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 M ycobacterium spp., is a powerful immunostimulant, TDM primes murine ma crophages (M phi) to produce nitric oxide (NO) and to develop antitumo ral activity upon activation with low doses of lipopolysaccharide (LPS ). In this study, we investigated the ability of TDM to induce interle ukin 12 (IL-12) aod the role of this cytokine in TDM-induced activatio n of murine M phi. RNA isolated from peritoneal exudate cells (PEC) co llected at different times after TDM injection was used to determine I L-12 (p35 and p40 subunits) and gamma interferon (IFN-gamma) mRNA leve ls by semiquantitative reverse transcriptase-PCR. Constitutive express ion 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 al most undetectable in control PEC but was dramatically upregulated in P EC from TDM-injected mice, IL-12p40 expression peaked at 8 h and subsi ded to baseline levels at 39 h postinjection, TDM was also able to ind uce 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 addi tion, pretreatment of mice with neutralizing monoclonal antibodies dir ected against IL-12 (C15,6,7 and C15,1.2) blocked IFN-gamma mRNA induc tion in PEC from TDM-treated mice, We further determined if the induct ion of IL-12 and/or IFN-gamma contributes to the in vivo priming effec t of TDM on peritoneal M phi. TDM-injected mice were treated in vivo w ith 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 thei r ability to produce NO and to develop cytostatic activity toward cocu ltivated L1210 tumor cells. Priming of M phi by TDM was completely blo cked by in vivo neutralization of either IL-12 or IFN-gamma as demonst rated by an absence of tumoricidal activity and NO production by TDM-e licited M phi in the presence of LPS, Taken together our results show that TDM, a defined molecule from M. tuberculosis, induces in vivo pro duction of IL-12, Moreover, synthesis of IL-12 mediates TDM priming of mouse peritoneal M phi through IFN-gamma induction.