Interleukin-12 synthesis is a required step in trehalose dimycolate-induced activation of mouse peritoneal macrophages

Trehalose dimycolate (TDM), a glycolipid present in the fell wall of Mycobacterium spp., is a powerful immunostimulant, TDM primes murine macrophages (M phi) to produce nitric oxide (NO) and to develop antitumoral activity upon activation with low doses of lipopolysaccharide (LPS). In this study, we investigated the ability of TDM to induce interleukin 12 (IL-12) aod the role of this cytokine in TDM-induced activation of murine M phi. RNA isolated from peritoneal exudate cells (PEC) collected at different times after TDM injection was used to determine IL-12 (p35 and p40 subunits) and gamma interferon (IFN-gamma) mRNA levels by semiquantitative reverse transcriptase-PCR. Constitutive expression of IL-12p35 was observed in PEC from untreated as well as from TDM-injected mice, In contrast, expression of the IL-12p40 subunit was almost undetectable in control PEC but was dramatically upregulated in PEC from TDM-injected mice, IL-12p40 expression peaked at 8 h and subsided to baseline levels at 39 h postinjection, TDM was also able to induce IFN-gamma expression; however, kinetics of induction of IFN-gamma was different from that of IL-12p40. Maximal levels of LFN-gamma mRNA were reached by 24 h and did not return to baseline by 4 days. In addition, pretreatment of mice with neutralizing monoclonal antibodies directed against IL-12 (C15,6,7 and C15,1.2) blocked IFN-gamma mRNA induction in PEC from TDM-treated mice, We further determined if the induction of IL-12 and/or IFN-gamma contributes to the in vivo priming effect of TDM on peritoneal M phi. TDM-injected mice were treated in vivo with anti-IL-12 or anti-IFN-gamma (XMG,1,6) monoclonal antibodies. TDM-primed M phi were then activated in vitro with LPS and tested for their ability to produce NO and to develop cytostatic activity toward cocultivated L1210 tumor cells. Priming of M phi by TDM was completely blocked by in vivo neutralization of either IL-12 or IFN-gamma as demonstrated by an absence of tumoricidal activity and NO production by TDM-elicited M phi in the presence of LPS, Taken together our results show that TDM, a defined molecule from M. tuberculosis, induces in vivo production of IL-12, Moreover, synthesis of IL-12 mediates TDM priming of mouse peritoneal M phi through IFN-gamma induction.