Hydroquinone, a reactive metabolite of benzene, enhances interleukin-4 production in CD4+ T cells and increases immunoglobulin E levels in antigen-primed mice

Abstract

Exposure to cigarette smoke is known to increase the risk of the development of allergic disease. The mechanism is not well understood. In this study, we determined the effect of hydroquinone (HQ), a major metabolite of benzene present in large quantities in cigarette tar, on interleukin-4 (IL-4) production by CD4+ T cells. HQ significantly enhanced IL-4 production by keyhole limpet haemocyanin (KLH)-primed CD4+ T cells in a dose-dependent manner. The enhancing effect of HQ on IL-4 production was maximal at a concentration of 50 micro m. It increased the level of IL-4 production approximately 10-fold. HQ enhanced IL-4 mRNA expression and also IL-4 gene promoter activity, suggesting that the enhancing effect of HQ on IL-4 production may occur at the transcriptional level. Furthermore, the injection of KLH-primed mice with HQ resulted in a significant increase in the levels of IL-4 and immunoglobulin E. These findings provide evidence that HQ, a major component of cigarette tar, may enhance allergic immune responses by inducing the production of IL-4 in CD4+ T cells.

Figure 1

Effect of HQ on IL-4 production in KLH-primed lymph node cells. Mice were injected into the footpad with KLH in alum. Seven days later, the lymph node cells were collected and stimulated in vitro for 4 days with KLH in the presence of varying amounts of HQ. The cell culture supernatants were harvested and assayed for IL-4 by ELISA. The values represent the means±SEM (n=3). *P<0·05, relative to the group without HQ.

Figure 2

Effect of HQ on IL-4 mRNA expression by KLH-primed lymph node cells. Lymph node cells were from KLH-primed mice, as described in the legend to Fig. 1, and re-stimulated for 24 hr with 5 µg/ml KLH in the presence of varying amounts of HQ (a), or in the presence of 10 µm for varying times (b). Cellular RNA from each treatment was extracted and the expression of IL-4 mRNA was determined by RT–PCR.

Figure 3

HQ-mediated the enhancement of IL-4 promoter activated by PMA/ionomycin. EL-4 cells were transiently transfected with the IL-4 promoter constructs followed by stimulation with PMA/ionomycin in the presence of HQ. The results are represented as induction fold over the value obtained with unstimulated EL-4 cells transfected with each of promoter constructs, given as an arbitrary value of 1. The data are representative of three independent experiments.

Figure 4

CD4⁺ T cells are the major cell-type responsive to HQ. Mice were injected in the footpad with KLH in alum. Seven days afterward, draining lymph node cells were incubated in vitro with anti-CD8 or anti-CD4 mAbs on ice for 30 min, followed by incubation with complement at 37° for 45 min After washing, cells from each treatment group were stimulated with KLH (50 µg/ml) in the presence of HQ (20 or 50 µm) for 4 days and IL-4 levels in the culture supernatants were analysed by ELISA. The data represent the means±SEM (n=3). *P<0·01, relative to a group treated with complement only.

Figure 5

IL-4 production by lymph node cells in KLH-primed mice treated in vivo with HQ. Mice (n=5) were injected in the footpad with KLH in alum. The mice were injected i.p. HQ (50 mg/kg) or saline every other day. One week after the last injection, lymph node cells were re-stimulated in vitro with KLH (0–100 µg/ml) for 4 days. IL-4 levels in the culture supernatants were analyzed by ELISA. The data represent the means±SEM (n=3). *P<0·01, relative to groups without HQ treatment.

Figure 6

Effect of HQ on serum IgE levels in KLH-primed mice. Mice (five per group) were treated with the same protocol as described in the legend of Fig. 5. One week after immunization, total IgE levels (a) and KLH-specific IgE levels (b) in sera were determined by ELISA. The bars (a) represent the average of IgE levels of individual mouse and the data (b) represent the means±SD from triplicate determinations. The experiment was repeated twice with similar results. *P<0·01, relative to other groups.