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. 2007 Aug;149(2):387-98.
doi: 10.1111/j.1365-2249.2007.03425.x. Epub 2007 Jun 12.

Lung macrophages from bacille Calmette-Guérin-vaccinated guinea pigs suppress T cell proliferation but restrict intracellular growth of M. tuberculosis after recombinant guinea pig interferon-gamma activation

A Jeevan  1 , K Majorov, K Sawant, H Cho, D N McMurray
Affiliations

Lung macrophages from bacille Calmette-Guérin-vaccinated guinea pigs suppress T cell proliferation but restrict intracellular growth of M. tuberculosis after recombinant guinea pig interferon-gamma activation

A Jeevan et al. Clin Exp Immunol. 2007 Aug.

Abstract

The guinea pig model of low-dose pulmonary tuberculosis has been used to study the pathogenesis of infection as well as the mechanisms of bacille Calmette-Guérin (BCG) vaccine-induced resistance. We investigated the function of lung cells from naive and BCG-vaccinated guinea pigs after enzymatic digestion of lung tissue with collagenase and DNase I. The total lung digest cells proliferated poorly to purified protein derivative (PPD) but comparatively better to ConA as assessed by [(3)H]-thymidine uptake. However, the non-adherent population obtained after plastic adherence of lung digests showed an enhanced response to concanavalin A (ConA) and PPD. Therefore, proliferation to ConA and PPD of nylon wool-purified T cells co-cultured with peritoneal (PMøs), alveolar (AMøs) or lung macrophages (LMøs) was assessed. Co-cultures of lung T cells and PMøs showed maximum proliferation to PPD, whereas proliferation was suppressed significantly by the addition of AMøs or LMøs. The response of T cells to ConA was unaffected in co-cultures. Incubation of co-cultures with recombinant guinea pig interferon-gamma (rgpIFN-gamma) did not reverse the suppression. In contrast, rgpIFN-gamma-treated plastic adherent LMøs that were non-specific esterase-positive were capable of reducing the intracellular growth of Mycobacterium tuberculosis. Similarly, total, non-adherent and adherent lung digest cells from BCG-vaccinated guinea pigs showed IFN-gamma and tumour necrosis factor (TNF)-alpha mRNA expression in response to ConA, lipopolysaccharide or PPD by reverse transcription-polymerase chain reaction followed by release of TNF protein but not IFN. These studies indicate that rgp-IFN-gamma-treated lung tissue macrophages from BCG-vaccinated guinea pigs are defective for inducing antigen-specific proliferation in T cells, but control the intracellular accumulation of virulent M. tuberculosis.

