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The Journal of Immunology, 2001, 167: 2547-2554.
Copyright © 2001 by The American Association of Immunologists

Bidirectional Negative Regulation of Human T and Dendritic Cells by CD47 and Its Cognate Receptor Signal-Regulator Protein-{alpha}: Down-Regulation of IL-12 Responsiveness and Inhibition of Dendritic Cell Activation1

Sylvain Latour2,*, Hiroyuki Tanaka{dagger}, Christian Demeure{dagger}, Véronique Mateo{dagger}, Manuel Rubio{dagger}, Eric J. Brown{ddagger}, Charles Maliszewski§, Frederik P. Lindberg, Anna Oldenborg, Axel Ullrich||, Guy Delespesse{dagger} and Marika Sarfati3,{dagger}

* McGill Cancer Center, McGill University and Institut de Recherches Cliniques, Montréal, Québec, Canada; {dagger} Allergy Research Laboratory, Centre de Recherche du Centre Hospitalier Université de Montréal, Notre-Dame Hospital, Montreal University, Québec, Canada; {ddagger} Program in Microbial Pathogenesis and Host Defense, University of California, San Francisco, CA 94143; § Immunex Research and Development Corporation, Seattle, WA 98101; Division of Infectious Diseases, Washington University School of Medicine, St. Louis, MO 63110; and || Department of Molecular Biology, Max Planck Institute, Martinsried, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Proinflammatory molecules, including IFN-{gamma} and IL-12, play a crucial role in the elimination of causative agents. To allow healing, potent anti-inflammatory processes are required to down-regulate the inflammatory response. In this study, we first show that CD47/integrin-associated protein, a ubiquitous multispan transmembrane protein highly expressed on T cells, interacts with signal-regulator protein (SIRP)-{alpha}, an immunoreceptor tyrosine-based inhibition motif-containing molecule selectively expressed on myelomonocytic cells, and next demonstrate that this pair of molecules negatively regulates human T and dendritic cell (DC) function. CD47 ligation by CD47 mAb or L-SIRP-{alpha} transfectants inhibits IL-12R expression and down-regulates IL-12 responsiveness of activated CD4+ and CD8+ adult T cells without affecting their response to IL-2. Human CD47-Fc fusion protein binds SIRP-{alpha} expressed on immature DC and mature DC. SIRP-{alpha} engagement by CD47-Fc prevents the phenotypic and functional maturation of immature DC and still inhibits cytokine production by mature DC. Finally, in allogeneic MLR between mDC and naive T cells, CD47-Fc decreases IFN-{gamma} production after priming and impairs the development of a Th1 response. Therefore, CD47 on T cells and its cognate receptor SIRP-{alpha} on DC define a novel regulatory pathway that may be involved in the maintenance of homeostasis by preventing the escalation of the inflammatory immune response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interleukin-12 is a potent proinflammatory molecule that provides a link between innate and adaptive immunity (1, 2). During the early stage of infections caused by bacteria, fungi, intracellular pathogens, and certain viruses, IL-12 is released by mononuclear and polynuclear phagocytes and by dendritic cells (DC).4 IL-12 rapidly triggers IFN-{gamma} production by NK cells and activated T cells. IFN-{gamma} enhances defense against pathogens, and both IFN-{gamma} and DC-derived IL-12 direct the differentiation of naive T cells into Th1 effectors, producing high levels of IFN-{gamma} and little or no IL-4. The IL-12/IFN-{gamma} proinflammatory loop is of short duration; uncontrolled IL-12 production and responsiveness are associated with some organ-specific autoimmune diseases, underscoring the requirement of potent negative regulatory feedback mechanisms (3). Engagement of phagocytic receptors (i.e., CR3, Fc{gamma}R, scavenger receptor), viral receptors (i.e., CD46), and extracellular matrix receptors (i.e., CD36) down-regulates IL-12 production (4, 5, 6, 7). Several negative regulators of IL-12 production, including IL-10 and TGF-{beta}, and certain biochemical mediators, such as dexamethasone and PGE2 (3, 8), reportedly down-regulate IL-12R expression and IL-12 responsiveness. However, cognate interaction of APC with T cells increases IL-12 release through CD40-CD40 ligand (CD40L) interactions, and IL-12 responsiveness is facilitated via up-regulation of costimulatory molecules on APC, including CD80 and CD86 (1).

We recently reported that ligation of CD47, by a mAb or its natural ligand thrombospondin, negatively regulates IL-12 production by APC and inhibits the development of naive T cells into Th1 effectors (9, 10, 11).

The CD47 Ag (integrin-associated protein), a multispan transmembrane protein expressed on all hemopoietic cells, is physically and functionally associated with {alpha}v{beta}3 integrin, the vitronectin receptor. CD47-deficient mice rapidly die from Escherichia coli peritonitis, a phenomenon associated with a reduction in leukocyte activation in response to {beta}3, but not {beta}2 integrins (12, 13).

