lectin transmembrane protein CD72 was identified as a low-affinity receptor for CD100/SEMA4D at the surface of murine B cells (15). This observation was recently extended to human tonsillar B cells, on which sCD100 binding was found to require functional CD72 (16). Finally, a member of the family of T-cell Ig domain and mucine domain, Tim-2, was described as being a receptor for SEMA4A (17).
In this report, we demonstrate that sCD100 strongly impairs the migration of both human monocytes and derived immature DCs. We show that while plexin C1 does not bind sCD100, it is strongly involved in sCD100-mediated inhibitory effect on monocyte migration. We further show that the differentiation of monocytes to immature DCs results in a switch in sCD100 receptor requirement. Indeed, while expression of plexin C1 is down-modulated on in vitro-derived DCs, expression of plexin B1 is induced along the differentiation process. Binding of sCD100 to plexin B1 then accounts for the inhibition of immature DC migration. Finally, exposure of monocytes or immature DCs to sCD100 resulted in a significant down-modulation of pro-inflammatory cytokine production.
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Methods |
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DCs were obtained by differentiation of freshly isolated human monocytes. Monocytes were grown at a concentration of 106 cells ml1 in RPMI 1640 medium supplemented with 2 mM glutamine, 1 mM penicillinstreptomycin and 10% heat-inactivated human serum, in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) (50 ng ml1; PeproTech, Rocky Hill, NJ, USA) and IL-4 (50 ng ml1; PeproTech). Every 2 days, fresh GM-CSF and IL-4 were added. Immature DCs were obtained after 7 days of differentiation under these culture conditions. At day 7 and every 2 days, tumor necrosis factor- (TNF-
, 10 ng ml1; PeproTech) was added to the culture for four additional days to allow DCs full maturation.
CD100 over-expressing Jurkat cells were generated by transfection of cells with a cDNA construct coding for the transmembrane form of CD100 molecule. Stable transfectants were selected by addition of the appropriate antibiotic (2 mg ml1 geneticin; Invitrogen, Cergy Pontoise, France) to RPMI 1640 medium containing 10% FCS, 2 mM glutamine and 1 mM penicillinstreptomycin. sCD100-enriched culture supernatant was obtained by incubation of 2 x 107 cells in 1 ml of medium without FCS overnight at 37°C. sCD100 purification was conducted using an anti-CD100 (BB18) immunoadsorbant, followed by an acidic release with 2 M HClglycine, pH 2.8, and neutralization by 1 M K2HPO4. The eluate was then concentrated and dialyzed against PBS, and CD100 concentration was estimated by silver staining of the gel. A similar procedure was conducted using an anti-CD8 mAb to generate an eluate referred to as control.
Antibodies
The following antibodies were used in flow cytometry: PEanti-CD14 (10 µl per test; Beckman Coulter, Villepinte, France), anti-CD1a (L544, IgG1, ascites dilution: 1/1000), anti-CD72 (J3.109, IgG1, 1 µg per test; Beckman Coulter), anti-CDw136 (IDII-C2, IgG2a, 1 µg per test) (kindly provided by A. von Agthoven, Beckman Coulter, Marseille, France), anti-plexin C1 (M460, IgG1, kindly provided by Amgen, Seattle, WA, USA; 1 µg per test), polyclonal goat anti-plexin B1 (N-18, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1 µg per test) and rabbit anti-neuropilin 1 (1 µg per test) (18). The corresponding mouse, rabbit or goat Igs were used as negative controls. Cell-surface immunostaining was performed according to standard procedure. Briefly, cells (2 x 105) were washed in PBS and incubated for 15 min with the primary antibody diluted in PBS. After washes, cells were incubated with the appropriate FITC-conjugated secondary antibody for 15 min and analyzed using an XL flow cytometer (Coulter, Miami, FL, USA).
