Neutralization of tumor necrosis factor alpha suppresses antigen-specific type 1 cytokine responses and reverses the inhibition of mycobacterial survival in cocultures of immune guinea pig T lymphocytes and infected macrophages

Abstract

Neutralization of tumor necrosis factor alpha (TNF-alpha) significantly down-regulated antigen-induced lymphoproliferation and the expression of interleukin-12 p40 and gamma interferon mRNA and enhanced the viability of intracellular attenuated and virulent mycobacteria in cocultures of immune T cells and macrophages obtained from Mycobacterium bovis BCG-vaccinated guinea pigs. This suggests the crucial role of TNF-alpha in the activation of a type 1 T-cell response against Mycobacterium tuberculosis infection.

FIG. 1.

Effect of guinea pig PM on the proliferation of splenic immune T cells to PPD (A) and ConA (B). Different numbers of adherence-purified PM (0 × 10⁵ to 4 × 10⁵ cells/well) were cocultured with the same number of splenic T cells (2 × 10⁵ cells/well) from BCG-vaccinated guinea pigs in the presence of PPD (12.5 μg/ml) or ConA (5 μg/ml). Proliferative responses are expressed as a stimulation index, which is counts per minute of stimulated cells divided by counts per minute of unstimulated cells. Results are displayed as the means ± standard errors of the means of four or five animals per group. Differences between stimulation indices from different numbers of PM in cocultures were tested for statistical significance by analysis of variance followed by Duncan's post hoc analysis (P < 0.05 [*]).

FIG. 2.

Effect of neutralizing endogenous TNF-α on the proliferation of splenic immune T cells induced by guinea pig PM infected with virulent M. tuberculosis H37Rv. Different numbers of PM (0 × 10⁵ to 4 × 10⁵ cells/well) were infected with a live virulent (H37Rv) strain of M. tuberculosis at an MOI of 1:10 and cocultured with the same number of splenic immune T cells (2 × 10⁵ cells/well) in the presence or absence of anti-rgpTNF-α antibody (1:1,000). Proliferative responses are expressed as a stimulation index, which is counts per minute of stimulated cells divided by counts per minute of unstimulated cells. Results are displayed as the means ± standard errors of the means of four or five animals per group. Differences between anti-rgpTNF-α-treated and untreated cultures were tested for statistical significance by analysis of variance followed by Duncan's post hoc analysis (P < 0.05 [*]).

FIG. 3.

Effect of neutralizing endogenous TNF-α on expression of TNF-α, IL-12 p40, and IFN-γ mRNA levels in cocultures following infection with virulent M. tuberculosis H37Rv. Levels of TNF-α (A), IL-12 p40 (B), and IFN-γ (C) mRNA were quantified in guinea pig splenic immune T-cell and peritoneal macrophage cocultures at 6 and 24 h after infection with a live virulent (H37Rv) strain of M. tuberculosis at an MOI of 1:10 in the presence or absence of anti-rgpTNF-α antibody (1:1,000). Fold induction was determined from the threshold cycle values normalized for hypoxanthine phosphoribosyltransferase expression and then normalized to the values derived from unstimulated cultures. Results are displayed as the means ± standard errors of the means of four or five animals. Differences between anti-rgpTNF-α-treated and untreated cultures were tested for statistical significance by analysis of variance followed by Duncan's post hoc analysis (P < 0.05 [*]).

FIG. 4.

Effect of neutralizing endogenous TNF-α on the growth of mycobacteria in cocultures of T cells and macrophages from BCG-vaccinated guinea pigs. Resident peritoneal macrophages were infected with live attenuated (H37Ra) (A and B) or virulent (H37Rv) (C and D) strains of M. tuberculosis at an MOI of 1:10 (A and C) or 1:1 (B and D). After 3 h, extracellular mycobacteria were washed away and autologous splenic immune T cells were added. The cocultures were incubated with or without anti-rgpTNF-α antibody until day 4. The counts per minute (cpm) of tritiated uracil taken up by mycobacteria in cultures were measured. Results are expressed as the means ± standard errors of the means of results from five experiments. Differences between the counts per minute from anti-rgpTNF-α-treated and untreated cultures were compared by Student's t test (P < 0.05 [*]).