Expression and secretion of CXCL-8 and CXCL-10 from mycobacterium bovis BCG-infected human epithelial cells: role of IL-4

Abstract

CXC chemokine release can be modulated by Th2-derived cytokines. Interleukin(IL)-4 is one of the cytokines that are the hallmark of the Th-2 response, and plays an important role in human tuberculosis. In the current study, we investigated the effect of IL-4 on chemokine production by human epithelial cells infected with Mycobacterium bovis bacillus calmette-guérin (BCG). Gene expression of CXCL-8 and CXCL-10 was determined by the reverse transcription (RT)-polymerase chain reaction method. The levels of immunoreactive CXCL-8 and CXCL-10 were determined by enzyme-linked immunosorbent assay. We found that, although M. bovis BCG induced gene expression of CXCL-8 and CXCL-10 in M. bovis BCG-infected human epithelial cells, CXCL-8 mRNA level was significantly reduced by IL-4, whereas no significant effect of IL-4 was observed on CXCL10 mRNA level. In addition, IL-4 decreased CXCL-8 (in a graded and significant manner) but not CXCL-10 secretion. These results were further confirmed, since a significant reversion was obtained with a neutralizing antibody to human IL-4, whereas an isotype-matched control antibody had no significant effect on CXCL-8 secretion. Furthermore, we found a similar effect of IL-4 on M. bovis BCG-induced CXCL-8 and CXCL-10 secretion by using other human epithelial A549 cell line. Collectively, these data demonstrate that M. bovis BCG-infected human epithelial cells can have an active role in a local inflammatory immune response via the secretion of CXC chemokines which can be selectively regulated by Th2-derived cytokines.

Figure 1

IL-4 has divergent effects on M. bovis BCG-induced chemokine gene expression. HEp-2 cells were treated with IL-4 (50 ng/mL) prior to infection with M. bovis BCG. After incubation total RNA was isolated and the levels of (a) CXCL-8 mRNA or (b) CXCL-10 mRNA were measured by RT-PCR method. PCR products were run on a 2% agarose gel containing ethidium bromide. The results depicted are representative of three independent experiments. GAPDH, glyceraldehhyde 3-phosphato dehydrogene probe was used to confirm equal RNA loading. The histograms represent relative transcription rates, which were calculated after normalization to the respective GAPDH signal.

Figure 1

IL-4 has divergent effects on M. bovis BCG-induced chemokine gene expression. HEp-2 cells were treated with IL-4 (50 ng/mL) prior to infection with M. bovis BCG. After incubation total RNA was isolated and the levels of (a) CXCL-8 mRNA or (b) CXCL-10 mRNA were measured by RT-PCR method. PCR products were run on a 2% agarose gel containing ethidium bromide. The results depicted are representative of three independent experiments. GAPDH, glyceraldehhyde 3-phosphato dehydrogene probe was used to confirm equal RNA loading. The histograms represent relative transcription rates, which were calculated after normalization to the respective GAPDH signal.

Figure 2

Effect of recombinant IL-4 on M. bovis BCG-induced CXCL-8 and CXCL-10 secretion in HEp-2 cells. HEp-2 cells were cultured without a stimulus (no stimuli) or infected by M. bovis BCG (5 : 1 bacteria/cell) after 2 h pretreatment with IL-4 (1–50 ng/mL). The (a) CXCL-8 and (b) CXCL-10 protein levels from cellular supernatants were measured by ELISA. Data are presented as mean ± SEM of five independent experiments. Reduction of M. bovis BCG-induced CXCL-8 secretion after addition of 30 or 50 ng IL-4 is statistically significant (*P < .01).

Figure 2

Effect of recombinant IL-4 on M. bovis BCG-induced CXCL-8 and CXCL-10 secretion in HEp-2 cells. HEp-2 cells were cultured without a stimulus (no stimuli) or infected by M. bovis BCG (5 : 1 bacteria/cell) after 2 h pretreatment with IL-4 (1–50 ng/mL). The (a) CXCL-8 and (b) CXCL-10 protein levels from cellular supernatants were measured by ELISA. Data are presented as mean ± SEM of five independent experiments. Reduction of M. bovis BCG-induced CXCL-8 secretion after addition of 30 or 50 ng IL-4 is statistically significant (*P < .01).

Figure 3

Neutralizing anti-IL-4 antibody significantly reverses the inhibitory effect of IL-4 on M. bovis BCG-induced CXCL-8 secretion. HEp-2 cells were pretreated with IL-4 (50 ng/mL) in the presence of different concentrations of anti-IL-4 or an isotype control antibody for 2 h prior to M. bovis infection for an additional 24 h at 37°C. CXCL-8 levels were measured by ELISA. The results are the means ± SEM for four separate experiments.

Figure 4

Effect of recombinant IL-4 on CXC chemokine secretion by human alveolar epithelial A549 cells infected with M. bovis BCG. Cells were treated with medium (no stimuli) or increasing doses of IL-4 for 2 h prior to infection with M. bovis BCG ( MOI = 5). After 24 h incubation, supernatants was collected and (a) CXCL-8 or (b) CXCL-10 were measured by ELISA. Data shown are the mean ± SD of four independent experiments. The percentage in parentheses indicates inhibition in the presence of IL-4 compared with M. bovis BCG cultures which did not receive IL-4.