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Abstract
In this study, we investigated the role of toll-like receptor (TLR) and mitogen-activated protein kinase (MAPK) pathways involved in the tumor necrosis factor (TNF)-α and interleukin (IL)-6 expression after stimulation with purified protein derivatives (PPD) or native 38-kDa protein antigen (Ag) of Mycobacterium tuberculosis H37Rv in human primary monocytes. Both PPD and 38-kDa Ag significantly induced TNF-α and IL-6 in human primary monocytes. MAPK [extracellular signal-regulated kinase (ERK) 1/2 and p38] are rapidly phosphorylated in human monocytes stimulated with the PPD or 38-kDa Ag. Both p38 and ERK 1/2 activation are essential for PPD- or 38-kDa-induced TNF-α and IL-6 production. The inhibition of TLR2 and TLR4 by specific antibodies significantly abrogated the 38-kDa-induced secretion of TNF-α and IL-6, whereas blockade of TLR2, but not TLR4, was responsible for the PPD-induced TNF-α and IL-6 production in human monocytes. Collectively, these data suggest that the PPD and 38-kDa Ag differentially interact with TLR2 and TLR4, which in turn mediate an essential role for the early inflammatory immune responses during human tuberculosis.
References
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Figure 1.
SDS-PAGE analysis of purified 38-kDa antigen (Ag) of M. tuberculosis culture filtrate. (A) The 38-kDa Ag was purified from unheated culture filtrate of M. tuberculosis H37Rv by ammonium sulfate precipitation and hydrophobic interaction chromatography, and anion exchange chromatography. The antigen was separated by SDS-PAGE and then analyzed by Coomassie blue staining. (B) Purified 38-kDa Ag separated by IEF on pH 4.7∼5.9 IPG strip in the first dimension and 12% SDS-PAGE in the second dimension. The second dimension gel was blotted onto nitrocellulose and immunoblot with rabbit anti-38 kDa polyclonal antibody.
Figure 2.
The PPD or 38-kDa Ag-induced TNF-α and IL-6 secretion in human monocytes. Human monocytes were stimulated with the PPD or 38-kDa Ag (5.0 μg/ml, for each) for 0, 6, 18, 48, and 96 h. The TNF-α (A) and IL-6 (B) production were measured by ELISA. The cytokine synthesis by cells stimulated with the 38-kDa Ag was statistically compared with those by unstimulated control. ∗, p<0.05; ∗∗, p<0.01; ∗∗∗, p<0.001. The error bars indicate SD.
Figure 3.
The PPD or 38-kDa Ag-induced MAPK activation from human monocytes. Human monocytes were stimulated with the PPD (A) or 38-kDa (B) Ag for the times indicated. The cells were lysed, and aliquots of the total cell lysates were separated by SDS-PAGE and immunoblotted as described. The blots were incubated overnight with specific anti-phospho-ERK 1/2, anti-phospho-p38, or control Ab each for the unphosphorylated form, followed by appropriate peroxidase-coupled secondary reagents, and were visualized using ECL. Similar data were obtained in six independent experiments. P, phosphorylated form of Ab; total, total form of Ab.
Figure 4.
Effects of MEK or p38 MAPK inhibitors on the PPD or 38-kDa-mediated TNF-α- and IL-6 production. The MEK inhibitor (U0126) or p38 MAPK (SB203580) was added to monocytes at concentrations ranging from 5 to 20 μM at 45 min before stimulation with the PPD (A) or 38-kDa Ag (B) of M. tbc (5.0 μg/ml). The supernatants were harvested after 18 h for cytokine assessment using ELISA. Similar data were obtained in five independent experiments. The mean levels (plus standard errors of the mean) of TNF-α or IL-6 following stimulation with the PPD or 38-kDa Ag were set to 100, and the relative loss of cytokine production in the presence of inhibitor is shown. The solvent control was 0.1% DMSO. SB, SB203580; U, U0126.
Figure 5.
Effects of anti-TLR2 or anti-TLR4 mAbs on the PPD or 38-kDa-mediated TNF-α and IL-6 production. Human monocytes were preincubated with the indicated amounts of anti-TLR2, anti-TLR4, or isotype control Abs (1 μg/ml) and challenged with the PPD (A) or 38-kDa (B) Ag subsequently for 30 minutes. The supernatants were harvested after 18 h for cytokine assessment using ELISA. Similar data were obtained in five independent experiments. The mean values (plus standard errors of the mean) of TNF-α or IL-6 following stimulation with the 38-kDa Ag were set to 100, and the relative loss of cytokine production in the presence of neutralizing Abs was shown. The solvent control was 0.1% DMSO.
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