CD47 is also involved in 1) platelet aggregation, 2) transendothelial and transepithelial leukocyte migration, and 3) integrin-independent T cell costimulation (14, 15, 16, 17, 18, 19). CD47 acts as a thrombospondin receptor (20), and more recently has been reported to be the ligand of signal-regulator protein (SIRP-{alpha}), also named SHPS-1, BIT, and p84 (21, 22, 23). SIRP-{alpha} is a transmembrane receptor selectively expressed in neurons and myeloid cells and serves as a substrate for activated receptor-tyrosine kinases (24, 25). The extracellular domains of SIRPs consist of three or one Ig-like domains, and are involved in cell-cell interactions (23, 24, 25, 26, 27). In humans, the SIRP family is divided in two subgroups differing by the absence (SIRP-{beta}) or the presence (SIRP-{alpha}) of immunoreceptor tyrosine-based inhibition motifs in the cytoplasmic tail (24). SIRP-{alpha} is an inhibitory receptor that regulates responsiveness to receptor-tyrosine kinase ligands such as epidermal growth factor or platelet growth factor and adhesion processes (24, 26, 27). Upon phosphorylation, immunoreceptor tyrosine-based inhibition motifs recruit Src homology 2 domain-containing phosphatases (including SHP-1), known to negatively regulate cell activation (28). By contrast, SIRP-{beta} is an activating receptor that associates with the immunoreceptor tyrosine-based activation motifs containing subunit KARAP/DAP12 (29, 30).

In the present study, we investigate the function of CD47 and its cognate receptor, SIRP-{alpha}, in the regulation of human IL-12 production and responsiveness.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell preparations and culture conditions

PBMCs. PBMCs were isolated by density gradient centrifugation of heparinized blood from healthy volunteers using Lymphoprep (Nycomed, Oslo, Norway) and cultured at 1 x 106/ml for 3 days with anti-CD3 (clone UCHT1; 1 µg/ml) in RPMI 10% FCS supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 10 mM HEPES, 100 IU penicillin, and 100 µg/ml streptomycin in the presence or absence of IL-12 (60 pM) (M. Gately, Hoffmann-LaRoche, Nutley, NJ).

DC. Enriched monocytes were prepared by cold aggregation (as reported in Ref. 9), followed by T and NK depletion. Monocyte purity was shown to be 95% CD14+ cells by flow cytometry. Human monocyte-derived immature DC (iDC) were prepared exactly as described (9, 10), except that two-thirds of culture medium were replaced by fresh medium containing GM-CSF and IL-4 every other day and nonadherent cells were harvested at day 5 to obtain iDC. Mature DC (mDC) were generated following stimulation of iDC (0.5 x 106/ml) for 2 days with Staphylococcus aureus Cowan I strain (SAC) at 0.01% (w/v; Pansorbin; Calbiochem-Behring, La Jolla, CA), LPS (10 ng/ml), or soluble CD40L (sCD40L) (0.5 µg/ml; Immunex, Seattle, WA) and IFN-{gamma} (500 U/ml) in complete RPMI 10% FCS. DC were cultured in the presence of soluble CD47-Fc (5 µg/ml), immobilized CD47-Fc (5 µg/ml) on plastic-coated goat anti-human Ig (BioSource-Tago, Montreal, Canada), normal human IgG1 (NHIg), or Fc fragment from NHIg (NHIg-Fc), as indicated.

T lymphocytes and allogeneic MLR. Highly purified T cells were obtained from the monocyte-depleted PBMC or cord blood mononuclear cells by rosetting with 2-aminoethylisothiouronium bromide-treated SRBC, followed by treatment of rosette-forming cells with Lympho-Kwik T (One Lambda, Los Angeles, CA), according to the manufacturer’s recommendations. Cell purity was assessed by flow cytometry using PE-conjugated anti-CD3, anti-CD4, or anti-CD8 mAbs (Ancell, London, Ontario, Canada), and was shown to be 98%. T cells (1 x 106/ml) were stimulated with anti-CD3 (soluble clone 64.1 or immobilized UCHT-1) and IL-12 (60 pM) or IL-2 (50 U/ml) (D. Bron, Institut Bordet, Brussels, Belgium) in the presence of mitomycin-treated control L cells (L/pLXSN) or L-SIRP-{alpha} transfectants (25 x 103/ml) (24). CD4+ T cells were isolated using Lympho-kwik TH (One Lambda), and CD8+ T cells were positively selected using anti-CD8-coated Dynabeads (Dynal, Oslo, Norway), followed by negative selection using anti-CD4-coated Dynabeads (Dynal) to remove double-positive cells (CD4+CD8+ cells). All cultures were performed in RPMI 10% FCS. Culture supernatant was collected at day 6 for cytokine determination. Primary MLRs were conducted in 96-well U-bottom microplates (Falcon) by adding mitomycin C-treated mDC to allogeneic naive neonatal CD4+ T cells (106/ml) in complete culture medium at 1:4 stimulator (DC):responder (T cells) ratio. Expansion in IL-2 and restimulation of effector T cells were performed exactly as previously described (31).

