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Recent studies indicate that interleukin 8 (IL-8) production contributes to the host immune responses against mycobacterial infection. In this study, we were interested to determine whether induction of IL-8 in human monocytes infected with Mycobacterium bovis was regulated by other monocyte-derived cytokines important in antimycobacterial immunity: IL-10 and transforming growth factor ␤ (TGF-␤). Here, we report that IL-10 reduced, in a graded and significant manner, IL-8 production by M. bovis-infected human monocytes. Additionally, the specificity of the observed inhibition was further confirmed, since the addition of an anti-IL-10 neutralizing antibody completely reversed the inhibitory effect. In contrast, addition or neutralization of TGF-␤ appeared to have no significant effect on M. bovis-induced IL-8 secretion by human monocytes, whereas CD40 expression on M. bovis-infected monocytes was significantly inhibited by this cytokine. This was consistent with the finding by the reverse transcription-PCR method that pretreatment with IL-10, but not TGF-␤, potently inhibited IL-8 mRNA levels. Interestingly, neutralization of endogenous IL-10 did not significantly alter IL-8 secretion, suggesting that induction of IL-8 was not significantly affected by coexpression of IL-10 during infection of human monocytes with M. bovis. Collectively, these data indicate that IL-8 production may be regulated when human monocytes are exposed to IL-10 prior to activation with M. bovis BCG. These data will aid in our understanding of the mechanisms involved in regulating the protective immune response to stimulation with M. bovis BCG.

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CXC chemokine-interleukin-8 (IL-8) (CXCL-8) is a potent proinflammatory chemotactic factor that induces important immune responses for antimycobacterial defenses. However, little is known about the biochemical mechanisms by which the mycobacterial antigens upregulate the release of CXCL-8 from human monocytes. In this study, the mechanisms through which Mycobacterium bovis BCG induces CXCL-8 secretion in human monocytes were investigated. We found that M. bovis BCG induced the production of high levels of CXCL-8 by human monocytes. M. bovis-induced CXCL-8 secretion was unaffected by the protein kinase C (PKC) inhibitor bisindolylmaleimide. In contrast, preincubation of the monocytes with the protein tyrosine kinase (PTK) inhibitor genistein resulted in dose-dependent suppression of mycobacteria-induced CXCL-8 secretion. These results were further supported by the fact that treatment of monocytes with herbimycin-A, another well-described inhibitor of PTK activity with a different mechanism of action, significantly diminished the effect of M. bovis on CXCL-8 secretion. In addition, the specificity of this inhibition was demonstrated by the inability of herbimycin-A to block in a significant manner IL-1 beta induction of CXCL-8. Herbimycin-A significantly blocked tyrosine phosphorylation of p59(hck) in response to M. bovis. Finally, two specific NF-kappa B inhibitors, sulfasalazine and caffeic acid phenetyl ester (CAPE), strongly inhibited the production of CXCL-8 by human monocytes infected with M. bovis. These results show intracellular signaling pathways and a transcription factor involved in the M. bovis-mediated upregulation of CXCL-8 biosynthesis and release by human monocytes.