Figure 4. PI 3-K inhibitors restore IL-12 production. Monocytes (106/ml) were stimulated with SAC and IFN-g in the absence or presence of CD47 mAb (10 mg/ml) and titrated concentrations of wortmannin or Ly294002. IL-12p70 release was measured after 20 h. Results represent mean values from one of three independent experiments using three different donors. suppressed IL-12 regardless of the presence of divalent cat- ion chelating agents. CD47 antigen, also named integrin-associated protein (IAP), regulates the function and the binding of Vn to avb3, its associated integrin (13). However, Vn does not bind CD47, and the natural ligand of CD47 is reported to be TSP (16). TSP interacts with CD47 through its CBD, but binds other surface receptors such as CD36 or avb3 via distinct domains (17, 24). Since TSP reportedly activates latent TGF-b in vitro and in vivo (25) and ligation of CD36 by CD36 mAb down- regulates IL-12 and TNF-a production and increases IL-10 (26), we used 4N1K peptide encoding the CBD of TSP to demonstrate that TSP-mediated inhibition of IL-12 re- sulted from its direct effect on CD47 antigen. In addition, CD47 ligation did not decrease TNF-a nor increase IL-10 production. Note that TSP uses its properdin-like domain to activate TGF-b (17) and that TGF-b production was not upregulated after CD47 signaling (Fig. 1 C). IL-12 downmodulation, in the absence of TNF-a inhi- bition, was also observed in IFN regulatory factor (IRF-1 [27]) and IFN consensus sequence binding protein (ICSBP)- deficient mice (28). These two transcription factors belong to the IRF family and bind IL-12p40 promoter as well as other IFN-stimulated genes. As a direct consequence of the primary defect in IL-12 production, the deficient mice dis- played an impaired Th1 response and IFN-g–dependent host resistance to intracellular pathogens. Similarly, our un- published observations indicate that CD47 signaling strongly impaired IL-12–induced proliferative and IFN-g responses of activated T cells. The role of CD47 antigen, which is expressed by all leukocytes, has been demonstrated in the transendothelial migration of neutrophils, lymphocytes, and more recently, monocytes (15). Our present findings reveal that CD47 ligation by TSP modulates the function of mono- cytes. Therefore, we propose that engagement of CD47 is a novel and unexplored pathway to selectively downregulate IL-12 production. Inhibition of the IL-12 response is cru- cial for the treatment of organ-specific autoimmune diseases (1). For instance, administration of anti–IL-12 mAb com- pletely abrogated established colitis of trinitrobenzene sul- fonic acid (TNBS)-treated mice. IL-12 neutralization was also revealed to be an effective treatment of autoimmune en- cephalitis and insulin-dependent diabetes. Finally, in addition to its ability to downregulate IL-12 response through its CBD, TSP is known to display several important antiinflammatory activities via distinct domains. TSP facilitates elimination of apoptotic cells (29), activates TGF-b in vivo and in vitro (25), potently inhibits angio- genesis (17), and suppresses HIV infectivity through its binding to HIVg120 (24). Moreover, absence of TSP causes pneumonia (30). Therefore, since TSP is transiently expressed at high concentration in damaged and inflamed tissue, its selective suppression of the IL-12 response through interaction with the CD47 molecule may be rele- vant in limiting the duration and the intensity of the inflam- matory response. The importance of such a process awaits confirmation in CD47- and TSP-deficient mice. We are grateful to Dr. M. Gately (Hoffmann-LaRoche) for generously providing the IL-12 reagents. We fully appreciate the excellent secretarial assistance of Norma Del Bosco. This work is supported by Medical Research Council grants MT13311 and MT14432. 1179 Armant et al.