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

Abstract

Trehalose dimycolate (TDM), a glycolipid present in the cell wall of Mycobacterium spp., is a powerful immunostimulant. TDM primes murine macrophages (M) 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) and the role of this cytokine in TDM-induced activation of murine M. 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-␥) 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-␥ expression; however, kinetics of induction of IFN-␥ was different from that of IL-12p40. Maximal levels of IFN-␥ 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-␥ mRNA induction in PEC from TDM-treated mice. We further determined if the induction of IL-12 and/or IFN-␥ contributes to the in vivo priming effect of TDM on peritoneal M. TDM-injected mice were treated in vivo with anti-IL-12 or anti-IFN-␥ (XMG.1.6) monoclonal antibodies. TDM-primed M 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 by TDM was completely blocked by in vivo neutralization of either IL-12 or IFN-␥ as demonstrated by an absence of tumoricidal activity and NO production by TDM-elicited M 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 through IFN-␥ induction.

The taming of IL-12: suppressing the production of proinflammatory cytokines

“…Both LPS and lipoteichoic acid from gram-positive bacteria have been shown to induce IL-12 primarily through their interaction with CD14 [7]. Trehalose dimycolate, a glycolipid present in the cell wall of Mycobacterium tuberculosis [8], and glycoprotein fractions from Trypanosoma cruzi trypomastigotes [9] and Toxoplasma gondii tachyzoites [10] have been shown to induce IL-12. Other inducers of IL-12 include the recombinant Leishmania braziliensis antigen LeIF [11] and the CpG motifs contained in bacterial DNA [12].…”

Immunological Properties of Trehalose Dimycolate (Cord Factor) and Other Mycotic Acid‐Containing Glycolipids–‐A Review

“…TOM thus obviously stimulates important effector cells of innate immunity. Other works showed that TOM also supports an early adaptive immune response by inducing lL-12 and/or IFN-y (67,76). In particular the induction oflL-12 and the depletion of an NK T cell population implicated in the production of IL-4 (17), a cytokine promoting a Th2 response, prove that depending on the experimental conditions TOM can also produce a cytokine milieu supporting a Thl response (67,76).…”

Priming and activation of mouse macrophages by trehalose 6,6′-dicorynomycolate vesicles from Corynebacterium glutamicum

“…After 4 h, non-adherent cells were removed by washing with PBS and the properties of the macrophage monolayers were tested. Their ability to produce NO was measured by nitrite accumulation in the culture supernatants, and their antiproliferative activity was measured by their ability to inhibit the growth of P815 mastocytoma cells as described previously [14,15,20].…”

“…We have previously shown that 7 days after i.p. injec-tion of particles of TDM isolated from mycobacteria, the macrophages harvested ex vivo from the peritoneal cavity of mice are primed [14,15,20,26]; primed macrophages can acquire an NO-dependent antiproliferative activity after a 4-h exposure to ng concentrations of LPS in vitro. Moreover, unlike resident or thioglycollate-elicited macrophages, they exhibit an oxidative burst after priming with a phorbol ester (PMA).…”

“…Numerous subsequent studies have established the molecular mechanisms of this toxicity [1,10] and showed that the glycolipid possesses various additional biological activities [11] ; these latter include antitumoral activity [12], and immunostimulating properties [13] such as priming of murine macrophages to produce nitric oxide (NO), and development of an antitumoral activity upon exposure to low doses of lipopolysaccharide (LPS) [14]. TDM also triggers in vivo the synthesis of interleukin-12, which mediates a TDM-induced priming of mouse macrophages through interferon-Q (IFN-Q) induction [15]. In contrast, little is known on the biological activities of TDM analogs from other bacterial sources, except that they are less toxic for mice than the mycobacterial glycolipid [1].…”