Cytokine measurement

IL-12p70, TNF-{alpha}, IL-4, IL-5, IL-10, IL-13, and IFN-{gamma} release were assessed by a two-site sandwich ELISA or RIA, as described (9, 10, 31). The sensitivity of the assay was 6 pg/ml for IL-12 and 50 pg/ml for the other cytokines. IL-6, IL-8, IL-18, and TGF-{beta} ELISA kits were purchased from R&D Systems (Minneapolis, MN). All the measurements were performed in duplicate.

Flow cytometry analysis

IL-12R expression was performed at day 3 by a three-step procedure using rat mAb to human IL-12R{beta}1 or {beta}2 (D. H. Presky, Hoffmann-LaRoche, Nutley, NJ). All other FITC- or PE-conjugated mAbs were purchased from Ancell and used in direct staining. Binding of CD47-Fc was assessed using a two-step procedure. Briefly, cells were first incubated for 1 h at 4°C with a biotinylated CD47-Fc or NHIg-Fc (5 µg/ml). After washing, cells were incubated with PE-labeled streptavidin (Ancell) for 1 h at 4°C. Stained cells were analyzed using a FACSort (BD Biosciences, Mountain View, CA).

Soluble CD47-Fc preparation

The cDNA encoding the human soluble CD47-Fc fusion protein was constructed by PCR. It is composed of the extracellular domain of CD47 (aa residues 1–142) (12) fused to a modified human IgG1 Fc region in which 3 aa were mutated (L234A, L235E, and G237A) to lower the binding to FcR. The corresponding cDNA was cloned into the pCD409 plasmid (32). The resulting plasmid was transfected by DEAE-dextran method into COS-1 cells, and the fusion protein was purified from culture supernatants using protein A-Sepharose.

Immunoprecipitation and immunoblot

Human cells or mouse spleen tissue were lysed in 1x 50 mM Tris, pH 8, 2mM EDTA, pH 8, 1% Nonidet P-40 (TNE) buffer containing protease and phosphatase inhibitors exactly as described previously (26). Proteins were recovered by immunoprecipitation using CD47-Fc, NHIg, polyclonal anti-human SIRP-{alpha} (Santa Cruz Biotechnology, Santa Cruz, CA). Immune complexes were then collected with protein G- or protein A-Sepharose and washed in 1x TNE buffer containing 1 mM sodium orthovanadate. For deglycosylation, samples were treated for 2 h at 37°C with 1000 U peptide: N-glycosidase F (PNGase F; New England Biolabs, Beverly, MA), according to the manufacturer’s protocol. Proteins were eluted in sample buffer, boiled, and electrophoresed in 8% SDS-PAGE. Immunoblots were performed as described using HRP donkey anti-goat (Transduction Laboratories, Lexington, KY).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CD47 ligation down-regulates IL-12 responsiveness

Ligation of CD47 by a soluble mAb reportedly inhibited IL-12 responsiveness by PHA-activated neonatal mononuclear cells (11). In the course of this study, we confirmed and extended these observations to activated adult PBMC. As depicted in Fig. 1GoA, soluble CD47 mAb strongly inhibited IFN-{gamma} production by PBMC stimulated with or without soluble anti-CD3 mAb in the absence or presence of IL-12. Several reports have shown that immobilized anti-CD47 mAbs (B6H12, 2D3, or 1/1A4 mAbs) costimulate T cell activation (i.e., increased IL-2 production), whereas soluble CD47 mAb may exert inhibitory functions in allogeneic MLR (17, 18, 19). Therefore, we postulated that the suppression of IL-12 responsiveness observed in PBMC might result from either the delivery of a negative signal by CD47 mAb to T cells, or alternatively from a blockade by the mAb of a positive signal delivered by the newly described CD47 ligand, SIRP-{alpha} (22, 23).



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  		FIGURE 1. CD47 mAb inhibits IL-12 responsiveness. A, IFN-{gamma} production by resting or anti-CD3-activated human PBMC cultured for 3 days in the absence or presence of IL-12, with or without CD47 or isotype-matched control mAbs (10 µg/ml). Mean ± SEM of six experiments (*, p < 0.01). B, IFN-{gamma} production by purified T cells cultured for 5 days with anti-CD3 in the presence of IL-2 (50 U/ml) or IL-12 (60 pM) with CD47 or isotype-matched control mAbs (10 µg/ml). Mean ± SEM of six experiments (***, p < 0.0001). C, IFN-{gamma} production by purified CD4 or CD8 T cells stimulated as in B. Shown is one representative experiment of three.

