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. 2007 Apr;75(4):1870-7.
doi: 10.1128/IAI.00858-06. Epub 2007 Feb 5.

Guinea pig neutrophils infected with Mycobacterium tuberculosis produce cytokines which activate alveolar macrophages in noncontact cultures

Kirti V Sawant  1 , David N McMurray
Affiliations

Guinea pig neutrophils infected with Mycobacterium tuberculosis produce cytokines which activate alveolar macrophages in noncontact cultures

Kirti V Sawant et al. Infect Immun. 2007 Apr.

Abstract

The early influx of neutrophils to the site of infection may be an important step in host resistance against Mycobacterium tuberculosis. In this study, we investigated the effect of M. tuberculosis infection on the ability of guinea pig neutrophils to produce interleukin-8 (IL-8; CXCL8) and tumor necrosis factor alpha (TNF-alpha) and to activate alveolar macrophages. Neutrophils and alveolar macrophages were isolated from naïve guinea pigs, cultured together or alone, and infected with virulent M. tuberculosis for 3, 12, and 24 h. IL-8 protein production in cocultures, as measured by using an enzyme-linked immunosorbent assay, was found to be additive at 24 h and significantly greater in M. tuberculosis-infected cocultures than in uninfected cocultures and in cultures of the infected neutrophils or macrophages alone. The IL-8 mRNA levels, determined by real-time reverse transcription-PCR, were elevated at 24 h in infected cocultures and infected cells cultured alone. In order to elucidate the contributions of neutrophils and their soluble mediators to the activation of alveolar macrophages, neutrophils and alveolar macrophages were cultured in a contact-independent manner by using a Transwell insert system. Neutrophils were infected with virulent M. tuberculosis in the upper wells, and alveolar macrophages were cultured in the lower wells. The release of hydrogen peroxide from alveolar macrophages exposed to soluble products from infected neutrophils was significantly increased compared to that from unexposed alveolar macrophages. Significant up-regulation of IL-1beta and TNF-alpha mRNA levels in alveolar macrophages was observed at 24 and 30 h, respectively, compared to those in cells not exposed to soluble neutrophil products. Treatment with anti-guinea pig TNF-alpha polyclonal antibody completely abolished the response of alveolar macrophages to neutrophil products. This finding suggests that TNF-alpha produced by infected neutrophils may be involved in the activation of alveolar macrophages and hence may contribute to the containment of M. tuberculosis infection during the early period of infection.