Polyclonal anti-plexin C1 antibodies were obtained by immunization of rabbits with specific human plexin C1 peptides corresponding to amino acids 7287 and 333347 (Eurogentec, Seraing, Belgium). Immune sera were affinity purified and used for immunoblotting at 2 µg ml1. Polyclonal rabbit anti-plexin B1 antibodies (IC2) were generated and used as previously reported (19).
Immunoprecipitation and immunoblotting
Monocytes or immature DCs (day 7) were lyzed in lysis buffer (1% Nonidet P-40, 150 mM NaCl, 20 mM TrisHCl, pH 7.5, 10 mM NaF, 1 mM phenylmethylsulfonylfluoride, 1 mM sodium vanadate, 1 µg ml1 aprotinin and 1 µg ml1 leupeptin) for 1 h at 4°C. Immunoprecipitations were performed using anti-plexin C1 mAb (M460) or anti-plexin B1 antibodies (IC2). After washes, immune complexes were separated by SDSP and transferred onto a nitrocellulose membrane. Western blots were conducted with anti-plexin B1 (IC2) or anti-plexin C1 antibodies followed by anti-rabbit HRP-conjugated secondary antibodies, and developed with an enhanced chemiluminescence detection system according to the manufacturer's procedure (Amersham Biosciences, Orsay, France).
cDNA constructs, RNA isolation, reverse transcription and PCR
Plexin C1 construct was purchased from the Origene clone collection (Clinisciences SA, Montrouge, France). Carboxy-terminal Flag-tagged cDNA of human CD72 was generated by PCR amplification of the full-length CD72-coding region with the forward 5'-ATGGCTGAGGCCATCACCTATGCAG-3' and reverse 5'-TTACTTGTCATCGTCGTCCTTGTAGTCGGAAGGAGGGGCACAGGTTCTTGTTG-3' specific primers. The resulting product was ligated into the pcDNA3.1/V5-His vector according to the manufacturer's procedure (Invitrogen). Plexin B1-coding construct has been described elsewhere (19).
Total RNAs were isolated from monocytes and DCs (day 7) and subjected to reverse transcription using the Power Script reverse transcriptase kit from Clontech (BD Biosciences, Palo Alto, CA, USA). PCR amplification was then performed using the following specific primers: forward 5'-AAGAACCCCAAGCTGATGCTGC-3' and reverse 5'-CCTTCACGGGCACGCCCTGGGC-3' for plexin B1 and forward 5'-CCACATCGTATTTTCTGATTGTGCTCC-3' and reverse 5'-GCTTACATCCACTTGCATTTCTTCTTTTC-3' for plexin C1. Amplified products were resolved by electrophoresis on a 1% agarose gel.
Migration assays and semaphorin-binding assays
Assays were performed using a Transwell system (Costar, Cambridge, MA, USA) comprising two chambers separated by a 5-µm filter insert. Cells (2 x 105), re-suspended in a final volume of 100 µl, were added to the upper chamber together with the indicated purified semaphorin. In some experiments, monocyte chemoattractant protein-3 (Tebu-Bio, Le Perray en Yvelines, France) was added to the lower well at 50 ng ml1. For competition assays, purified recombinant viral semaphorin A39R (1 µg per well, a gift from M. Spriggs, Amgen), SEMA7A (50 ng ml1) or receptor-specific blocking antibody (5 µg per well) was added to the cells. Alternatively, cells were pre-incubated for 1 h at 37°C with sCD100, recombinant A39R, SEMA7A or blocking antibodies before migration. Migration assays were performed in duplicate. After 6 h of incubation at 37°C, cells in suspension in the upper and lower chambers were enumerated.
For semaphorin-binding assays, a soluble form of CD100 or SEMA7A fused to placental secreted alkaline phosphatase (CD100AP and SEMA7AAP) was prepared from transiently transfected COS cells, as described (11, 13). Binding assays were performed on COS cells transiently transfected with plexin B1, C1 and/or CD72 according to standard procedures (8, 13).
