Recombinant guinea pig tumor necrosis factor alpha stimulates the expression of interleukin-12 and the inhibition of Mycobacterium tuberculosis growth in macrophages

Abstract

Tumor necrosis factor alpha (TNF-alpha) plays an important role in the host immune response to infection with the intracellular pathogen Mycobacterium tuberculosis. It is essential for the formation of protective tuberculous granulomas and regulates the expression of other cytokines which contribute to a protective immune response. Interleukin-12 (IL-12) is known to promote a Th1 response, which is essential for antimycobacterial resistance. Recombinant guinea pig TNF-alpha (rgpTNF-alpha) protein (17 kDa) was purified, and its bioactivity was confirmed by its cytotoxicity for L929 fibroblasts. High titers of polyclonal anti-gpTNF-alpha antibody were obtained by immunization of rabbits. Resident alveolar and peritoneal macrophages were isolated from guinea pigs and infected with either the H37Ra or H37Rv strain of M. tuberculosis. The mRNA levels for TNF-alpha and IL-12 p40 were measured using real-time PCR. IL-12 p40 mRNA was up-regulated in a dose-dependent manner by rgpTNF-alpha alone. In infected macrophages, a lower dose of rgpTNF-alpha intensified the mRNA levels of TNF-alpha and IL-12 p40. However, higher doses of rgpTNF-alpha suppressed TNF-alpha and IL-12 p40 mRNA. The antimycobacterial activity of macrophages was assessed by metabolic labeling of M. tuberculosis with [3H]uracil. Resident alveolar and peritoneal macrophages treated with anti-gpTNF-alpha antibody to block endogenous TNF-alpha exhibited increased intracellular mycobacterial growth. These data suggest that the dose of TNF-alpha is crucial to the stimulation of optimal expression of protective cytokines and that TNF-alpha contributes to the control of mycobacterial replication to promote host resistance against M. tuberculosis.

FIG. 1.

rgpTNF-α bioactivity (percent L929 cell cytotoxicity) compared to recombinant human TNF-α (rhTNF-α) in the L929 bioassay. Bioactivities of 1, 5, 10, 50, 100, and 200 ng of rgpTNF-α/ml were assessed against a standard curve generated with commercial rhTNF-α. Results are given as the means ± standard errors of the means of results from three or four experiments. Differences between the bioactivities of different amounts of rgpTNF-α were compared by analysis of variance (*, P < 0.0001).

FIG. 2.

Neutralization of rgpTNF-α bioactivity (percent L929 cell cytotoxicity) by polyclonal anti-rgpTNF-α in the L929 bioassay. Fifty nanograms of rgpTNF-α per milliliter was incubated with serial dilutions of rabbit polyclonal anti-gpTNF-α antibody (1:20,000, 1:10,000, 1:5,000, and 1:2,500) for 30 min prior to the L929 bioassay. Results are given as the means ± standard errors of the means of the standard recombinant human TNF-α employed to construct the standard curve and represent the results from three experiments. Differences between the residual bioactivities by anti-gpTNF-α polyclonal antibody were compared by analysis of variance (*, P < 0.005).

FIG. 3.

Expression of TNF-α and IL-12 p40 mRNA in guinea pig resident alveolar or peritoneal macrophages stimulated by rgpTNF-α. Expression of TNF-α (A and C) and IL-12 p40 (B and D) mRNA was quantified in guinea pig alveolar (A and B) or peritoneal (C and D) macrophage cultures at 3, 6, and 12 h after stimulation with 10, 50, or 200 ng of rgpTNF-α/ml. Fold induction was determined from the Ct values normalized for HPRT expression and then normalized to the values derived from unstimulated macrophage cultures at 0 h. Results are given as the means ± standard errors of the means of results from three experiments. Differences between the fold inductions from rgpTNF-α treatment cultures were compared by analysis of variance (*, P < 0.05).

FIG. 4.

Expression of TNF-α and IL-12 p40 mRNA in guinea pig resident alveolar or peritoneal macrophages following infection with attenuated M. tuberculosis H37Ra alone or with rgpTNF-α. Expression of TNF-α (A and C) and IL-12 p40 (B and D) mRNA was quantified in guinea pig alveolar (A and B) or peritoneal (C and D) macrophage cultures at 3, 6, and 12 h after infection with a live attenuated (H37Ra) strain of M. tuberculosis alone at an infectivity ratio of 1:100 or in the presence of 50 or 200 ng of rgpTNF-α/ml. Fold induction was determined from the Ct values normalized for HPRT expression and then normalized to the values derived from unstimulated macrophage cultures at 0 h. Results are given as the means ± standard errors of the means of results from three experiments. Differences between the fold inductions from in vitro infected cultures were compared by analysis of variance (*, P < 0.05).

FIG. 5.

Expression of TNF-α and IL-12 p40 mRNA in guinea pig resident alveolar or peritoneal macrophages following infection with virulent M. tuberculosis H37Rv alone or with rgpTNF-α. Expression of TNF-α (A and C) and IL-12 p40 (B and D) mRNA was quantified in guinea pig alveolar (A and B) or peritoneal (C and D) macrophage cultures at 3, 6, and 12 h after infection with a live virulent (H37Rv) strain of M. tuberculosis alone at an infectivity ratio of 1:100 or in the presence of 50 or 200 ng of rgpTNF-α/ml. Fold induction was determined from the Ct values normalized for HPRT expression and then normalized to the values derived from unstimulated macrophage cultures at 0 h. Results are given as the means ± standard errors of the means of results from three experiments. Differences between the fold inductions from in vitro infected cultures were compared by analysis of variance (*, P < 0.05).

FIG. 6.

Effect of neutralizing endogenous TNF-α on attenuated M. tuberculosis H37Ra growth in alveolar or peritoneal macrophages. Resident alveolar (A and B) or peritoneal (C and D) macrophages were infected with a live attenuated (H37Ra) strain of M. tuberculosis at an infectivity ratio of 1:1 (A and C) or 1:10 (B and D). After 3 h, extracellular mycobacteria were washed away and the infected cultures were incubated with or without anti-gpTNF-α antibody until day 7. The counts per minute (cpm) of tritiated uracil taken up by mycobacteria in cultures were measured at days 1, 4, and 7. Results are given as the means ± standard errors of the means of results from three experiments. Differences between the counts per minute from anti-gpTNF-α-treated and untreated cultures were compared by Student's t test (*, P < 0.05).

FIG. 7.

Effect of neutralizing endogenous TNF-α on virulent M. tuberculosis H37Rv growth in alveolar or peritoneal macrophages. Resident alveolar (A and B) or peritoneal (C and D) macrophages were infected with a live virulent (H37Rv) strain of M. tuberculosis at an infectivity ratio of 1:1 (A and C) or 1:10 (B and D). After 3 h, extracellular mycobacteria were washed away and the infected cultures were incubated with or without anti-gpTNF-α antibody until day 7. The counts per minute (cpm) of tritiated uracil taken up by mycobacteria in cultures were measured at days 1, 4, and 7. Results are given as the means ± standard errors of the means of results from three experiments. Differences between the counts per minute from anti-gpTNF-α-treated and untreated cultures were compared by Student's t test (*, P < 0.05).