 
We next evaluated the ability of CD47 mAb and L-SIRP-{alpha} transfectants to directly regulate IL-12 responsiveness of purified adult T cells. We showed that CD47 mAb decreased IL-12-induced IFN-{gamma} production in nonfractionated (Fig. 1GoB), CD4+ and CD8+ T cell preparations (Fig. 1GoC). Note that the secretion of IFN-{gamma} in response to IL-2 remained unaffected. These data strongly suggested that engagement of CD47 on T cells selectively inhibited their IL-12 responsiveness. In support of this hypothesis, we found that L-SIRP-{alpha}, but not control transfectants significantly decreased IL-12 and not IL-2-induced IFN-{gamma} production of purified CD3+ T cells (Fig. 2GoA). The inhibition of IL-12 responsiveness was associated with a significant reduction in both IL-12R{beta}1 and {beta}2 expression on anti-CD3- and IL-12-stimulated T cells (Fig. 2B). Our unpublished observations further indicated that CD47 ligation by soluble mAb or L-SIRP-{alpha} transfectants concomitantly decreased IL-12 and not IL-2-induced T cell proliferation.



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  		FIGURE 2. CD47/SIRP-{alpha} interaction inhibits IL-12 responsiveness and IL-12R expression. A, IFN-{gamma} production by purified T cells cultured for 5 days with anti-CD3 in the presence of control L cells or L-SIRP-{alpha} transfectants, with IL-12 or IL-2. Mean ± SEM of six experiments (***, p < 0.0001). B, IL-12R expression (plain histograms) by IL-12- and anti-CD3-stimulated T cells cultured for 2 days in the presence of untransfected or L-SIRP-{alpha} transfectants. Background staining (dotted line). MFI, Mean fluorescence intensity. Similar results were obtained in three independent experiments.

 
Taken together, our results provide evidence that the CD47 ligation by mAb or its natural ligand, SIRP-{alpha}, negatively regulates IL-12 responsiveness, a phenomenon that is correlated with a decrease in IL-12R expression.

CD47 binds SIRP-{alpha} on DC

In turn, we postulated that CD47 molecule might engage SIRP-{alpha} expressed on monocytes and DC (22, 25, 30) and regulate their function. To test this hypothesis, we first prepared a soluble CD47-Fc fusion protein composed of the extracellular domain of CD47 fused to the Fc portion of human IgG1 and demonstrated its binding to human SIRP{alpha}. As shown in Fig. 3GoA, biotinylated soluble CD47-Fc stained L cells transfected with SIRP-{alpha}, but not control transfectants. This binding was inhibited by CD47 mAb (clone B6H12), but not by two CD47 mAbs directed against two different epitopes (clone 2D3 or 10G2). Of interest, CD47-Fc inhibited the binding of either three CD47 mAbs to CD47 transfectants (data not shown). These results confirmed and extended recent data indicating that human CD47 interacted with SIRP-{alpha} (33), and that B6H12 mAb, but not 2D3 mAb, blocked the binding of soluble biotinylated SIRP-{alpha}-GST to CD47 (22).



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  		FIGURE 3. Cellular distribution of human SIRP-{alpha} and CD47. A, Flow cytometry analysis of biotinylated CD47-Fc (open histograms) binding to L cells, L-SIRP-{alpha} in the presence of IgG1 (1), or CD47 mAb (2D3) (2), (10G2) (3), (B6HI2) (4). Background staining with biotinylated NHIg-Fc (plain histograms). B, CD47 expression by mononuclear cells using 2D3 mAb. C, Binding of biotinylated CD47-Fc (open histograms) or NHIg-Fc (plain histograms), followed by PE-streptavidin staining. Shown is one representative experiment of four.

 
Second, we evaluated the cellular distribution of CD47 and CD47 ligand. As expected, CD47 was expressed on a broad array of hemopoietic cells, with T cells expressing the highest levels (Fig. 3GoB). By contrast, CD47-Fc only stained cells of the myelomonocytic lineage (Fig. 3GoC). Indeed, we showed by flow cytometric analysis that CD47-Fc bound iDC and mDC, but not lymphoid cells. Similar cellular distribution was observed with cell lines (not detailed). The selective expression of SIRP-{alpha} on myeloid cells was confirmed by immunoprecipitation and Western blot analysis using anti-SIRP-{alpha} polyclonal Ab. SIRP-{alpha} protein was undetectable in T cell lysates (Fig. 4GoA, lane 1). In the lysates of monocytes, iDC, and mDC, SIRP-{alpha} molecules corresponded to glycosylated proteins of 70- to 105-kDa (gp90) and 35–40 kDa (gp40) (Fig. 4GoA, lanes 2, 3, and 4), which, upon deglycosylation, displayed the expected molecular mass of 65 and 35 kDa, respectively (S. Latour and M. Sarfati, unpublished observations). The smaller form of 35 kDa is likely to be the human equivalent of the murine isoform of SIRP-{alpha} containing only one Ig-like domain in the extracellular region (26).



