Opsonizing antibodies (IgG1) up-regulate monocyte proinflammatory cytokines tumour necrosis factor-alpha (TNF-alpha) and IL-6 but not anti-inflammatory cytokine IL-10 in mycobacterial antigen-stimulated monocytes-implications for pathogenesis

Abstract

Cachexia is one of the prominent features of advanced tuberculosis (TB) seen in association with increased expression of the monokine TNF-alpha. Several mycobacterial proteins, including PPD, stimulate TNF-alpha secretion from monocytes. Host factors that may play a role in cytokine expression from monocytes remain largely unknown. One such factor is the opsonizing antibodies. Monocytes have high-affinity receptors (FcgammaI and FcgammaIII) for IgG1 and IgG3 antibodies that mediate antigen uptake. We have reported selective up-regulation of IgG1 (which bind to Fcgamma receptors) in advanced TB and have recently shown the ability of PPD-specific IgG1 antibodies to augment TNF-alpha expression in PPD-stimulated monocytes. These observations have now been extended to other cytokines with semipurified fractions from secreted antigens of Mycobacterium tuberculosis (containing 30 kD and 58 kD) that were devoid of lipids, glycolipids and carbohydrates. In the presence of heat-inactivated TB plasma containing known amounts of antigen-specific IgG1 antibodies, these fractions induced significantly increased TNF-alpha, IL-6 and IL-10 secretion. Absorption of IgG1 with Protein 'A' removed the augmenting activity for TNF-alpha and IL-6 secretion from the TB plasma samples. In the case of IL-10, removal of IgG1 resulted in increased rather than decreased IL-10 secretion. These results suggest a possible pathogenic role for antibodies in TB by enhancing proinflammatory and blocking down-regulatory cytokines such as IL-10 cytokines during the chronic phase of TB.

Fig. 2

Kinetics of cytokine (TNF-α (•), IL-6 (▪) and IL-10 (Δ)) release from antigen-stimulated monocytes. Purified adherent cells (1 × 10⁶ cells in 500 μl) were stimulated with various concentrations of PPD, Fx10 and Fx24. Cytokines were assessed in the supernatants after 24 h of antigen stimulation using ELISA assays.

Fig. 1

Fractionation of TNF-α stimulating fractions. Separation of culture filtrate antigens using Prep cell as described. Pooled fractions were run on a 10% acrylamide gel, transferred to nitrocellulose paper and stained with aurodye. Each fraction was also monitored for its capacity to directly stimulate TNF-α in purified adherent cell population. All fractions were tested at a concentration of 0·1 μg/ml.

Fig. 3

Functional assessment of adherent cell assay for TNF-α release from two skin test-negative donors (TE and DH). Purified adherent cells (1 × 10⁶ cells in 500 μl) were stimulated with either lipopolysaccharide (LPS; 0·1 μg), Fx10 (0·1 μg) or Fx24 (0·1 μg) in the presence or absence of Polymyxin B (PMB; 10 μg).

Fig. 4

Augmentation of TNF-α secretion in the presence of tuberculosis plasma from purified adherent cells. Purified adherent cells (1 × 10⁶ cells in 500 μl) were stimulated with either Fx10 (0·1 μg) or Fx24 (0·1 μg) in the presence of control plasma or TB plasma or antigen alone. TNF-α was assessed in the supernatants after 24 h of stimulation by ELISA. Spontaneous release of TNF-α by adherent cells was also assessed for plasma samples without any added stimulant (spontaneous), and was <10 pg/ml for all plasma samples.

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 (□) or protein ‘A’-treated plasma (▪) to Fx10 (top panel) and to Fx24 (bottom panel). Antibody activity is expressed as optical density (OD) units/ml (OD × dilution of the plasma).