The production of tumour necrosis factor-alpha is decreased in peripheral blood mononuclear cells from multidrug-resistant tuberculosis patients following stimulation with the 30-kDa antigen of Mycobacterium tuberculosis

Abstract

The clearance of intracellular bacteria requires the appropriate induction of proinflammatory cytokines and chemokines to recruit macrophages and T cells to the site of infection. In this study, we investigated the production of tumour necrosis factor (TNF)-alpha, interleukin (IL)-8 and interferon (IFN)-gamma by the peripheral blood mononuclear cells (PBMC) of patients with multidrug-resistant tuberculosis (MDR-TB) in response to in vitro stimulation with the 30-kDa antigen of Mycobacterium tuberculosis. The results were compared with those from cases of newly diagnosed TB (N-TB) and TB with treatment failure (TF-TB), and healthy tuberculin reactors (HTR). The most significantly depressed TNF-alpha levels were found in MDR-TB patients. IFN-gamma production was depressed significantly in all groups of TB patients compared with the HTR group. TNF-alpha secretion in response to the 30-kDa antigen was unchanged by coculturing with recombinant human interferon (rhIFN)-gamma, and was increased dramatically following IL-10 neutralization with an anti-human IL-10 antibody. The IL-8 levels were depressed significantly in MDR-TB patients compared with N-TB patients, but were similar to the IL-8 levels in TF-TB patients. Furthermore, rhTNF-alpha directly increased IL-8 secretion, and neutralizing antibody to TNF-alpha inhibited IL-8 production by the PBMC of MDR-TB patients that were stimulated with the 30-kDa antigen. Taken together, these data suggest that the PBMC of MDR-TB patients typically show TNF-alpha depression in response to the 30-kDa antigen, and this effect is modulated by IL-10. In addition, we highlight the role of TNF-alpha in IL-8 secretion in MDR-TB patients.

Fig. 1

TNF-α and IFN-γ production by PBMCs from TB patients and HTR after in vitro stimulation with the 30-kDa Ag of M. tuberculosis. The PBMCs were stimulated with the 30-kDa Ag, and the supernatant harvested after 18 and 96 h, respectively. Secreted TNF-α and IFN-γ levels in the culture supernatants were measured by ELISA, as described in Materials and methods. The TNF-α (a), IFN-γ (b) and IL-10 (c) levels in PBMCs from each group of TB patients and HTR were determined after in vitro stimulation with the 30-kDa Ag. The values are shown as the mean ± s.d. of triplicate supernatant samples. The data are from one representative experiment. *P < 0·05; **P < 0·01; ***P < 0·001 (Student's t-test).

Fig. 2

The effect of rhIFN-γ or endogenous IL-10 on TNF-α induction by the 30-kDa Ag of M. tuberculosis. PBMC from HTR (n = 5) and MDR-TB patients (n = 5) were cultured with or without rhIFN-γ, neutralizing antibody to IL-10 (2 µg/ml), or control antibody (2 µg/ml). The 30-kDa Ag (1 µg/ml) was added to each of the cultures. Immunoreactivity for TNF-α was assessed in the culture supernatants at 18 h. The data are the mean ± s.d. of a representative result of three independent experiments. The percentage increase in TNF-α immunoreactivity relative to the TNF-α level of a culture that was treated with the 30-kDa Ag alone (100%) is shown. □, 30-kDa only; , 30-kDa + rhIFN-γ; ▪, 30-kDa + α-IL-10; , 30-kDa + control IgG.

Fig. 3

IL-8 production by PBMC from TB patients and HTR in response to the 30-kDa Ag of M. tuberculosis. (a) IL-8 production in TB patients and HTR. The supernatants were prepared after 96 h, and the cytokine concentrations were measured by ELISA. The values shown are the mean ± s.d. of triplicate supernatant samples. The data are from one representative experiment. (b) There was significant correlation between the IL-8 and TNF-α levels in 30-kDa Ag-stimulated PBMCs from TB patients (n = 57, r = 0·58, P < 0·001), but not those from HTR controls (n = 20, r = 0·17, P > 0·1). •, HTR; —, HTR reg; r, TB; ···, TB reg.

Fig. 4

Effect of IFN-γ and TNF-α on 30-kDa Ag-induced IL-8 production by PBMC of MDR-TB patients. (a) Effect of rhIFN-γ or rhTNF-α (each 10 ng/ml) on IL-8 production induced by the 30-kDa Ag. PBMC from HTR (n = 7) were cultured with or without rhIFN-γ, rhTNF-α, rhIFN-γ+ rhTNF-α, rhIFN-γ+ anti-TNF-α antibody or rhIFN-γ+ control antibody. Then 30-kDa Ag (1 µg/ml) was added to each of the cultures. The culture supernatants were collected at 96 h and assayed for IL-8 production by ELISA. The percentage increase in IL-8 immunoreactivity relative to the IL-8 level of a culture that was treated with 30-kDa Ag alone (100%) is shown. (b) Effect of endogenous TNF-α on IL-8 production that was induced by the 30-kDa Ag. PBMC from HTR (n = 7) were stimulated with the 30-kDa Ag and cultured with or without neutralizing antibody to TNF-α (2 µg/ml) or control antibody (2 µg/ml). The percentage increase in IL-8 immunoreactivity relative to the IL-8 level of a culture that was treated with 30-kDa Ag alone is shown. (c) Effects of rhTNF-α on 30-kDa Ag-induced IL-8 production by PBMC from MDR-TB patients (n = 7). The data are representative of three separate experiments. ▪, 30-kDa; □, 30-kDa + rhTNF-α.