Induction of nitric oxide release from the human alveolar epithelial cell line A549: an in vitro correlate of innate immune response to Mycobacterium tuberculosis

Abstract

In view of the presence of a large number of epithelial cells in the alveoli of the lung and their ability to produce various cytokines and chemokines, the possible role of alveolar epithelial cells in the innate immune response to tuberculosis was examined. The human alveolar epithelial cell line A549 was used as a model. The ability of A549 cells to induce nitric oxide (NO) in response to Mycobacterium tuberculosis infection was taken as an in vitro correlate of innate immunity. M. tuberculosis infection induced A549 cells to produce significant levels of NO and to express inducible nitric oxide synthase mRNA at 48 hr of infection. However, the amount of NO released at this point was not mycobactericidal. Cytokine stimulation (interferon-gamma, tumour necrosis factor-alpha, interleukin-1beta, alone or in combination) of the infected A549 cells induced a higher concentration of NO. The study of colony-forming units (CFU) as a measure of the mycobactericidal capacity of A549 cells revealed a reduction in CFU of M. tuberculosis by 39.29% (from 10.62 +/- 0.48 - 6.392 +/- 0.54) following cytokine stimulation of the infected cells. Interestingly gamma-irradiated M. tuberculosis H37Rv could also induce higher than basal level of NO. Therefore we examined mycobacterial antigenic components for their possible role in NO production. We observed that A549 cells produced significantly higher amounts of NO at 48 hr when treated with mycobacterial whole cell lysates, cell wall or cell membrane preparations. The release of NO and the resultant mycobactericidal activity could be further enhanced by simultaneously conditioning the M. tuberculosis infected A549 cells with cytokine and mycobacterial components. These results suggest that alveolar epithelial cells respond to their microenvironment, which is constituted of various cytokines and macrophage-processed antigens and may contribute to the innate immune response to tuberculosis.

Figure 1

RT–PCR result for iNOS gene (322 bp product) mRNA expression at 48 hr. (a) Lane 1: 100 bp marker. Lane 2: A549 cells treated with IFN-γ. Lane 3: A549 cells infected M. tuberculosis H37Rv and treated with IFN-γ. Lane 4: A549 cells treated with cytokine mixture. Lane 5: A549 cells treated with TNF-α. Lane 6: A549 cells infected with M. tuberculosis H37Rv and treated with TNF-α. Lane 7: A549 cells infected with M. tuberculosis H37Rv and treated with cytokine mixture. Lane 8: Untreated A549 cells. (b) β-actin gene mRNA (control, 687 bp product) demonstrating equal loading in all the corresponding lanes as in (a).

Figure 2

M. tuberculosis H37Rv cell components induce NO production by A549 cells. A549 cells were stimulated with different mycobacterial components. Cell culture supernatant was harvested at 24 and 48 hr following stimulation. Results were the mean ± SEM of three independent experiments (n = 3). *P < 0·001 when paired t-test were performed between A549 cells alone and cells stimulated with mycobacterial components at 24 hr. φP < 0·001 when paired t-tests were performed between A549 cell alone and cells stimulated with mycobacterial components at 48 hr. WCL, whole cell lysate of H37Rv; CW, cell wall of H37Rv; MEM, membrane fraction of H37Rv; CYT, cytosol fraction of H37Rv; LAM, lipoarabinomannan of H37Rv.

Figure 3

RT–PCR result for iNOS gene (322 bp product) mRNA expression at 48 hr. (a) Lane 1: 100 bp marker. Lane 2: A549 cells stimulated with whole cell lysate of H37Rv. Lane 3: A549 cells stimulated with cytosol of H37Rv. Lane 4: A549 cells stimulated with LAM of H37Rv. Lane 5: A549 cells stimulated with cell wall fraction of H37Rv. Lane 6: A549 cells stimulated with membrane fraction of H37Rv. Lane 7: A549 cells only. (b) β-actin gene mRNA (control, 687 bp product) demonstrating equal loading in all the corresponding lanes as in (a).

Figure 4

M. tuberculosis components induced NO production from A549 cells infected with M. tuberculosis followed by stimulation with a cytokine mix along with different mycobacterial components. For estimation of NO, cell culture supernatants were harvested at 24, 48, 72 and 100 hr following stimulation. Results were the mean ± SEM of three independent experiments (n = 3). When paired t-tests were performed between A549 cells alone and infected A549 cells stimulated with the cytokine mixture and mycobacterial components, the P-value was <0·001 in the case of all components. Paired t-tests were also performed between infected A549 cells stimulated with cytokine mixture only and infected A549 cells simultaneously treated with the cytokine mixture and different antigenic components. The P-value (ϕ) was < 0·05 in experiments using WCL and CW only. WCL, whole cell lysate of H37Rv; CW, cell wall of H37Rv; MEM, membrane fraction of H37Rv; CYT, cytosol fraction of H37Rv; LAM, lipoarabinomannan of H37Rv. (Infection with H37Rv and cytokine stimulation was common in all experiments shown here as a bar diagram.)

Figure 5

RT–PCR result for IL-8 gene (300 bp product) mRNA expression at 48 hr. (a) Lane 1: 100 bp pair marker. Lane 2: Cytokine mixture stimulated A549 cells. Lane 3: IFN-γ stimulated A549 cells. Lane 4: IFN-γ + TNF-α stimulated A549 cells. Lane 5: TNF-α stimulated A549 cells. Lane 6: A549 cells infected with H37Rv. Lane 7: A549 cells infected with H37Rv and stimulated with IFN-γ. Lane 8: A549 cells infected with H37Rv and stimulated with cytokine mixture. Lane 9: A549 cells only. (b) β-actin gene mRNA (control, 687 bp product) demonstrating equal loading in all the corresponding lanes as in (a).

Figure 6

Concentration of IL-8 produced by A549 cells stimulated with cytokines, infected with live M. tuberculosis (H37Rv) and different mycobacterial components. Cell culture supernatant was harvested at 48 hr following stimulation. Results are mean ± SEM of three independent experiments (n = 3). Paired t-tests were performed between A549 cells alone and A549 cells after various treatment. The P-value (ψ) was < 0·001. Lane 1: A549 cells only. A549 treated with IFN-γ (lane 2), TNF-α (lane 3), cytokine mixture (lane 4), Live H37Rv (lane 5), mycobacterial WCL (lane 6), mycobacterial cell wall component (lane 7), mycobacterial membrane preparation (lane 8), mycobacterial cytosol (lane 9), LAM (lane 10).