Cytokine measurements by ELISA
To measure IL-6, IL-8, IL-10 and TNF- produced by monocytes and DCs, cells (103) were incubated for 18 h with a control eluate or with sCD100 in round-bottomed 96-well microtiter plates. Where indicated, cells were pre-incubated for 1 h at 37°C with anti-plexin B1 (N-18) or C1 (M460) antibody prior to contact with sCD100. Following incubation, cytokine levels in culture supernatants were determined from duplicates using ELISA kits (Diaclone, Besançon, France).
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Results |
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Switch from plexin C1 to plexin B1 expression during the differentiation process of monocytes to immature DCs
Although we demonstrated that sCD100 down-modulates the migration of monocytes and immature DCs, the molecular ligandreceptor interactions mediating this inhibition process remained to be elucidated. Expression cloning identified the C-type lectin transmembrane protein CD72 as a low-affinity receptor for sCD100 in the immune system (15). Binding of sCD100 through CD72 was further demonstrated on CHO-transfected cells and tonsillar B cells (16). Previously, the use of COS cell transfectants expressing various members of the human plexin family revealed a high-affinity binding of sCD100 to plexin B1, and of CD108/SEMA7A to plexin C1 (13). Finally, plexin C1 was initially shown to bind the soluble viral semaphorins SEMAVA (A39R) and SEMAVB (AHV) on monocytes, leading to the induction of cytokine production (14). However, the molecular basis of an interaction between sCD100 and these receptors had not yet been investigated on human monocytes and DCs.
To investigate this issue, cell-surface expression of the above receptors was analyzed on monocytes and along their differentiation into DCs (Fig. 2A). Immunofluorescence labeling revealed the presence of plexin C1 and CD72 at the surface of freshly isolated monocytes (Fig. 2A). In contrast, neither plexin B1 nor neuropilin 1 was detected. Membrane labeling of CDw136 (also known as Ron or macrophage-stimulating protein receptor), a protein tyrosine kinase receptor reported as being involved in the induction of cell migration processes (20), was also investigated. CDw136 was present on monocytes, and was used as a control in the following antibody-binding experiments (see below). Notably, while purified human monocytes expressed plexin C1, its expression was decreased during DC differentiation in vitro (Fig. 2A). A similar down-modulation of CD72 and CDw136 surface expression was observed during the differentiation process. In contrast, we observed that neuropilin 1 expression was up-regulated during the differentiation into DCs, as previously reported (21). Remarkably, plexin B1 also became detectable at the surface of immature DCs, reaching maximum expression level at day 7 of differentiation (Fig. 2A). Finally, plexin B1 surface expression was completely lost following the induction of DC full maturation by TNF-.
Because our immunostaining results were in part contradictory with previous studies reporting the absence of plexin B1 mRNA in monocytesderived DCs (16, 22), plexin B1 and C1 mRNAs analysis was conducted on monocytes and derived immature DCs (day 7) obtained from three independent donors. As shown in Fig. 2(B), plexin B1 and C1 mRNAs were detected in both monocytes and derived DCs, independent of the donor. These first observations prompted us to further analyze the expression of the proteins according to the cell type, by western blot analysis following immunoprecipitation with specific anti-plexin antibodies (Fig. 2C). In agreement with our cell-surface immunostaining data, we observed the presence of plexin C1 in monocytes. In contrast, it was no longer detected in immature DCs. Conversely, plexin B1 was not immunoprecipitated from monocytes, but was observed in immature DCs under a full-length and a proteolytically cleaved form (Fig. 2C), as previously reported (19). Thus, using different analysis procedures, we constantly observed the expression of plexin B1 in immature DCs after 7 days of in vitro differentiation. These data supported the view that a switch from plexin C1 to plexin B1 expression occurs during the differentiation process of monocytes into immature DCs.
