Effect of Mycobacterium bovis BCG vaccination on Mycobacterium-specific cellular proliferation and tumor necrosis factor alpha production from distinct guinea pig leukocyte populations

Abstract

In this study, we focused on three leukocyte-rich guinea pig cell populations, bronchoalveolar lavage (BAL) cells, resident peritoneal cells (PC), and splenocytes (SPC). BAL cells, SPC, and PC were stimulated either with live attenuated Mycobacterium tuberculosis H37Ra or with live or heat-killed virulent M. tuberculosis H37Rv (multiplicity of infection of 1:100). Each cell population was determined to proliferate in response to heat-killed virulent H37Rv, whereas no measurable proliferative response could be detected upon stimulation with live mycobacteria. Additionally, this proliferative capacity (in SPC and PC populations) was significantly enhanced upon prior vaccination with Mycobacterium bovis BCG. Accordingly, in a parallel set of experiments we found a strong positive correlation between production of antigen-specific bioactive tumor necrosis factor alpha (TNF-alpha) and prior vaccination with BCG. A nonspecific stimulus, lipopolysaccharide, failed to induce this effect on BAL cells, SPC, and PC. These results showed that production of bioactive TNF-alpha from mycobacterium-stimulated guinea pig cell cultures positively correlates with the vaccination status of the host and with the virulence of the mycobacterial strain.

FIG. 1.

Morphology of the cell subtypes identified in BAL cells (A), SPC (B), and PC (C). A total of 7 × 10⁴ cells from each subpopulation were mounted onto silanated slides by means of a cytospin. After brief air drying, the slides were fixed and stained with Dif-Quik, and a coverslip was applied with mounting medium. Representative photographs depict individual cell types as determined by morphology. Eo, eosinophil; L, lymphocyte; N, neutrophil; KC, Kurloff cell; Eb, erythroblast; Pe, proerythrocyte (Pe). Magnifications, ×100 (A and C) and ×40 (B).

FIG. 2.

Proliferative responses of leukocyte-rich guinea pig cell populations. BAL cells (A), SPC (B), and PC (C) from both unvaccinated (open bars) and BCG-vaccinated (closed bars) guinea pigs were prepared as described in the text and incubated for 4 days in the presence of mitogenic stimuli (ConA or LPS) or mycobacterium-specific stimuli, including a live attenuated (H37Ra) or virulent (H37Rv) strain of M. tuberculosis at an infectivity ratio of 1:100 or hkRv. The SI is defined as the ratio of counts per minute of tritiated thymidine taken up by stimulated cells to counts per minute of unstimulated cells from the same source. Results are given as the means ± standard errors of the means for four animals per group. Differences between the vaccination groups were compared by Student's t test, assuming equal variances. P values of <0.05 (*) were considered significant.

FIG. 3.

Total bioactive TNF-α production from BAL cells, SPC, and PC and effect of BCG vaccination. BAL cells (A), SPC (B), and PC (C) from both vaccinated (closed bars) and unvaccinated (open bars) guinea pigs were cultured with either live or heat-killed mycobacteria for 1, 2, and 3 days. At the indicated time points, supernatants were collected and tested for levels of bioactive TNF by the L929 bioassay. Results are means ± standard errors of the means for three animals per treatment group. Differences between vaccination treatment groups were compared by Student's t test, assuming equal variances. P values of <0.05 (*) were considered significant.

FIG. 4.

Spontaneous bioactive TNF production from BAL cells, SPC, and PC. BAL cells (A), SPC (B), and PC (C) from both vaccinated (closed bars) and unvaccinated (open bars) guinea pigs were cultured in medium alone for the indicated time intervals. At the indicated time points, supernatants were collected and tested for levels of total bioactive TNF. The limit of detection for this assay was routinely 40 pg/ml; consequently, 20 pg/ml was used for determining the averages when results fell below the threshold of detection. Results are means ± standard errors of the means for four animals per treatment group.