Induction of tumor necrosis factor-alpha (TNF-alpha) by interleukin-12 p40 monomer and homodimer in microglia and macrophages

The present study was undertaken to explore the role of interleukin-12 (IL-12) p40 in the expression of TNF-alpha in microglia. Interestingly, we have found that IL-12 p70, p402 (the p40 homodimer) and p40 (the p40 monomer) dose-dependently induced the production of TNF-alpha and the expression of TNF-alpha mRNA in BV-2 microglial cells. In addition to BV-2 microglial cells, p70, p402 and p40 also induced the production of TNF-alpha in mouse primary microglia and peritoneal macrophages. As the activation of both NF-kappaB and CCAAT/enhancer binding protein beta (C/EBPbeta) is important for the expression of TNF-alpha in microglial cells, we investigated the effect of p40 on the activation of NF-kappaB as well as C/EBPbeta. Activation of NF-kappaB as well as C/EBPbeta by p40 and inhibition of p40-induced expression of TNF-alpha by Deltap65, a dominant-negative mutant of p65, and DeltaC/EBPbeta, a dominant-negative mutant of C/EBPbeta, suggests that p40 induces the expression of TNF-alpha through the activation of NF-kappaB and C/EBPbeta. In addition, we show that p40 induced the activation of both extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). Interestingly, PD98059, an inhibitor of ERK, inhibited p40-induced expression of TNF-alpha through the inhibition of C/EBPbeta, but not that of NF-kappaB, whereas SB203580, an inhibitor of p38 MAPK, inhibited p40-induced expression of TNF-alpha through the inhibition of both NF-kappaB and C/EBPbeta. This study delineates a novel biological function of p40 in inducing TNF-alpha in microglia and macrophages.     

Tolerance induced by the lipopeptide Pam3Cys is due to ablation of IL-1R-associated kinase-1

Stimulation of the human monocytic cell line Mono Mac 6 with the synthetic lipopeptide (S)-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser(S)-Lys(4)-OH, trihydrochloride (Pam(3)Cys) at 10 microg/ml induces a rapid expression of the TNF gene in a TLR2-dependent fashion. Preculture of the cells with Pam(3)Cys at 1 microg/ml leads to a reduced response after subsequent stimulation with Pam(3)Cys at 10 microg/ml, indicating that the cells have become tolerant to Pam(3)Cys. The CD14 and TLR2 expression is not decreased on the surface of the tolerant cells, but rather up-regulated. Analysis of the NF-kappaB binding in Pam(3)Cys-tolerant cells shows a failure to mobilize NF-kappaB-p50p65 heterodimers, while NF-kappaB-p50p50 homodimers remain unchanged. Pam(3)Cys-tolerant cells showed neither IkappaBalpha-Ser(32) phosphorylation nor IkappaBalpha degradation but MyD88 protein was unaltered. However, IRAK-1 protein was absent in Pam(3)Cys-induced tolerance, while IRAK-1 mRNA was still detectable at 30% compared with untreated cells. In contrast, in LPS-tolerized cells, p50p50 homodimers were induced, IRAK-1 protein level was only partially decreased, and p50p65 mobilization remained intact. It is concluded that in Mono Mac 6 monocytic cells, inhibition of IRAK-1 expression at the mRNA and protein levels is the main TLR-2-dependent mechanism responsible for Pam(3)Cys-induced tolerance, but not for TLR-4-dependent LPS-induced tolerance.