sCD100 inhibitory effect on monocyte migration requires functional plexin C1
Because monocytes expressed plexin C1 and CD72, the involvement of these potential sCD100 receptors in the generation of signals leading to the inhibition of cell motion was evaluated. Migration assays were performed using previously described function-blocking antibodies specific for these semaphorin receptors (14, 15, 23). The data presented in Fig. 3(A) demonstrated that monocytes pre-incubated with the anti-plexin C1 mAb, and subsequently allowed to migrate in the presence of sCD100, did not respond anymore to the inhibitory effect of sCD100. Conversely, cells pre-incubated with sCD100 and then placed in contact with the blocking anti-plexin C1 mAb still exhibited a decreased level of migration (Fig. 3A). In contrast, pre-incubation of the cells with the anti-CD72 or anti-CDw136 blocking mAb did not interfere with sCD100 inhibitory function on cell migration (data not shown). To further confirm that the inhibitory function of sCD100 on monocyte migration required functional plexin C1, similar competition assays using two different plexin C1 ligands, namely A39R and SEMA7A, were conducted. While A39R did not affect the ability of monocytes to spontaneously migrate (Fig. 3B), incubation of the cells with SEMA7A led to an increased level of migration (Fig. 3C), as previously reported (24). We additionally observed that the inhibitory effect of sCD100 on cell migration was abolished in the presence of recombinant A39R or SEMA7A (Fig. 3B and C). Indeed, cells pre-incubated with A39R or SEMA7A before migration in the presence of sCD100 showed spontaneous migration levels identical to the one obtained under control conditions.
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Discussion |
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In contrast, our results favor the involvement of plexin C1 in the generation of the negative signals, leading to a significant diminution of monocyte movement. As reported elsewhere, we did not observe any direct interaction of sCD100 with plexin C1 on transfected cells (Fig. 4; 13). However, we established that a plexin C1 function-blocking antibody prevented sCD100-induced inhibition of monocyte migration. Similarly, engagement of plexin C1 by one of its identified ligand (A39R or SEMA7A) resulted in sCD100 loss of function (Fig. 3). Thus, sCD100 signaling on monocytes apparently requires non-engaged and functionally available plexin C1 at their cell surface.
The involvement of plexin C1 in mediating sCD100-induced inhibition on monocytes favors the hypothesis of the existence of a multimeric receptor complex relaying sCD100 functions. We excluded the possibility of a direct cooperation between plexin C1 and CD72 for sCD100 binding since co-transfection of both molecules did not improve the low-affinity interaction of CD72 towards CD100 (Fig. 4). Other potential candidates might be integrins, which are known to associate to a large number of distinct proteins (25). It has been recently shown that, despite the ability of SEMA7A to interact with plexin C1, SEMA7A-mediated axon growth did not require plexin C1 but is dependent on ß1-integrins (11). Similarly, ß1-integrins may represent necessary co-receptors associating with yet unidentified sCD100-binding proteins on monocytes. In agreement with this hypothesis, it is important to mention that we did not detect any expression of ß1-integrins on COS cells (data not shown). This might explain the absence of sCD100 interaction on plexin C1 transient transfectants. The possibility of an association of plexin C1 with integrins is currently under investigation.
In contrast, the conclusion regarding sCD100 receptor appears clear on immature DCs which express plexin B1. Indeed, sCD100 was shown to directly interact with plexin B1 (Fig. 4; 13), and sCD100-mediated effects on immature DCs were abolished when plexin B1 blocking antibodies were added to the cells (Fig. 5, Table 1). Thus, plexin B1 appears to be sufficient by itself to drive sCD100-related inhibition on DCs.