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  		FIGURE 4. CD47-Fc binds and immunoprecipitates SIRP-{alpha} in human DC. A and B, Immunoblotting by polyclonal anti-human SIRP-{alpha} Ab. A, SIRP-{alpha} expression in cell extracts of monocytes, iDC, mDC, and T cells. B, Immunoprecipitation of SIRP-{alpha} by polyclonal anti-SIRP-{alpha} Ab (lane 1), NHIg (lane 2), or CD47-Fc (lanes 3–6). iDC (lanes 1, 2, and 3); no lysate (lane 4); mDC (lane 5); T cell lysate (lane 6). C, Binding of biotinylated CD47-Fc in the presence of IgG1 (thin line) or anti-human SIRP-{alpha} mAb (dark line), followed by PE-streptavidin staining. Biotinylated NHIg-Fc (dotted line).

 
To further show that CD47-Fc directly interacted with SIRP-{alpha} on DC, we performed immunoprecipitation experiments using CD47-Fc-coupled protein A-Sepharose (Fig. 4GoB). CD47-Fc precipitated gp90 and gp40 proteins from iDC (Fig. 4GoB, lane 3) and mDC (Fig. 4GoB, lane 5), which both reacted in Western blot with anti-SIRP-{alpha} Ab. NHIg (Fig 4GoB, lane 2) or mouse CD47-Fc (data not shown) did not precipitate proteins reacting with anti-SIRP-{alpha} Ab in DC, nor did CD47-Fc precipitate protein in T cell lysate (Fig. 4GoB, lane 6).

Finally, the data indicating that anti-human SIRP-{alpha} mAb (clone mSIRP130) abrogated CD47-Fc binding to both mDC and L-SIRP-{alpha} transfectants (Fig. 4GoC) formally demonstrate that the CD47-Fc molecule has the appropriate molecular form to bind SIRP-{alpha} on DC.

SIRP-{alpha} engagement down-regulates DC function

Therefore, we explored the possibility that engagement by CD47-Fc of SIRP-{alpha}, which has a predictive structure to deliver a negative signal, may regulate DC functions. As shown in Fig. 5GoA, CD47-Fc potently suppressed IL-12 and TNF-{alpha} release by monocyte-derived DC stimulated by SAC. The inhibitory effect was dose dependent, and significant suppression was seen with as little as 10 ng/ml CD47-Fc (Fig. 5GoB). Other cytokines, including IL-6 and IL-10, were also suppressed, whereas the production of IL-8 and IL-18 remained unaffected. The suppression of IL-12 and TNF-{alpha} was not mediated via FcR engagement nor by endogenous IL-10 or TGF-{beta} production, because addition to the cultures of neutralizing mAbs to these cytokines or of excess amount of NHIg did not overcome the inhibitory effect of CD47-Fc (data not shown). Preincubation of CD47-Fc with monovalent Fab of CD47 mAb completely abrogated the suppression (Fig. 5GoB). The same Fab blocked the binding of CD47-Fc to L-SIRP-{alpha} transfectants (data not shown). Shown in the same Fig. 5GoB, anti-SIRP-{alpha} mAb displayed similar inhibitory activity as CD47-Fc, and therefore did not restore TNF-{alpha} release by SAC-activated DC. As shown in Table IGo, CD47-Fc also decreased TNF-{alpha} in response to LPS or CD40 ligation. Similarly, IL-12p75 was suppressed following sCD40-L and IFN-{gamma} stimulation.



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  		FIGURE 5. CD47-Fc inhibits cytokine production by DC. iDC were cultured in the absence (white bars) or presence of SAC (0.01%) with soluble NHIg-Fc (black bars) or CD47-Fc (5 µg/ml) (gray bars). Culture supernatants were collected after 24 h for cytokine measurement, and phenotype was analyzed by flow cytometry after 48 h. A, IL-12, TNF-{alpha}, IL-6, IL-8, IL-10, and IL-18 production. Mean ± SD of six independent experiments. B, Preincubation of NHIg-Fc (filled bars), CD47-Fc (gray bars) with monovalent Fab of anti-CD47 mAb (CD47 Fab), or control Fab (cont Fab) in SAC-activated DC; dose-response inhibitory effect of soluble CD47-Fc on SAC-induced TNF-{alpha} release. Preincubation of DC with control mAb (cont mAb) or anti-human SIRP-{alpha} mAb (SIRP mAb), followed by activation with SAC in the presence of NHIg-Fc (filled bars) or CD47-Fc (gray bars) (100 ng/ml). One representative experiment of three is shown.

