Regulation of interleukin-8 by interleukin-10 and transforming growth factor beta in human monocytes infected with mycobacterium bovis

Abstract

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 beta (TGF-beta). 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-beta 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-beta, 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.

FIG. 1.

Inhibition of M. bovis-induced IL-8 secretion in human monocytes by IL-10. Monocytes (10⁵/well) were treated with BCG alone (solid bar) or with increasing concentrations of IL-10 (shaded bars) for 4 h at 37°C; 10⁵ M. bovis organisms were added, and the cells were incubated for an additional 24 h at 37°C. The cell culture supernatants were collected and analyzed for IL-8 protein by ELISA as described in Materials and Methods. The results are the means + SEM of five independent experiments. The number in parentheses indicates the percentage of inhibition by IL-10 with respect to M. bovis cultures.

FIG. 2.

Neutralizing anti-IL-10 antibody significantly reverses the inhibitory effect of IL-10 on M. bovis-mediated IL-8 production. Monocytes (10⁵/well) were incubated with IL-10 (2 ng/ml) in the presence of different amounts of an anti-IL-10 antibody or 10 μg of an isotype control antibody/ml for 4 h prior to M. bovis stimulation for an additional 24 h at 37°C. IL-8 levels were measured by ELISA. The numbers in parentheses indicate the percentages of inhibition by IL-10 with respect to M. bovis cultures. The results are the means + SEM for four separate experiments.

FIG. 3.

Effect of exogenous recombinant TGF-β on M. bovis-mediated IL-8 secretion. (A) Monocytes (10⁵/well) were cultured in the absence (solid bar) or presence of increasing concentrations of TGF-β (shaded bars) for 4 h at 37°C; 10⁵ M. bovis organisms were added, and the cells were incubated for an additional 24 h at 37°C. The cell culture supernatants were harvested, and the concentrations of IL-8 were determined by ELISA. The results are the means + SEM of five independent experiments. (B and C) CD40 surface expression on monocytes was analyzed by flow cytometry. Monocytes were cultured in the absence (B) or the presence (C) of TGF-β for 4 h and then infected with 10⁵ M. bovis organisms overnight. The cells were washed twice, stained with FITC-conjugated anti-CD40 antibody, and analyzed by one-color flow cytometry. One representative experiment of three is shown. FL1-height represents FITC fluorescence. The thin lines indicate the basal levels of fluorescence with isotype control antibody, and the thick lines indicate monocyte CD40 expression.

FIG. 4.

Cytokine regulation of IL-8 mRNA expression in M. bovis-infected monocytes. (A) Cells were pretreated with IL-10 or TGF-β for 4 h and then infected with 10⁵ M. bovis organisms overnight (IL-10 +M.bovis and TGF-β+M.bovis). Controls were treated either with medium (Med) or with M. bovis alone (M.bovis). Total RNA was isolated from the cells and subjected to reverse transcription-PCR analysis. GAPDH served as an internal control. (B) IL-8 levels were quantified by densitometry relative to GAPDH. The results are from one of three independent experiments with similar results. +, present; −, absent.

FIG. 5.

Effects of anti-IL-10 and anti-TGF-β on M. bovis-induced IL-8 production. Monocytes were preincubated in culture medium in the presence of different amounts of an anti-IL-10 antibody (A) or anti-TGF-β (B). An isotype-matched control antibody was used as a negative control. M. bovis organisms (10⁵) were added to single cultures, and incubation continued for 24 h. The supernatants were collected and filtered, and IL-8 levels were measured by ELISA. The results are the means + SEM of five different experiments. +, present; −, absent.