Figure 2. 4N1K selectively suppresses the production of IL-12 by monocytes. Monocytes (106/ml) were stimulated with SAC and IFN-g in the presence of various concentrations of 4N1K (peptide of COOH-ter- minal domain of TSP) or 4NGG (mutant peptide). IL-12p70 and TNF-a release were measured after 20 h. Data are mean values of duplicate cul- tures of one representative of three independent experiments using three different donors. Moreover, addition of EDTA or EGTA in the absence of mAb did not result in inhibition of TNF-a or IL-12 release. Since TSP signaling of focal adhesion in endothelial cells requires activation of phosphoinositide 3-kinase (PI 3-K [20]), we explored the involvement of this pathway in CD47 mAb–mediated suppression of IL-12 release by us- ing two PI 3-K inhibitors, wortmannin and Ly294002. We found that both PI 3-K inhibitors restored IL-12 release in a dose-dependent manner (Fig. 4) with no effect on TNF-a secretion (data not shown). However, neither protein ki- nase C (e.g., H7, HA1004) nor protein tyrosine kinase in- hibitors (e.g., herbimycin) prevented CD47 mAb–mediated inhibition of IL-12 release (not shown). Taken collectively, our results suggest that engagement of CD47 on monocytes by mAb, its natural ligand TSP, and 4N1K peptide specifi- cally suppressed IL-12 secretion through an Fc and Ca21– independent but PI 3-K–dependent pathway. Discussion The critical role of IL-12–mediated protection against mycobacterial infection was recently confirmed in IL-12 receptor–deficient patients (21). Nevertheless, uncontrolled production of IL-12 is pathogenic in some organ-specific autoimmune diseases (1) and endotoxic shock (22), under- scoring the requirement of potent negative regulatory feed- back mechanisms. The present findings demonstrate that en- gagement of CD47 by its natural ligand, TSP, or by CD47 mAb, represents a novel pathway to selectively downregulate IL-12 production. Inhibition of IL-12 release by CD47 ligation occurred in activated monocytes through a PI 3-K–dependent pathway. Indeed, two specific inhibitors of PI 3-K (wortmannin and Ly294002) overcame CD47-mediated IL-12 suppression with no effect on TNF-a production. Although these data are not a definite proof of the involvement of PI 3-K, they are supported by the observations that TSP-mediated func- tions such as cell spreading or focal adhesion disassembly required activation of PI 3-K (20). Besides inhibitory cy- tokines, including IL-4, IL-10, and TGF-b, which suppress the production of a large number of proinflammatory products (e.g., TNF-a, IL-1, IL-6, and IL-12) by activated monocytes, several monocyte surface receptors reportedly downregulate IL-12 production in a somewhat selective manner. Histoplasma capsulatum and measles virus have been shown to decrease IL-12 release by signaling through CD11b and CD46, respectively (3, 4). IL-12 downmodulation that resulted from scavenger receptor and FcgR ligation is due to extracellular Ca21 influx (2) and increase in IL-10 pro- duction in the case of FcgRI (23). In the present study, the CD47 mAb inhibition was Fc and Ca21 independent since intact, divalent, and monovalent fragments of CD47 mAb Figure 3. Suppression of IL-12 production by CD47 mAb is Fc and Ca21 independent. Monocytes (106/ml) were stimulated with SAC and IFN-g and titrated concentrations of F(ab9)2 anti-CD47 mAb (A) or 20 mg/ml of Fab anti-CD47, EDTA, and EGTA (5 mM) (B). IL-12p70 and TNF-a release were measured after 20 h. Results in A represent mean values of one out of three separate experiments using three different donors. Results in B are expressed as means 6 SD of percent inhibition of control response (SAC- and IFN-g–treated monocytes) of four separate experiments using four different donors. 1178 CD47 Regulation of IL-12 Production