 

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  		Table I. Inhibitory effect of CD47-Fc on cytokine release1

 
CD47-Fc not only suppressed cytokine release by maturing DC, but also largely prevented DC phenotypic maturation. As indicated in Fig. 6Go, CD83 and CD86 up-regulation were strongly reduced regardless of the stimulus used. For CD80, the inhibition was almost complete in response to CD40 signaling, moderate in response to SAC, and modest, but significant in response to LPS. CD40 expression was weakly up-regulated during DC maturation. In all three conditions, SIRP-{alpha} ligation by CD47-Fc abrogated the CD40 increase. Note only a slight reduction in HLA-DR expression. All together, these results demonstrate that engagement of SIRP-{alpha} largely inhibits DC maturation.



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  		FIGURE 6. CD47-Fc prevents functional maturation of DC. Surface staining of DC for CD40, CD80, CD83, CD86, and HLA-DR expression on iDC and activated DC. Soluble CD47-Fc (5 µg/ml) was used for SAC stimulation and immobilized CD47-Fc (5 µg/ml) for LPS (10 ng/ml) and sCD40L (1 µg/ml) and IFN-{gamma} stimulation (500 U/ml). DC cultured with NHIg-Fc (open histograms), CD47-Fc (plain histograms). Background staining (control mAb) (dotted line). One representative experiment of three is shown.

 
Interestingly, we observed that mDC restimulated by sCD40L and IFN-{gamma} (which mimicked DC/naive T cell interactions) were still sensitive to inhibitory effect of CD47-Fc on TNF-{alpha} (mean ± SEM (n = 5): 8.43 ± 1.6 ng/ml (NHIg-Fc) and 0.75 ± 0.26 ng/ml (CD47-Fc)) and IL-12 p75 release (mean ± SD (n = 3): 0.70 ± 0.5 ng/ml (NHIg-Fc) and 0.17 ± 0.14 ng/ml (CD47-Fc)). Because increased allostimulation by mDC is partly IL-12 dependent (34), we considered the possibility that SIRP-{alpha} ligation by CD47-Fc during allogeneic MLR affected IL-12 production in primary culture, and therefore regulated the development of naive T cells into Th effectors. We recently reported that human monocyte-derived mDC induce neonatal naive T cell differentiation into Th1 and Th2 effectors at high stimulator-responder ratio (31). Results in Fig. 7Go indicated that immobilized CD47-Fc during mDC/naive CD4+ T MLR (1:4 ratio) significantly inhibited IFN-{gamma} production after 5 days of priming without decrease in cell proliferation (data not shown). Allogeneic T cells were expanded in IL-2-containing medium for 9–12 days and restimulated by anti-CD3 immobilized on L-CD32 fibroblasts. Results showed an impairment of naive T cell maturation into IFN-{gamma}-producing cells (p < 0.001) with no effect on T cell proliferation and IL-2 production and no immune deviation toward Th2.



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  		FIGURE 7. CD47/SIRP-{alpha} interactions regulate allogeneic MLR. Human naive CD4+ T cells were cocultured for 5 days with allogeneic DC at 1:4 stimulator-responder ratio. IFN-{gamma} was measured after primary MLRs. After IL-2 expansion, T cells were counted and restimulated with anti-CD3 immobilized on L-CD32/B7 transfectants. Cell proliferation was assessed after 16-h incorporation of [3H]thymidine. IFN-{gamma}, IL-2, IL-4, IL-5, and IL-13 were measured in 24-h culture supernatant. Mean ± SEM of five independent experiments. **, p < 0.001.

 
Taken together, our results demonstrate that SIRP-{alpha} ligation by CD47-Fc strongly inhibits phenotypic and functional maturation of DC, down-regulates residual cytokine production by mDC, and impairs the development of naive T cells into Th1 effectors.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present findings establish that CD47 and its cognate receptor SIRP-{alpha} deliver a bidirectional negative signal to both T cells and DC. L-SIRP-{alpha} transfectants decrease IL-12, but not IL-2 responsiveness by activated T cells, whereas CD47-Fc engages SIRP-{alpha} on DC to inhibit IL-12 production and more generally to prevent phenotypic and functional maturation of DC. We cannot exclude a possible role of SIRP-{beta} engagement in this model. However, this is unlikely, as SIRP-{beta} was shown to be rather involved in the activation of cellular function (29) and is weakly expressed on human DC (30). Moreover, a recent report indicated that SIRP-{beta} does not bind CD47 (35).

