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BACKGROUND: The variable efficacy of bacillus Calmette-Guérin (Mycobacterium bovis BCG) in protecting humans against tuberculosis has prompted a search for the mechanisms through which BCG induces chemokines. In this study, our experiments were designed to determine the role of the transcription factor nuclear factor-kappaB (NF-kappaB) and intracellular calcium in the production of interleukin (IL)-8, a main chemotactic factor, by human-derived monocytic cell line U937 and by a human epithelial HEp-2 cell line infected with M. bovis BCG. METHODS: The concentrations of IL-8 in culture supernatants of U937 cells or HEp-2 cells infected with M. bovis BCG were determined by enzyme-linked immunosorbent assay. We used sulfasalazine and curcumin, which are well-described inhibitors of NF-kappaB activity, and we used ethylenediamine tetraacetic acid to deplete extracellular Ca2+ or used the cell-permeable agent 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester to chelate releasable intracellular stores of Ca2+ in order to investigate the mechanisms through which M. bovis BCG induces IL-8 secretion in our system. RESULTS: The enzyme-linked immunosorbent assay showed that IL-8 protein secretion was elevated in M. bovis-infected cell lines. This effect was statistically significant (p < 0.01). When calcium influx was suppressed in M. bovis-infected cell lines, IL-8 secretion was inhibited. Notably, specific inhibitors of NF-kappaB (sulfasalazine and curcumin) inhibited M. bovis-induced IL-8 secretion from U937 cells or HEp-2 cells. CONCLUSIONS: Collectively, these results indicate that activation of NF-kappaB is an important signal transduction pathway in M. bovis-induced IL-8 secretion in monocytic or epithelial cells. Furthermore, the results showed that calcium influx had a direct effect on IL-8 secretion in U937 cells or HEp-2 cells infected with M. bovis.

In this study, we found evidence that the interleukin-10 (IL-10) protein is functionally relevant in Mycobacterium bovis BCG-induced cytokine synthesis, as neutralization of endogenously synthesized IL-10 in human cells activated with BCG resulted in a two-to threefold increase in the level of IL-1␤. When exogenous recombinant human IL-10 was added to human mononuclear cells, a significant reduction of BCG-induced IL-1␤ secretion was observed. This inhibitory effect was not attributed to a cytotoxic effect, since trypan blue exclusion studies indicated no loss of cell viability in the presence of IL-10, and it was specific, as it was completely abolished in the presence of anti-IL-10 neutralizing monoclonal antibody while an irrelevant antibody used as a control had no effect. Taken together, these are the first studies that demonstrate that the depletion of endogenous IL-10 via anti-IL-10 antibody results in a very significantly enhanced BCG-induced IL-1␤ secretion and that the addition of exogenous IL-10 to human mononuclear cells stimulated with BCG inhibits IL-1␤ production. Further experimental work is needed to determine if the neutralization of IL-10 activity via anti-IL-10 antibody indeed enhances cytokine synthesis in vivo. However, the present results may be of importance, since the use of anti-IL-10 antibody could presumably contribute to the protective immunity induced by BCG against tuberculosis via an increase in cytokine synthesis that would amplify antimicrobial systems.

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