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Figures

Fig. 1
Fig. 1
Phenotypic analysis of guinea pig lung digest cells. Lung digest cells obtained by enzymatic digestion from naive and bacille Calmette–Guérin (BCG)-vaccinated guinea pigs was characterized by flow cytometry (a), by Diff-quik staining (b) and by non-specific esterase staining (c). Proportions of major histocompatibility complex (MHC) class II+ cells, T cells and their subsets were determined by fluorescence activated cell sorter (FACS) analysis after staining the cells with monoclonal antibodies (mAb) directed against the surface markers of MHC class II+ cells, T cells (CT5), CD4+ T cells (CT7) and CD8+ T cells (CT6). Results are expressed as percentage of positive cells. †P < 0·04 and *P < 0·007 compared with naive group as determined by Student's t-test. The Diff-Quik staining of the cytospins of total lung digest cells from BCG-vaccinated guinea pigs (b) show abundant macrophages (Møs) and Kurloff cells (KC) with occasional lymphocytes (L), neutrophils and eosinophils (b). (c) Lung macrophages obtained after plastic adherence by non-specific esterase staining. Cells from naive animals show similar staining properties.
Fig. 2
Fig. 2
Proliferation of total and non-adherent cells from guinea pig lung digests. Lung digest cells from naive and bacille Calmette–Guérin (BCG)-vaccinated guinea pig cells (2 × 106/ml) were cultured for 4 days in the presence of concanavalin A (ConA) (10 µg/ml) and purified protein derivative (PPD) (12·5 and 25 µg/ml). The cells were harvested after the addition of [3H]-thymidine (1 µg/well) 6 h earlier. The results are expressed as stimulation index calculated by dividing the counts per minute (cpm) of stimulated cells by the cpm of unstimulated cells. The results are the mean and standard error of the mean from three experiments. There were five animals per group. *P < 0·005 in comparisons between groups by Student's t-test.
Fig. 3
Fig. 3
Proliferation in co-cultures of guinea pig lung T cells and macrophages from different anatomical sites. Total and lung T (LgT) cell proliferation in the presence of macrophages from peritoneal (PMøs), lung (LMøs) and alveolar (AMøs) sites. Lung T cells (3 × 105/well) purified on nylon wool columns from bacille Calmette–Guérin (BCG)-vaccinated guinea pigs were co-cultured with peritoneal, lung or alveolar macrophages (1 × 105/well) in the presence of concanavalin A (ConA) (a) or purified protein derivative (PPD) (b) for 4 days and the [3H]-thymidine uptake was assessed. The cell populations were also treated with recombinant guinea pig interferon-γ (rgpIFN-γ) (200 and 500 ng/ml) at the beginning of the culture period and these results are shown in (c). The results represent mean ± standard error of the mean (s.e.m.) from five to 10 animals. Results are expressed as stimulation index. *P < 0·01 and **P < 0·005 when compared with total cells as determined by analysis of variance.
Fig. 4
Fig. 4
Effect of recombinant guinea pig interferon-γ (rgpIFN-γ) on intracellular growth of Mycobacterium tuberculosis in lung macrophages. LMøs from bacille Calmette–Guérin (BCG)-vaccinated guinea pigs were treated with varying doses (50–1000 ng/ml) of rgpIFN-γ for 24 h. The RPMI-1640 medium was removed and fresh medium without antibiotics was added and the cultures were infected with M. tuberculosis (multiplicity of infection 1 : 1) for 3 h. The extracellular bacteria were removed and some cultures were treated with 50–1000 ng/ml also after infection. The [3H]-uracil uptake by viable M. tuberculosis was measured on day 7 and is expressed as counts per minute (cpm). The results represent mean ± standard error of the mean (s.e.m.) from five experiments. The differences between unstimulated and stimulated cultures were examined by a Student's t-test. *P < 0·005 when compared with the untreated cultures.
Fig. 5
Fig. 5
Cytokine mRNA expression in guinea pig lung digest cells. Total (a), non-adherent (b, c) and adherent lung digest cells (d, e) from bacille Calmette–Guérin (BCG)-vaccinated guinea pigs were stimulated with concanavalin A (ConA) (10 µg/ml), lipopolysaccharide (LPS) (1 µg/ml) or purified protein derivative (PPD) (25 µg/ml) for 3, 12 or 24 h. Interferon (IFN)-γ (b, d) and tumour necrosis factor (TNF)-α mRNA (c, e) expression was quantified using real-time polymerase chain reaction (PCR). Fold induction of mRNA was calculated from the threshold cycle values (Ct) normalized to hypoxanthine-quanine phosphoribosyl transferase (HPRT) Ct values and then to unstimulated cultures. The results are expressed as mean ± standard errors of means (s. e.m.) from four to six animals. The differences in the fold induction between unstimulated and stimulated cultures were determined by a Student's t-test or by analysis of variance. †P < 0·05–0·01.
Fig. 6
Fig. 6
Production of tumour necrosis factor (TNF)-α protein in guinea pig lung digest cells. Total and non-adherent lung digest cells (a) were stimulated with purified protein derivative (PPD) (25 µg/ml) for 24 h and the adherent population (b) was stimulated with lipopolysaccharide (LPS) (1 µg/ml) or PPD for 3, 12 or 24 h. The culture supernatants were collected and assayed for the presence of TNF-α protein production by the L929 cytotoxicity assay. The results are expressed as concentration in ng/ml and denote mean and standard errors of means from four animals. *P < 0·006 and †P < 0·05 when compared to the response in untreated cultures determined by a Student's t-test [results in a)] or by analysis of variance [results in (b)].
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References

    1. Kaufmann S. Towards new leprosy and tuberculosis vaccines. Microbiol Sci. 1987;4:324–8. - PubMed
    1. Comstock GW. Epidemiology of tuberculosis. Am Rev Respir Dis. 1982;125:8–15. - PubMed
    1. Young LS, Inderlied B, Berlin OG, Gottlieb MS. Mycobacterial infections in AIDS patients, with an emphasis on the Mycobacterium avium complex. Rev Infect Dis. 1986;8:1024–33. - PubMed
    1. Canetti G. The tubercle bacillus in the pulmonary lesion in man. New York: Springer Publishing Co.; 1955.
    1. Stewart GR, Robertson BD, Young DB. Tuberculosis: a problem with persistence. Nat Rev Microbiol. 2003;1:97–105. - PubMed

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