We demonstrated that, besides its influence on the migration of monocytes and DCs, sCD100 also markedly affects their cytokine production. A significant increase in IL-10 secretion was observed while, on the other hand, the level of the pro-inflammatory cytokines IL-6, IL-8 and TNF- was significantly reduced upon exposure of the cells to sCD100. These results were contradictory to the one reported by Ishida et al. (16), who showed an increased production of IL-6, IL-8 and TNF-
following the treatment of monocytes with a CD100Fc soluble molecule. However, this discrepancy might derive from the use of distinct experimental procedures. Indeed, in their study, higher doses of sCD100 (420 µg ml1) than the one we used (5080 ng ml1) were added to the cells. This ligand excess might lead to the recruitment of additional signaling pathways and consequently overcome more tightly regulated cellular responses. Another explanation for the differences observed might come from the mode of cell purification. Our purification process of monocytes was based on a negative selection of the cells (all peripheral blood cells excepted monocytes are retained on the column), and not on the depletion of the T- and B-cell populations, which resulted in an enrichment of both monocytes and NK cells. In this latter case, one can then consider that the presence of NK cells together with monocytes, and along the differentiation process into DCs, might interfere with the cytokine production of the cells. In this regard, it has been reported that the co-culture of NK cells with DCs resulted in an increased proliferation of the NK population, and in the enhancement of its cytolytic activity (26). An increased secretion of IL-12 by the DCs, and of IFN-
by NK cells, was also evidenced which may promote both NK cells and DCs activation through the cytokine network involving IL-12 and IFN-
. Other factors such as the number of cells used in each assay (105 versus 103), the length of incubation time (48 versus 18 h) or the origin of sCD100 (sCD100Fc fusion protein versus a constitutively released soluble protein) might also represent non-negligible sources of variation. However, further analyses will be required to clarify this point.
Finally, our data suggested that sCD100 has a distinct role in cytokine production and migration of monocytes, when compared with A39R or SEMA7A. Recombinant soluble SEMA7A was described as a stimulator of cytokine production and chemotaxis for monocytes (24). However, the role of the membrane-anchored form of SEMA7A on T cells, and the possibility of its release in the extracellular environment, remains to be determined. We established that sCD100, that can be proteolytically cleaved as a result of T-cell activation, exerted a strong inhibitory biological activity on these monocyte functions. This suggests that, depending on the ligand, the engagement of plexin C1 alone or as part of a receptor complex might result in the generation of opposite signaling pathways, most likely through the recruitment of distinct transducing pathways. It is therefore interesting to mention that A39R, while enhancing cytokine release (14), had no detectable effect on monocyte migration (Fig. 3B). This might reflect the potential of the viral semaphorin to only partially mimic the functional activity of its physiological counterparts through binding to their common (co-)receptor. This represents an attractive model of differential cellular response towards closely related ligands, which although involving a common receptor (plexin C1), will see their functional specificity driven by the need or absence of associated co-receptors.
Altogether this work demonstrated that sCD100 might be critically involved in the cellular events leading to the generation of an immune response. Indeed, the release of sCD100 by T cells upon activation might represent an important step in the processes regulating the correct location of antigen-presenting cells with regard to T cells. In addition, sCD100-mediated cell paralysis might be a key event in keeping a prolonged cellcell contact, thus allowing the generation of efficient cellular responses. Numerous studies suggested that semaphorins and their receptors might play a role in both the neural and immune systems (see 23, 27, for review). We demonstrated that sCD100 exerts its functional activity in the immune system through plexin receptors initially identified in the nervous system. However, sCD100 apparently fulfills distinct functions in each system. Thus, while interfering with cell migration and enhancing anti-inflammatory cytokine secretion on immune cells, sCD100 was shown to induce the collapse of oligodendrocyte process extensions and to trigger neural cell apoptosis, most likely through plexin B1 (28). In this context, the maintenance of sCD100 in the immune compartment would represent an important factor in keeping the functional integrity of the nervous system.
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Acknowledgements |
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Abbreviations |
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AP | alkaline phosphatase |
DC | dendritic cell |
GM-CSF | granulocyte macrophage colony-stimulating factor |
KO | knockout |
sCD100 | soluble CD100 |
TNF | tumor necrosis factor |
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Notes |
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Transmitting editor: T. Tedder
Received 13 September 2004, accepted 19 January 2005.
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References |
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