Soluble CD100 functions on human monocytes and immature dendritic cells require plexin C1 and plexin B1, respectively

Isabelle Chabbert-de Ponnat1,*, Anne Marie-Cardine1,*, R. Jeroen Pasterkamp2, Valérie Schiavon1, Luca Tamagnone3, Nicole Thomasset4, Armand Bensussan1 and Laurence Boumsell1

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


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
CD100 represents the first semaphorin described in the immune system. It is expressed as a 300-kDa homodimer at the surface of most hematopoietic cells, but is also found in a soluble form following a proteolytic cleavage upon cell activation. We herein established that soluble CD100 (sCD100) impaired the migration of human monocytes and immature dendritic cells (DCs), but not of mature DCs. Performing competition assays, we identified plexin C1 (VESPR/CD232) as being involved in sCD100-mediated effects on human monocytes. Interestingly, we observed a complete down-regulation of plexin C1 expression during the in vitro differentiation process of monocytes to immature DCs, while concomitantly the surface expression of plexin B1 was induced. The latter receptor then binds sCD100 on immature DCs, mediating its inhibitory effect on cell migration. Finally, we showed that sCD100 modulated the cytokine production from monocytes and immature DCs. Together these results suggest that sCD100 plays a critical role in the regulation of antigen-presenting cell migration and functions via a tightly regulated process of receptor expression.

Keywords: cell migration, semaphorin receptors


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Semaphorins are a large family of secreted and transmembrane signaling proteins that were initially identified through their ability to regulate axonal guidance in the developing nervous system (1). CD100/SEMA4D represents the first semaphorin characterized within the immune system (2). Cloning of CD100 cDNA has revealed in its extracellular portion the presence of both a semaphorin and an Ig-like domain (3). CD100 is a leukocyte cell-surface molecule expressed as a disulfide-linked homodimer, but is also found as a soluble molecule, released from the membrane-associated form through a metalloprotease-dependent proteolytic process (4). The generation of CD100 transgenic or knockout (KO) mice demonstrated a role for soluble CD100 (sCD100) in the establishment of a humoral immune response. Thus, B cells from CD100-deficient mice showed reduced in vitro proliferative response and Ig production (5). Additionally, CD100–KO mice display severe defects in the antigen priming of T cells (5). More recently, it has been demonstrated that sCD100 enhances the maturation of dendritic cells (DCs), which is an essential step for the activation and differentiation of antigen-specific T cells (6). Accordingly, CD100–KO mice fail to develop experimental autoimmune encephalomyelitis.

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 plexin–neuropilin 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.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cells
PBMCs were obtained from healthy volunteers, and monocytes were purified using a monocyte isolation kit (Miltenyi Biotech, Bergish Gladbach, Germany). Finally, a member of the family of T-cell density gradient separation (Lymphoprep, Nycomed, Oslo, Norway) were labeled with a mixture of hapten-conjugated mAbs directed against T, B and NK cells (namely anti-CD3,-CD7,-CD16,-CD19,-CD56,-CD123 and glycophorin A mAbs). After washes, cells were incubated with anti-hapten microbeads and separated on a magnetic ab cell sorting (MACS) column. Monocytes were collected from the effluent, and purity, evaluated by CD14 surface staining, was >95%.

DCs were obtained by differentiation of freshly isolated human monocytes. Monocytes were grown at a concentration of 106 cells ml–1 in RPMI 1640 medium supplemented with 2 mM glutamine, 1 mM penicillin–streptomycin and 10% heat-inactivated human serum, in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) (50 ng ml–1; PeproTech, Rocky Hill, NJ, USA) and IL-4 (50 ng ml–1; 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-{alpha} (TNF-{alpha}, 10 ng ml–1; 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 ml–1 geneticin; Invitrogen, Cergy Pontoise, France) to RPMI 1640 medium containing 10% FCS, 2 mM glutamine and 1 mM penicillin–streptomycin. 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 HCl–glycine, 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: PE–anti-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 72–87 and 333–347 (Eurogentec, Seraing, Belgium). Immune sera were affinity purified and used for immunoblotting at 2 µg ml–1. 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 Tris–HCl, pH 7.5, 10 mM NaF, 1 mM phenylmethylsulfonylfluoride, 1 mM sodium vanadate, 1 µg ml–1 aprotinin and 1 µg ml–1 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 ml–1. For competition assays, purified recombinant viral semaphorin A39R (1 µg per well, a gift from M. Spriggs, Amgen), SEMA7A (50 ng ml–1) 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 (CD100–AP and SEMA7A–AP) 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-{alpha} 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).