Based on these in vitro data, we propose that this pair of molecules is involved in the down-regulation or termination of the inflammatory response. For instance, in peripheral tissues, iDC are activated by external agents (pathogens) or internal injury (necrotic cells), but not by apoptotic cells (36, 37). The recently activated DC produce large amount of IL-12 that stimulates peripheral tissue homing NK, macrophages, and memory T cells (38). The present study indicated that engagement of SIRP-{alpha} during DC activation (possibly by hemopoietic and/or nonhemopoietic CD47-expressing cells) strongly inhibited the production of proinflammatory cytokines, whereas CD47 ligation (by APC) down-regulated IL-12 responsiveness of TCR- and non-TCR-activated T cells. One may envision that CD47/SIRP-{alpha} interaction occurs in resting state, is disrupted after an environmental insult, and rapidly restored to prevent escalation of the ensuing inflammatory reaction and maintain homeostasis. Recent reports proposed a similar role for the newly described pair of molecules, CD200/CD200R (39). Like CD47/SIRP-{alpha}, CD200 has a broad expression pattern, whereas CD200R expression is quite restricted to the myeloid cell lineage (40). Engagement of CD200R led to macrophage and granulocyte deactivation, and most importantly, CD200-/- mice displayed increased susceptibility to collagen-induced arthritis compared with the wild-type mice, which are resistant to the disease (41).

Further observations strongly supported the existence of CD47/SIRP-{alpha} interaction in vivo in resting tissue. In rodents, SIRP-{alpha} was demonstrated to be constitutively tyrosine phosphorylated and associated with the phosphatase SHP-1 in resting wild-type spleen tissue (26). However, we failed to detect SIRP-{alpha} tyrosine phosphorylation as well as association with SHP-1 in spleen tissue of CD47-deficient mice (S. Latour, F. P. Lindberg, and M. Sarfati, unpublished observations). In addition, Oldenborg et al. (42) observed that engagement of SIRP-{alpha} by CD47-expressing erythrocytes induced tyrosine phosphorylation of SIRP-{alpha} in rodent macrophages. This phosphorylation was dramatically decreased with CD47-/- erythrocytes. Furthermore, it was proposed that CD47 was a marker of self on erythrocytes, and that CD47/SIRP-{alpha} pathway negatively regulated the clearance of RBCs by macrophages (42). In macrophages, an inhibitory role for SIRP-{alpha} has been recently established. SIRP-{alpha} was shown to inhibit Fc{gamma}R-dependent and independent phagocytosis (43, 44).

Upon inflammatory stimulation, iDC release several cytokines and undergo maturational changes that involve down-regulation of endocytic capacity, up-regulation of surface immunogenic MHC-peptide complexes, and increased expression of costimulatory molecules. These maturational changes cause DC to become efficient stimulators of naive T cells and favor the development of a Th1 response (45). We found that engagement of SIRP-{alpha} by CD47-Fc on iDC largely prevented their phenotypic maturation. We previously reported that thrombospondin, at least via its CD47-binding moiety, impaired DC maturation. SIRP-{alpha} and CD47 are coexpressed by monocytes, iDC, and mDC, and the cross-talk between the two molecules on the same APC is under current investigation.

Other inhibitors of functional DC maturation include IL-10 and TGF-{beta}, Plasmodium falciparum, measles virus, glucocorticoids, and 1{alpha},25-dihydroxyvitamin D3 (3, 8, 46, 47). Moreover, several of these negative regulators of IL-12 production concomitantly down-regulate IL-12R expression and/or signaling pathways (8, 48). We observed that CD47/SIRP-{alpha} interaction not only suppressed IL-12 production by maturing DC, but also inhibited IL-12 responsiveness by anti-CD3-activated T cells, a phenomenon associated with decreased IL-12R expression. We (10) and others (38, 49) reported that mDC, considered as exhausted DC, released limited amount of IL-12. We have shown that this residual IL-12 production by mDC was further suppressed by CD47-Fc. Therefore, we postulate that the inhibitory activity of CD47-Fc on mDC (inhibition of IL-12 secretion) dominates over its blocking effect on DC/T interactions. As a consequence, we found an impairment in the development of naive T cells into Th1 effectors.

The mechanism by which SIRP-{alpha} inhibits functional maturation of DC in response to LPS, SAC, and CD40 signaling is not yet elucidated. SIRP-{alpha} reportedly recruited the tyrosine phosphatases SHP-1 and/or SHP-2, which are negative regulators of protein tyrosine kinase (PTK)-dependent signals (24, 26, 50). LPS and SAC signal through Toll-like receptors via MyD88/TNFR-associated factor 6-dependent pathways (51). A Myd88-independent pathway leading to the up-regulation of costimulatory molecules has been described in DC; whether it involves PTK or not is not known. In the past, it has been speculated that PTK activity was important in LPS signal transduction process (52, 53). However, LPS-induced TNF-{alpha} production remained unchanged in deficient mice for Src-family kinases, Hek, Fgr, and Lyn, despite their resistance to septic shock (54, 55). In the case of CD40 signaling, Vidalain et al. (56) nicely demonstrated that CD40-induced tyrosine phosphorylation of intracellular substrates was initiated by membrane raft-associated Lyn kinase. Therefore, SIRP-{alpha} engagement may inhibit either directly or indirectly signals provided by LPS, SAC, or CD40L. Also, SIRP-{alpha} may regulate cell adhesion and/or tyrosine kinase receptor signaling, two processes in which SIRP-{alpha} has been clearly implicated (24, 27, 54). Interestingly, recent data reported that LPS induces in monocytes cell adhesion, actin reorganization, and tyrosine phosphorylation of the tyrosine kinase Pyk 2, a related focal adhesion tyrosine kinase (57).

