Association of interleukin-10 cytokine expression status with HLA non-DRB1*02 and Mycobacterium bovis BCG scar-negative status in south Indian pulmonary tuberculosis patients

Abstract

HLA DRB1*02 and its subtypes predispose individuals for a far-advanced sputum-positive pulmonary tuberculosis transcending ethnic boundaries. Mycobacterium bovis BCG does not afford the desired protection against adult pulmonary tuberculosis, and a spectrum of immune reactivity exists in controls and hospital contacts. All of these findings have been identified and demonstrated in areas of endemicity. Skewing of immunity from protective to pathogenic may involve a shift in the Th1-Th2 paradigm. To elaborate these ideas, we studied gamma interferon (IFN-gamma), interleukin-4 (IL-4), and IL-10 cytokine expression in 71 adult pulmonary tuberculosis patients and 74 controls from areas of endemicity in south India by 48-h microculture and reverse transcription-PCR. Most of the patients and controls expressed IFN-gamma de novo, and in the presence of purified protein derivative (PPD), all of them expressed significantly higher levels of IFN-gamma, suggesting a PPD-specific recall memory. HLA DRB1* allele-dependent IFN-gamma expression was identified only in controls, suggesting a skewing of the immune response in patients. In contrast to the case for IFN-gamma, only some patients and controls expressed IL-4 or IL-10 (Th2 profile); thus, the Th1 profile was identifiable only by a nonexpression of IL-4 or IL-10 in this area of endemicity. The Th2 profile was associated with HLA non-DRB1*02 and BCG scar-negative status in patients, attributing a significant risk (odds ratio = 2.074; 95% confidence interval = 0.612 to 7.07). It is possible that Mycobacterium tuberculosis (PPD)-specific IL-10 is expressed preemptively in unvaccinated (BCG scar-negative) individuals with a non-DR2 genetic background by chronic exposure in this area of endemicity and leads to pulmonary tuberculosis of adults.

FIG. 1

Photograph of a 1.5% agarose gel showing β-actin, IFN-γ, IL-4, and IL-10 bands identified by RT-PCR. Fifty microliters of whole blood from each patient was diluted to 200 μl using RPMI 1640 and cultured in duplicate in the absence (lanes 1 to 4) or in the presence (lanes 5 to 8) of PPD-RT23 (100 U/ml) at 37°C in a CO₂ incubator. After 48 h, cultures were harvested, duplicates were pooled, total RNA was extracted, and cDNA was synthesized and diluted to 100 μl. Five microliters each of this cDNA was used as a template to amplify the β-actin, IFN-γ, IL-4, or IL-10 gene using sequence-specific primers. The results were documented in a Kodak EDAS 120 system, and band intensity was measured as pixels. The cytokine band intensity was normalized to the β-actin band intensity using the formula net intensity of cytokine/net intensity of β-actin × 100. Lane M, standard marker (φ174 digested with HaeIII).

FIG. 2

Comparison of basal (no-antigen control) and PPD-RT23-induced IFN-γ, IL-4, and IL-10 expression in pulmonary tuberculosis patients (n = 43) and controls from areas of endemicity (n = 44) from south India, studied by the whole-blood culture method. Fifty microliters of whole blood diluted to 200 μl with RPMI medium was incubated for 48 h in the presence or absence of antigen in a CO₂ incubator. Ninety-one percent of the controls and 71% of the patients expressed IFN-γ in the no-antigen control mixture, while all of the patients and controls expressed IFN-γ in the presence of PPD. In contrast, only certain individuals (responders, positives), produced IL-4 and/or IL-10 in either the presence or absence of PPD. The values of cytokine expression (percentage of β-actin expression) of a sample in the absence and presence of PPD are connected by lines, and the paired t test (PT) or WRT was applied to determine the significance.