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
sCD100 inhibits the migration of human monocytes and derived immature DCs
We previously established that sCD100 inhibited the migration of cells from the monocytic and B-cell lineages (7). sCD100, produced by a spontaneous proteolytic cleavage of the membrane-associated homodimer (4), was purified by immunoaffinity from culture supernatant of an over-expressing CD100 Jurkat stable transfectant. The effect of sCD100 on the migration of freshly isolated PBMCs was then tested. The results from a representative dose–response experiment are shown in Fig. 1(A) and demonstrate the negative effect of sCD100 on monocyte migration. Maximal inhibition was observed at a concentration of 80 ng ml–1, a plateau being obtained thereafter.



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Fig. 1. sCD100 inhibits the spontaneous and chemokine-induced migration of human monocytes and derived immature DCs. (A) The spontaneous migration of freshly isolated human monocytes was evaluated using a Transwell migration assay. Cells were exposed for 6 h to various concentrations of purified sCD100. After migration, cells present in the upper and lower chambers were counted. Results are presented as the percentage of inhibition of migration (means of duplicates). (B) Migration of monocytes and in vitro-derived DCs was performed as in (A) in the presence of sCD100 (50 ng ml–1). Results are presented as the percentage of migrating cells (means of duplicates) for one representative experiment. (C) To evaluate the effect of sCD100 on the chemokine-induced migration of monocytes and immature DCs, monocyte chemoattractant protein-3 (MCP-3, 50 ng ml–1) was added to the lower chamber, where indicated.

 
Alternatively, monocytes were further induced to differentiate into DCs by incubation for 3 or 7 days in the presence of IL-4 and GM-CSF. As previously described, CD14 expression was down-regulated whereas CD1a expression was up-regulated during in vitro differentiation (Fig. 2A). Migration assays demonstrated that at sub-optimal inhibiting doses (50 ng ml–1), sCD100 also exerted a negative effect on the spontaneous migration of in vitro-derived DCs (Fig. 1B). Thus, DCs obtained after 3 or 7 days of differentiation showed a decrease of 35 and 28%, respectively, in their spontaneous migration ability. In contrast, DCs forced to fully mature in the presence of TNF-{alpha} presented a low ability to spontaneously migrate, which was not affected by sCD100 (Fig. 1B).



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Fig. 2. (A) Expression of semaphorin receptors during the in vitro differentiation of monocytes to DCs. Immature DCs were obtained after culture of purified monocytes for 3 or 7 days in the presence of GM-CSF and IL-4. Maturation of DCs was induced by incubation of the cells with TNF-{alpha} for 4 additional days. At the indicated time points, the cells were analyzed by flow cytometry for the expression of the monocyte marker CD14, the DC marker CD1a, the semaphorin receptors CD72, plexin C1 and plexin B1, the protein tyrosine kinase receptor CDw136 and neuropilin 1. One representative experiment, out of six, is shown. ND: not determined. (B) Expression of plexin B1 and C1 mRNAs by monocytes and derived DCs. Reverse transcription–PCR was performed on total RNA isolated from monocytes (M) and derived immature DCs (day 7) from three healthy donors (13). Reverse transcribed RNAs from the IL-2-dependent T-cell clone JAR or the melanoma cell line A375 were used as negative controls for the amplification of plexin B1 and C1 products, respectively. Positive controls were obtained by using the cDNA from PBMCs as template. The expected plexin B1 and C1 PCR products migrated at 200 bp and 1.8 kb, respectively. Amplification of the same reverse transcribed products with ß-actin primers was used as a control. (C) Detection of plexin B1 and C1 protein expression on monocytes or immature DCs. Immunoprecipitation was performed on total cell lysates with anti-plexin C1 mAb (M460) or anti-plexin B1 polyclonal antibodies (IC2). Immune complexes were resolved by SDSP and western blot analysis was conducted using anti-plexin B1 (IC2) or affinity-purified polyclonal anti-plexin C1 antibodies.

