Profiles of IFN-gamma and its regulatory cytokines (IL-12, IL-18 and IL-10) in peripheral blood mononuclear cells from patients with multidrug-resistant tuberculosis

Abstract

This study investigated the profiles of IFN-gamma and its regulatory cytokines (IL-12, IL-18 and IL-10) in response to a purified protein derivative (PPD) antigen in peripheral blood mononuclear cells (PBMC) from 18 HIV-negative patients with multidrug-resistant tuberculosis (MDRTB), and compared them with those from 19 healthy tuberculin reactors (HTR). ELISA results showed that following stimulation with PPD, IFN-gamma production was significantly reduced, whereas production of both IL-18 and IL-10 was significantly elevated in MDRTB patients compared with HTR. Three out of 18 patients with MDRTB of greater than 4 years duration showed significantly elevated IL-12 p70 production, induced by in vitro PPD stimulation of their PBMC, when compared with data from HTR. However, when taken as a group, MDRTB patients were similar to HTR in their IL-12 p70-producing capacity. IL-12 p70 protein paralleled IL-12 p40 protein expression. In addition, the production of IL-12 p40 was significantly correlated with IL-10 in all patients, but was not correlated with IFN-gamma. Neutralization of IL-10 increased IL-12 p40 about twofold, but did not significantly alter IFN-gamma induction in MDRTB. IFN-gamma in MDRTB was highly correlated with lymphoproliferation and CD4 counts, but was not correlated with IL-12, IL-18 or IL-10 production. Our findings suggest that patients with MDRTB have dysregulated IL-12, IL-18 and IL-10 production during Mycobacterium tuberculosis infection, and the cytokine profiles are similar to those in patients with drug-sensitive advanced TB previously reported in the literature. In addition, IL-10 may not have a dominant role in defective IFN-gamma production in patients with MDRTB.

Fig.1

Lymphoproliferative responses, CD4 counts and IFN-γ production in PBMC from patients with MDRTB and HTR in response to the PPD antigen of Mycobacterium tuberculosis. (a) PBMC were isolated and FACS analysis performed. CD4 count was calculated based on a concomitant complete blood count. (b) PBMC were stimulated for 5 days with the PPD antigen of M. tuberculosis at a concentration of 1·0 μg/ml. Proliferative responses were assessed as ³H]-thymidine incorporation in PBMC from healthy controls and TB patients. Incorporation of ³H]-thymidine occurred during the last 18 h of a 5 day culture; unstimulated PBMC served as controls. (c) PBMC were stimulated for 96 h with PPD antigen at a concentration of 1·0 μg/ml. Supernatant fluids were prepared following a 96 h stimulation with PPD, and IFN-γ production was measured using ELISA. Values are the mean ± s.d. of triplicate supernatant samples. *P < 0·05; **P < 0·01; ***P < 0·001 (Student’s t-test). Significant correlations were found between (d) IFN-γ and CD4 counts (n = 18, r = 0·85, P < 0·001), and (e) between IFN-γ and lymphoproliferative responses in TB patients (n = 18, r = 0·68, P < 0·01).

Fig.2

IL-18 and IL-10 production in PBMC from patients with MDRTB in response to the PPD antigen of M. tuberculosis. (a) IL-18 and (b) IL-10 production in PBMC were determined after in vitro stimulation with PPD. Supernatant fluids were prepared following a 96 h stimulation with PPD, and cytokine production was measured using ELISA. Values are the mean ± s.d. of triplicate supernatant samples. *P < 0·05; **P < 0·01; ***P < 0·001 (Student’s t-test). No significant correlations were found (c) between IFN-γ and IL-18 (n = 18, r = –0·06, P > 0·05), or (d) between IFN-γ and IL-10 production in TB patients (n = 18, r = 0·34, P > 0·05).

Fig. 3

IL-12 production in PBMC from patients with MDRTB in response to the PPD antigen of M. tuberculosis. IL-12 p40 and p70 production in PBMC were determined after in vitro stimulation with PPD antigen. Supernatant fluids were prepared after 18 h and cytokine concentrations were measured using ELISA. Values are the mean ± s.d. of triplicate supernatant samples. (a) IL-12 p40 production in MDRTB patients. (b) IL-12 p70 production in MDRTB patients. A significant correlation was found between (c) IL-12 p40 and IL-10 (n = 18, r = 0·94, P < 0·001), whereas no significant correlation was found between (d) IFN-γ and IL-12 p40 production in TB patients (n = 18, r = 0·24, P > 0·05).

Fig.4

Effect of endogenous IL-10 on PPD-induced IL-12 p40 and IFN-γ production by PBMC. PBMC from HTR (n = 10) and MDRTB patients (n = 9) were cultured with or without neutralizing antibody to IL-10 (2 μl). PPD (1 μg/ml) was added to all cultures. The immunoreactivity for IL-12 p40 and IFN-γ was assessed in culture supernatant fluids at 18 and 96 h, respectively. The percent increase in IFN-γ and IL-12 p40 immunoreactivity compared with those of PPD alone (100%) is shown. (□) PPD only; (▪) a-IL-10 (HTR); (▪) a-IL-10 (MDRTB).