Figure 1. CD47 mAb selectively inhibits IL-12 release by purified monocytes. (A) Monocytes (106/ml) were cultured with SAC (0.01%; n 5 5), SAC plus IFN-g (500 U/ml; n 5 12), or sCD40L (1 mg/ml) plus GM-CSF (25 ng/ml) plus IFN-g (500 U/ml; n 5 6) in the absence or presence of CD47 mAb (IgG1) or isotype-matched control mAb (cont; 10 mg/ml). IL-12p70 was measured in the culture supernatants after 20 h. (B) Mono- cytes were stimulated with SAC and IFN-g (n 5 6) in the presence of CD47 or control mAb. TNF-a, IL-1b, IL-6, and GM-CSF were mea- sured after 20 h. Data are mean values 6 SEM of n separate experiments from n different donors. ***P , 0.005; *P , 0.05. (C) Monocytes were stimulated with SAC and IFN-g in the presence of CD47 or control mAb; IL-10 and TGF-b were measured after 20 h. Data are mean values 6 SEM from six experiments using six different donors. (D) Monocytes were stimulated with SAC and IFN-g in the presence of CD47 or control mAb; neutral- izing anti–IL-10 (10 mg/ml), anti–TGF-b (30 mg), or isotype-matched control mAbs were added to the cultures. Data are mean values of duplicate cul- tures and represent one of three experiments using three different donors. anti–IL-10 or anti–TGF-b antibodies added alone or simul- taneously did not overcome CD47 mAb–mediated inhibi- tion of IL-12 production by SAC- and IFN-g–stimulated monocytes (Fig. 1 D, and data not shown). Thrombospondin Inhibits IL-12 Release. We next evaluated the effect of TSP, the natural ligand of CD47 (16), on monokine release. As shown in Table I, TSP significantly reduced IL-12 production by SAC- and IFN-g–stimulated monocytes, whereas it slightly enhanced TNF-a release. Note that monocytes coexpressed two other TSP receptors (CD36 and avb3 [15]). Since the site of interaction be- tween CD47 and TSP is limited to the cell-binding domain (CBD) of TSP, we examined the role of a synthetic peptide (4N1K) encoding this particular domain (16). We found that 4N1K, but not control mutant peptide 4NNG, dose- dependently inhibited IL-12 release in response to SAC and IFN-g without affecting TNF-a production (Fig. 2). CD47 Ligation Suppresses IL-12 Release via an Fc and Ca21– independent but Phosphoinositide 3-Kinase–dependent Pathway. This specific inhibition of IL-12 release after CD47 en- gagement was reminiscent of recent studies reporting that FcgR cross-linking selectively downregulated IL-12 secre- tion in monocytes via an increase in extracellular calcium influx (2). We analyzed whether this mechanism might be involved in the CD47 mAb–mediated IL-12 suppression. First, F(ab9)2 fragments of CD47 mAb dose-dependently inhibited IL-12p70 release, demonstrating that the inhibi- 1177 Armant et al. tory effect was not Fc mediated (Fig. 3 A). Furthermore, monovalent Fab fragments still suppressed IL-12 secretion (Fig. 3 B), suggesting that CD47 mAb either prevented monocyte/monocyte interactions and the delivery of a positive signal, or directly delivered to the monocytes a negative signal for IL-12 production. Second, IL-12 down- regulation was observed in monocyte cultures supplemented with a fivefold excess of the Ca21 and Mg21 chelating agents EDTA or EGTA, demonstrating that the suppressive effect was independent of extracellular Ca21 influx (Fig. 3 B). Table I. TSP Selectively Inhibits IL-12 Production by Monocytes SAC 1 IFN-g SAC 1 IFN-g 1 TSP Percent inhibition IL-12 (pg/ml) TNF-a (ng/ml) Exp. 1 168 79 53 Exp. 2 Exp. 3 Exp. Exp. Exp. 1 2 3 1,020 2,120 746 1,090 27 49 120 147 – 64 83 – 87 105 – Monocytes (106/ml) were cultured for 20 h with SAC (1:10,000) and IFN-g (500 U/ml) with or without plastic-coated TSP (10 mg/ml). Culture supernatants were harvested for cytokine determination as de- scribed. Exp., experiment.