PPD-specific IgG1 antibody subclass upregulate tumour necrosis factor expression in PPD-stimulated monocytes: possible link with disease pathogenesis in tuberculosis

Abstract

Cachexia is a prominent feature of advanced tuberculosis, in association with increased expression of the monokine tumour necrosis factor (TNF)-alpha. Monocytes, have high affinity receptors (mannose, complement and Fc gamma1 and gamma111) which mediate antigen uptake and subsequent cytokine activation. Several mycobacterial proteins, including PPD, can stimulate TNF-alpha secretion from monocytes. However, the role of various receptors in stimulating or regulating TNF-alpha secretion is still unclear. We have previously shown selective augmentation of opsonic antibodies (IgG1 and IgG3) in tuberculosis patients with advanced pulmonary disease. We now analyse the role of opsonizing antibodies in modulating TNF-alpha expression in antigen stimulated monocytes. PPD was used as the prototypic mycobacterial antigen to stimulate monocytes from PPD skin test negative donors (n = 7) in the presence of plasma from tuberculosis patients (n = 8), containing known amounts of IgG1 and IgG3 anti-PPD antibodies. TNF-alpha secretion was enhanced in the presence of TB plasma (4/8) but not in the presence of control plasma. Using Spearman Rank analysis (two-tailed Fisher exact test), a significant correlation (rho = 0.762; P = 0. 04) was observed between IgG1 antibodies and enhancement of TNF-alpha secretion. No significant association was observed with IgG2 (rho = 0.310; P = 0.41), IgG3 (rho = 0.089; P = 0.81) or IgG4 (rho = - 0.357; P = 0.347) subclass antibodies. Absorption of IgG1 with protein 'A' removed the enhancement of TNF-alpha secretion activity from the plasma samples. Our results therefore indicate that IgG1 antibodies may enhance the chronic release of TNF-alpha in TB patients with progressive disease and, for the first time, show a direct link between disease pathogenesis and raised antibody levels.

Fig. 1

Functional assessment of adherent cell assay for TNF release from a single PPD skin test negative donor (DR). Purified adherent cells (1 × 10⁶ cells in 500 μl) were stimulated with either LPS (0.1 μg) or PPD (10 μg) in the presence and absence of polymyxin B (10 μg). Spontaneous release of TNF-α by adherent cells was also assessed without any added stimulant (spontaneous), and was <10 pg/ml for all plasma samples tested.

Fig. 2

Enhancement of TNF-α release in the presence of a panel of plasma from patients with active pulmonary tuberculosis and healthy PPD skin test donors. Purified adherent cells (1 × 10⁶ cells in 500 μl) were stimulated with PPD at 10 μg/ml. TNF-α release was assessed in antigen stimulated adherent cells in the presence of either control plasma, or plasma from patients with active pulmonary tuberculosis. All plasma samples were heat inactivated and added at a dilution of 1:500. Spontaneous release of TNF-α by adherent cells was also assessed in the presence of plasma samples without any added stimulant, and was <10 pg/ml for all plasma samples tested. □, Control plasma; , patient plasma.

Fig. 3

Enhancement of TNF-α secretion in seven PPD skin test donors. Results are expressed as concentration of TNF-α release in the presence of control antibodies or patient antibodies compared to PPD stimulated TNF-α release in the absence of added plasma. Vertical bars indicate mean of the group and horizontal lines indicate the standard error around the group mean. All other parameters were the same as those in Fig. 2. ▪, Stimulation with PPD alone; □, addition of control plasma; hatched bars indicate addition of TB plasma.

Fig. 4

Relationship of antibodies to PPD in plasma samples and enhancement of TNF-α secretion. Spearman Rank correlation analysis (two-tailed exact Fisher test) was carried out to determine the correlation. between TNF-α secretion and the concentrations of IgG1 and IgG3 antibodies to PPD in plasma samples (n = 8). Each • represents the average of results obtained from seven donors (± SEM), shown as vertical lines. Antibody concentration is given as units of activity. (a) Antibody activity for IgG1. (b) Antibody activity for IgG3 anti PPD antibodies. The significance of correlation (P-values) is given for antibody subclasses.

Fig. 5

Absorption of IgG1 antibodies from plasma samples obtained from tuberculosis patients. Plasma samples were subjected to protein ‘A’ absorption. IgG1 and IgG3 antibodies were determined in untreated (hatched bars) or protein ‘A’ treated plasma (dotted bars).