 
Experiments performed on cells incubated in the presence of a chemoattractant demonstrated that sCD100 also strongly impaired the chemokine-induced migration of both monocytes and immature DCs (Fig. 1C).

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 ligand–receptor 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-{alpha}.

Because our immunostaining results were in part contradictory with previous studies reporting the absence of plexin B1 mRNA in monocytes–derived 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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Fig. 3. CD100 inhibitory effect on monocyte migration involved plexin C1. (A) Monocyte migration assays were performed in duplicates as described in Fig. 1(A). For competition assays, cells were simultaneously exposed to sCD100 (50 ng ml–1) and blocking anti-plexin C1 mAb (M460, 1 µg per well), or alternatively pre-incubated with the blocking anti-plexin C1 mAb (1 µg per well) prior to migration with sCD100. Reverse experiment (pre-incubation of cells with sCD100 followed by migration in the presence of anti-plexin C1 mAb) was also done. Similar competition assays as in (A) were conducted using a purified recombinant A39R protein (1 µg per well, B), or SEMA7A (50 ng ml–1, C), instead of blocking anti-plexin C1 mAb. Cells were enumerated in the upper and lower chambers and results expressed as the percentage of migrating cells. Shown is one representative experiment out of five.

 
By performing semaphorin-binding assays on plexin C1-transfected COS cells, we confirmed previous results (13) indicating no direct interaction of sCD100 with plexin C1, even at high ligand concentration (110 nM, Fig. 4D). In contrast, binding of SEMA7A to plexin C1 was detected at a ligand concentration of 45 nM (Fig. 4E). Additionally, sCD100 interaction with CD72 was detected when elevated semaphorin concentration was used (110 nM, Fig. 4F), thus reflecting CD72 low affinity for the semaphorin. The possibility of an association between CD72 and plexin C1, leading to the generation of a higher affinity receptor complex, was finally tested. Co-transfection of both receptors resulted in a level of sCD100 binding identical to the one observed with CD72 alone (Fig. 4G). Taken together these results indicate that even if plexin C1 molecules do not directly bind sCD100, they must be available and not engaged at the cell surface to allow sCD100 negative signals delivery, leading to the inhibition of monocyte migration.



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Fig. 4. sCD100 binding to CD72, plexin B1 and plexin C1. Binding assays were performed to assess the interaction of sCD100–AP on COS cells transiently expressing plexin B1 (A–C), plexin C1 (D), CD72 (F) or plexin C1 and CD72 (G). Similarly, the interaction of SEMA7A–AP with plexin C1 (E) or CD72 (H) transient transfectants was tested. The concentration of semaphorin used is indicated on each micrograph.

 
Plexin B1 mediates sCD100 inhibitory effect on immature DC migration
Our observation that the loss of plexin C1 paralleled the appearance of plexin B1 at the surface of immature DCs suggested that a switch in CD100 receptor requirement occurred along the differentiation of monocytes to DCs. As mentioned previously, a direct high-affinity interaction between sCD100 and the semaphorin receptor plexin B1 has been described (13, Fig. 4A–C). We therefore tested the hypothesis of sCD100 binding to plexin B1 as inducing the observed inhibition of immature DCs migration. Subsequent experiments were performed on DCs obtained after 7 days of differentiation, since these cells no longer expressed plexin C1, CD72 or CDw136 but exhibited substantial levels of plexin B1 and neuropilin 1 at their cell surface (Fig. 2A). We first established, by performing sCD100-binding assays on plexin B1-transfected COS cells, that the commercially available anti-plexin B1 antibodies strongly impaired the interaction of the semaphorin with its receptor (data not shown). Immature DCs (day 7) were then incubated with the anti-plexin B1 antibodies and their migration in the presence of sCD100 subsequently analyzed (Fig. 5). As reported previously, a 30% reduction of cell migration was observed in the presence of sCD100. However, when cells were pre-incubated with the anti-plexin B1 antibodies, no more activity of sCD100 was observed. Note that the use of an anti-CD72 or neuropilin 1 function-blocking antibody, as control, did not interfere with sCD100 negative effect on DC migration (data not shown). Therefore, this identifies plexin B1 as the receptor mediating sCD100 negative effect on immature DC migratory ability.



