glycans (10). Consequently, molecules that mediate the physical interactions between the immune cells and the ex- tracellular environment may play a critical role by imposing a context in which biological and inflammatory processes take place. For instance, low molecular weight hyaluronan was shown to induce IL-12 production by binding CD44 on inflamed macrophages (11). Also, the b1 and b2 inte- grin family of adhesive receptors, expressed by PBMCs, mediates ECM–cell interactions that deliver costimulatory signals to the incoming cells. Engagement of b1 integrins by fibronectin increases adhesion of T lymphocytes to im- mobilized ligands (12) and promotes TCR signaling. CD47 antigen, also known as integrin-associated protein (IAP), is a widely expressed multispan transmembrane protein which is physically and functionally associated with avb3 integrin, the vitronectin (Vn) receptor (13). Indeed, CD472 cell lines expressed avb3 but failed to bind Vn-coated beads. Moreover, CD47-deficient mice rapidly die of Escherichia coli peritonitis, a phenomenon directly associated with a re- duction in leukocyte activation in response to b3, but not b2, integrin ligation (14). More recently, CD47 has been implicated in leukocyte transendothelial migration (15), whereas its potential role in immune regulation has not yet been explored. Its newly described natural ligand, thrombo- spondin (TSP), is transiently expressed at high concentra- tion in damaged and inflamed tissues (16, 17). TSP is a homo- trimeric ECM protein mainly produced by platelets; it may also be synthesized by monocytes and alveolar macrophages. In addition to CD47, TSP binds heparan sulfate proteogly- cans, the avb3, a4b1, a5b1 integrins, and CD36 (16–18). We here examine the potential role of CD47 and TSP in the regulation of cytokine synthesis by monocytes. We re- port that ligation of CD47 antigen, by mAb or TSP, specif- ically and selectively inhibits IL-12 production by purified monocytes in response to T cell–dependent or –indepen- dent stimuli. Materials and Methods Reagents. Recombinant GM-CSF was provided by Dr. D. Bron (Institut Bordet, Brussels, Belgium). IFN-g was obtained from Genzyme. sCD40L trimeric protein was provided by Drs. C. Maliszewski and R. Armitage (Immunex Corp., Seattle, WA). Staphylococcus aureus Cowan I strain (SAC) was used at 0.01% (wt/vol) (Pansorbin; Calbiochem-Behring) and LPS at 10 mg/ml (Sigma Chemical Co.). Anti-CD47 mAbs B6H12 (mouse IgG1) and BRIC126 (mouse IgG2b) were purchased from Serotec. Isotype-matched negative control mAb (mouse IgG1) was pre- pared in our laboratory. Anti-CD18 mAb (mouse IgG1; Ameri- can Type Culture Collection) was used at 10 mg/ml. Neutral- izing anti–IL-10 mAb (clone 19 F1.1; American Type Culture Collection) and neutralizing anti–TGF-b (polyclonal chicken Ig) were used at 10 and 30 mg/ml, respectively. TSP was purchased from GIBCO BRL, and EDTA and EGTA were from Sigma Chemical Co. The 4N1K peptide (KRFYVVMWKK) was ob- tained from Genosys and corresponds to the COOH-terminal domain of TSP (16); 4NGG (KRFYGGMWKK) was a gift from W. Frazier (Washington University, St. Louis, MO). Cell Purification and Culture Conditions: Monocytes. PBMCs were isolated by density gradient centrifugation of heparinized blood from healthy volunteers (total n 5 30) using Lymphoprep (Nyco- med). Enriched monocytes were prepared by cold aggregation as described (9), followed by one cycle of rosetting with S-(2-amino- ethyl) isothiouronium bromide (Aldrich)–treated SRBCs to de- plete residual T and NK cells. Monocyte purity was shown to be .95% by flow cytometry (FACScan™; Becton Dickinson) using PE-conjugated anti-CD14 mAb (Ancell). Monocytes were cultured at 106/ml in 96-well round-bottomed Falcon plates (Becton Dickinson). Cultures were performed in quadruplicate in complete serum-free HB101 medium (Irvine Scientific) containing Ca21 (600 mM) and Mg21 (490 mM) and supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 10 mM Hepes, 100 IU penicillin, and 100 mg/ml streptomycin. Cytokine Measurement. IL-12p70 release was assessed by a two- site sandwich ELISA using clone 20C2 as the capture mAb and clone 4D6 as the second mAb as described (9). Both mAbs were provided by Dr. M. Gately (Hoffmann-LaRoche, Nutley, NJ). The sensitivity of the assay was 6 pg/ml. TNF-a was measured using a sandwich ELISA as described previously (19), and IL-10 was de- termined by a sandwich solid-phase RIA using anti–IL-10 mAb (clone 9D7) as the capture mAb and 125I-labeled anti–IL-10 (clone 12G8) as the detecting probe. IL-1b, IL-6, GM-CSF, and TGF-b ELISA kits were purchased from R&D Systems. All of the measurements were performed in duplicate with a coefficient variation of ,10%. Statistical Analysis. The paired t test was used to determine statistical significance of the data. *P , 0.05; **P , 0.01; ***P , 0.005. Results CD47 mAb Selectively Suppresses IL-12 Release by Mono- cytes. We examined the effect of soluble CD47 mAb on IL-12 release by purified monocytes costimulated by IFN-g and T cell–dependent (sCD40L and GM-CSF) or –indepen- dent (SAC) signals. As depicted in Fig. 1 A, CD47 mAb significantly suppressed IL-12 secretion in response to either stimuli, whereas isotype-matched IgG1 mouse mAb had no effect. In agreement with Marth and Kelsall (3), anti-CD18 mAb, used as a cell-binding irrelevant mAb, did not sup- press IL-12 production (data not shown). We noted an in- hibition of IL-12 release even when monocytes were cul- tured with SAC alone, indicating that CD47 mAb did not simply impair the enhancing effect of IFN-g on IL-12 pro- duction. Our unpublished observations revealed a similar suppressive effect by CD47 mAb after LPS and IFN-g co- stimulation. The blockade of IL-12 production in response to SAC and IFN-g was rather selective, since the release of other monocyte products (TNF-a, IL-1, IL-6, and GM-CSF) remained largely unaffected (Fig. 1 B). Note a threefold in- crease in IL-6 secretion, which did not reach statistical sig- nificance. Cell viability, as determined by trypan blue dye exclusion, was unchanged. Also, the inhibition of IL-12 was probably not mediated via an increase of autocrine antiinflammatory cytokines (IL-10 and TGF-b), known to efficiently downregulate bacteria-induced monokine release (1). As shown in Fig. 1 C, IL-10 and TGF-b levels remained unaffected by CD47 mAb treatment. Moreover, neutralizing 1176 CD47 Regulation of IL-12 Production