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Figures

FIG. 1.
FIG. 1.
Expression of IL-8 mRNA in cultures of neutrophils and alveolar macrophages, alone or together, following infection with M. tuberculosis H37Rv. The levels of expression of IL-8 mRNA in cocultures of alveolar macrophages (5 × 105/well) and neutrophils (5 × 105/well) in a ratio of 1:1 (A) and in cultures of neutrophils (B) and alveolar macrophages (C) alone at 3, 12, and 24 h after infection with M. tuberculosis H37Rv (MtbH37Rv) at an MOI of 0.1 are indicated. The level of induction (n-fold) was determined from the CT values normalized for HPRT expression and then normalized to values for uninfected cells for each respective time point. Results are expressed as the means ± standard errors of the means of results for three animals. The IL-8 mRNA levels in infected cells were not found to be statistically significant compared to those in the uninfected cells in the cocultures or the individual cultures.
FIG. 2.
FIG. 2.
Concentrations of IL-8 protein in cultures of neutrophils and alveolar macrophages, either together or alone, following infection with M. tuberculosis H37Rv. Shown are the concentrations of IL-8 protein in cocultures of alveolar macrophages and neutrophils in a ratio of 1:1 (A) and cultures of neutrophils (B) and alveolar macrophages (C) alone at 3, 12, and 24 h after infection with M. tuberculosis H37Rv (MtbH37Rv) at an MOI of 0.1. Protein concentrations were determined by ELISA. Results are expressed as the means ± standard errors of the means of results for three animals. Differences between the infected and uninfected cultures at different time points were compared by using ANOVA followed by Duncan's post hoc analysis. *, P < 0.05.
FIG. 3.
FIG. 3.
Concentrations of TNF-α and IL-8 protein in supernatants of neutrophils infected with M. tuberculosis H37Rv (MtbH37Rv) in a Transwell insert. Neutrophils (2 × 105/well) were infected with M. tuberculosis at an MOI of 5 or 10, and the supernatants were collected at 12 and 24 h. The supernatants were then assayed for TNF-α protein by the L929 bioassay and for IL-8 protein by ELISA. Cell viability at MOIs of 5 and 10 was found to be >80% and 70%, respectively. Results are expressed as the means ± standard errors of the means of results from three experiments. There were three animals per group, and the differences between infected and uninfected cultures were compared by using ANOVA followed by Duncan's post hoc analysis. *, P < 0.05.
FIG. 4.
FIG. 4.
Production of hydrogen peroxide by alveolar macrophages treated with supernatants from infected neutrophils. Alveolar macrophages (2 × 105/well) were treated with supernatants from neutrophils infected with M. tuberculosis H37Rv (MOI of 5) for 24 h (Sup), and H2O2 production was measured by using horseradish peroxide-dependent oxidation of phenol red. Alveolar macrophages treated with supernatants from uninfected neutrophils were used as controls (Un Sup). Some of the alveolar macrophages were treated with anti-recombinant guinea pig TNF-α antibody (1:600 dilution; data not shown). PMA and catalase (Cat) were used by macrophages as a stimulant and an inhibitor of H2O2 production, respectively. The other controls used were uninfected, untreated macrophages (Un AM); macrophages infected with M. tuberculosis H37Rv at an MOI of 0.1 (Rv); and alveolar macrophages treated with supernatants from infected neutrophils and also infected with M. tuberculosis at an MOI of 0.1 (Rv + Sup). H2O2 concentrations (Conc) were expressed in nanomoles. The levels of production of H2O2 by different treatment groups (three animals per group) were compared with that by the untreated control by using ANOVA followed by Duncan's post hoc analysis. *, P < 0.05.
FIG. 5.
FIG. 5.
Expression of IL-1β and TNF-α mRNA in alveolar macrophages cocultured with M. tuberculosis-infected or uninfected neutrophils in a contact-independent manner. Alveolar macrophages (5 × 105/well) were exposed to supernatants from infected neutrophils (2 × 105/well; MOI of 5; PMN+MtbH37Rv) or uninfected neutrophils (PMN) in a Transwell insert coculture system in a contact-independent manner for 24 h. The expression of IL-1β and TNF-α mRNA in alveolar macrophages was measured at 24 and 30 h, and the level of induction (n-fold) was determined from the CT values normalized for HPRT expression and then normalized to values for treated alveolar macrophages at 0 h. The results are expressed as the means ± standard errors of the means of results for four animals per group. Differences between the infected and uninfected cocultures were compared by using ANOVA followed by Duncan's post hoc analysis. *, P < 0.05.
FIG. 6.
FIG. 6.
Effect of anti-guinea pig TNF-α on levels of TNF-α and IL-1β mRNA in alveolar macrophages cocultured with M. tuberculosis-infected neutrophils in a contact-independent manner. Alveolar macrophages were exposed to supernatants from infected neutrophils (MOI of 5; PMN + MtbH37Rv) or uninfected neutrophils (PMN) in a Transwell insert coculture system in a contact-independent manner for 24 h, and some cocultures were treated with anti-guinea pig TNF antibody (anti gp TNFAb). Levels of IL-1β (A) and TNF-α (B) mRNA in alveolar macrophages were quantified at 24 and 30 h, and the level of induction (n-fold) was determined from the CT values normalized for HPRT expression and then normalized to values for cells at 0 h. Results are expressed as the means ± standard errors of the means of results for four animals. Differences between the cultures receiving the various treatments were compared by using ANOVA followed by Duncan's post hoc analysis. *, P < 0.05.
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