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Fig. 5. Ligation of sCD100 to plexin B1 is responsible for the inhibition of immature DCs migration. Immature DCs were assayed for migration after 7 days of differentiation. Cells were left un-treated or pre-incubated with anti-plexin B1 blocking antibody (N-18) before migration. Migration assays were conducted in the presence of sCD100 as indicated in the figure. The percentage of migrating cells was evaluated as described in Fig. 3. The data presented are representative of four independent experiments.

 
Modulation of cytokine production by sCD100
Monocytes are cells known to secrete several pro-inflammatory cytokines in response to activating stimuli. It has been demonstrated that treatment of monocytes with the viral semaphorin A39R enhanced IL-6 and IL-8 production (14). More recently, a soluble version of the glycosyl-phosphatidylinositol-linked semaphorin SEMA7A/CD108 has been identified as a potent monocyte stimulator according to its ability to up-regulate pro-inflammatory cytokine production (24). Although the generation of transfected cells allowed the identification of plexin C1 as a low-affinity binding receptor for SEMA7A (13), a direct SEMA7A binding through plexin C1 on monocytes has not been demonstrated yet. Because plexin C1 seems to be involved in the generation of sCD100-mediated signals on monocytes, we were prompted to test the cytokine production from sCD100-treated cells. As shown in Table 1, exposure of monocytes to sCD100 resulted in an increased IL-10 production and a significant down-regulation of IL-6, IL-8 and TNF-{alpha} secretion. Basal levels of cytokine production were detected when monocytes were incubated with the anti-plexin C1 blocking mAb before contact with sCD100, showing that sCD100-mediated modulation of cytokine production required functional plexin C1 receptors. Similarly, an up-regulation of IL-10 release and a down-modulation of the pro-inflammatory cytokines IL-6, IL-8 and TNF-{alpha} were observed on sCD100-treated immature DCs. This modulation of cytokine production was abolished in the presence of the anti-plexin B1 antibodies (Table 1).


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Table 1. Inhibition of monocytes and immature DCs cytokine production upon exposure to sCD100

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this study, we extend our previous observation that sCD100 exerts a negative function on the migration of immune cell lineages to human monocytes and derived immature DCs. In an attempt to better characterize the molecular basis underlying such observation, we performed a protein expression analysis of known potential receptors, namely CD72, plexin B1 and plexin C1 (Fig. 2). Using several detection methods, we observed the expression of CD72 and plexin C1 on freshly isolated human monocytes. While it has been previously reported that sCD100 binding to tonsillar B cells was abolished in the presence of a blocking anti-CD72 mAb (16), sCD100 function on monocyte migration was not modified by the addition of the same mAb (data not shown). Furthermore, the difference observed between the biological activity of sCD100 on monocyte motion and cytokine production (50–80 ng ml–1, corresponding to ~3 x 10–10 M) and the affinity of sCD100 for CD72 (KD = 3 x 10–7 M) strongly suggested that CD72 might not mediate all sCD100 functions on monocytes.

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-{alpha} 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-{alpha} following the treatment of monocytes with a CD100–Fc soluble molecule. However, this discrepancy might derive from the use of distinct experimental procedures. Indeed, in their study, higher doses of sCD100 (4–20 µg ml–1) than the one we used (50–80 ng ml–1) 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-{gamma} by NK cells, was also evidenced which may promote both NK cells and DCs activation through the cytokine network involving IL-12 and IFN-{gamma}. 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 (sCD100–Fc 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 cell–cell 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.


    Acknowledgements
 
We gratefully acknowledge Amgen for providing anti-plexin C1 mAb and recombinant A39R protein. This work was supported by INSERM and CNRS funds, and grants from the Association pour la Recherche sur le Cancer (contract no. 2094 to A.M.-C.).


    Abbreviations
 
AP   alkaline phosphatase
DC   dendritic cell
GM-CSF   granulocyte macrophage colony-stimulating factor
KO   knockout
sCD100   soluble CD100
TNF   tumor necrosis factor

    Notes
 
* These authors equally contributed to this work. Back

Transmitting editor: T. Tedder

Received 13 September 2004, accepted 19 January 2005.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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