The current explanations for the shutdown of the immune response include 1) a negative signal delivered to T cells via CTLA-4, 2) the activation-induced cell death of T cells through Fas/Fas ligand (or TNF), and 3) the elimination of DC by activated T cells (58, 59, 60, 61). The selective down-regulation of IL-12 responsiveness by CD47 may be involved in the attenuation of type 1 immune response. The molecular mechanisms underlying a selective inhibition of IL-12 responsiveness by CD47 ligation are still difficult to unravel because of the particular structure of CD47. CD47 molecule is composed of an Ig-like extracellular domain, five putative transmembrane domains, and a short intracytoplasmic tail without any specific known signaling motif (12). However, CD47 was reported to localize in the membrane rafts, where it regulates the activation of heterotrimeric G proteins (62). Recent findings provided evidence that CD47 is implicated in T cell activation by the ability of CD47 mAb to trigger actin cytoskeleton rearrangement, protein kinase C{theta} translocation in a TCR-dependent and independent manner, and to cooperate with TCR signaling for IL-2 secretion (63). Importantly, biological consequences of CD47 stimulation seemingly depend on the way CD47 is engaged. Of note, opposite effect of the two molecular forms of CD47 mAb was demonstrated in granulocyte function (64). When immobilized, CD47 mAb costimulated IL-2 production by CD3-activated normal or transformed adult T cells (17, 18), whereas, in soluble form, it inhibited allogeneic MLR (19). It also decreased IL-2 production and IL-12R{beta}2 expression by IL-12- and PHA-activated naive cells; the latter is completely restored by addition of exogenous IL-2 at priming (11). Similarly, IL-2 abrogated the CD47 mAb-mediated inhibition of IL-12 responsiveness in anti-CD3-activated adult T cells (M. Sarfati, data not shown). It was recently reported that thrombospondin, at least through its CD47-binding moiety, inhibited TCR-mediated T cell activation and IL-2 production by PBMC, further supporting the view that CD47 ligation may deliver a negative signal to T cells (65). Theoretically, SIRP-{alpha} and thrombospondin might compete for CD47 on T cells, assuming they would be expressed simultaneously during the immune response.

It has been recently postulated that CD200/CD200R interaction was included in a spatial organization resembling the immunological synapse that would allow the delivery of efficient negative signals by direct cell-cell contact. This hypothesis was based on the structure of CD200/CD200R complexes that would consist of four tandem Ig-like domains (40, 66). Similarly, CD47/SIRP-{alpha} complexes are likely to be arranged in four tandem Ig-like domains, as the amino-terminal V-like domain of SIRP-{alpha} is sufficient to interact with CD47 (33), and as such, might take place in a spatial organized structure related to the immunological synapse. This idea is further supported by the fact that, in T cells, at least 65% of the CD47 molecule is localized in lipid rafts (62, 63), another level of organization required for efficient Ag-mediated TCR activation (67). Note that lipid rafts localization of CD47 is necessary for both TCR-dependent and independent CD47 signaling (63).

In conclusion, CD47/SIRP-{alpha} may participate in the down-regulation of the inflammatory response, the termination of Ag-specific immune responses initiated by the contact between mDC and naive T cells, and/or the inhibition of undesired Th1 responses. Further exploration of the mechanisms underlying the bidirectional negative regulatory response of CD47/SIRP-{alpha} in vivo and in vitro may lead to development of novel strategies for autoimmune diseases, organ transplantation (prevention of undesired Th0/Th1 response), and allograft rejection (transient DC inactivation) (68).


   Footnotes
 
1 This work was supported by Medical Research Council of Canada (MRC/CIHR) grants. S.L. was supported by a Medical Research Council postdoctoral fellowship. Back

2 Current address: Institut National de la Santé et de la Recherche Médicale Unité 429, Hôpital Necker, 75015 Paris, France. Back

3 Address correspondence and reprint requests to Dr. Marika Sarfati, Allergy Research Laboratory, Centre de Recherche du Centre Hospitalier Université de Montréal, Notre-Dame Hospital, Montreal University, Quebec, H2L 4M1, Canada. E-mail address: sarfatm{at}poste.umontreal.ca Back

4 Abbreviations used in this paper: DC, dendritic cell; CD40L, CD40 ligand; iDC, immature DC; mDC, mature DC; NHIg, normal human IgG1; PTK, protein tyrosine kinase; SAC, Staphylococcus aureus Cowan I; sCD40, soluble CD40; SIRP, signal-regulator protein. Back

Received for publication March 13, 2001. Accepted for publication June 25, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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