A Study On The Effect Of Exposure To Allergen At Different Time On A Rat Asthma Model And Its Mechanisms

In recent 20 years"hygiene hypothesis"was proposed to explain the pathogenesis of asthma. The prior study by our lab discovered that injection with ovalbumin into the Sprague-Dawley rats within the first day after birth could obviously inhibit the asthmatic airways inflammation in their adulthood. However, the adult rats can be sensitized and provoked into asthma model by allergen exposure. Therefore we speculate that there is a"time-window"in the protective role induced by above early exposure to prevent the development of asthma. In order to illuminate the effect of exposure to allergen at different time on asthma, and to explore the role of regulatory T cells and costimulatory molecules in it, we performed such experiments as follows.1. The effect of exposure to allergen at different time on the rat asthma model (accomplished with AN yun)Neonate rats were randomly divided into asthma group, allergen exposure groups (day 3 group, day 7 group and day 14 group) and control group. Each allergen exposure group was injected with ovalbumin 1 mg (containing aluminum hydroxide 5 mg) subcutaneously at day 3, day 7 and day 14 after birth respectively. When all rat were kept to adulthood (6 weeks of age), all groups but control group were immunized and provoked via OVA to make asthma model. The pathologic changes of lung tissue stained with H&E and periodic acid-Schiff (PAS) were observed, and the asthmatic inflammation of different groups was evaluated and the number of mucous cells per bronchiole was calculated.The results revealed that the airways inflammation at day 3 and day 7 groups was apparently alleviated and the number of mucous cells at both groups was decreased compared with asthma group (2.29±0.49 vs 3.50±0.76, 1.25±0.46 vs 3.50±0.76, P<0.01). But there was not any improvement of airways inflammation and the hyperplasia of mucous cells at day 14 group in comparison with asthma group (3.00±1.16 vs 3.50±0.76, P>0.05).2. The relationship between the role of allergen exposure at different time and the regulatory T cells in spleen and thymus of adult ratsThe percentage of CD4+CD25+T cells among CD4+T cells in spleen and thymus of different groups mentioned above was calculated by flow cytometry. The mRNA expression of forkhead box P3 (Foxp3) transcription factor at different groups was measured by Real-time PCR. The serum levels of interleukin 10 (IL-10) and transformating growth factorβ1 (TGF-β1) in different groups were also tested by Enzyme-Linked ImmunoSorbent Assay (ELISA).The results showed that the percentage of CD4+CD25+T and Foxp3 mRNA in spleen at day 3 group [(13.68±3.54)% vs (7.33±3.39)%, 18.46±5.01 vs 5.49±1.99, P<0.01] and day 7 group [(16.65±4.91)% vs (7.33±3.39)%, 26.37±4.68 vs 5.49±1.99, P<0.01] were increased significantly compared with control group, so was the Foxp3 mRNA in thymus at day 7 group (18.73±3.66 vs 11.13±1.75, P<0.05). But there were no statistically significant differences of the CD4+CD25+T cells and Foxp3 mRNA in spleen between day 14 group and control group [(11.04±2.88)% vs (7.33±3.39)%, 8.71±2.19 vs 5.49±1.99, P>0.05]. The serum level of TGF-β1 at day 3 group was increased compared with control group [(87.30±9.20)μg/L vs (68.03±4.36)μg/L, P<0.05]. The median value of TGF-β1 at day 7 group was also increased [(75.05±9.14)μg/L vs (68.03±4.36)μg/L], but was not of statistical significance (P=0.069). The TGF-β1 level at day 14 group was not changed compared with control group [(66.41±4.85)μg/L vs (68.03±4.36)μg/L, P>0.05]. The serum level of IL-10 at all groups had no significant difference (P>0.05).3. The rule of the regulatory T cells and costimulatory molecule CD28 changes in thymus and spleen of young rats after exposure to allergen at different timeNeonate SD rats were randomly divided into allergen exposure groups (day 7 group and day 14 group) and control group with the same age accordingly. The allergen exposure groups were injected with ovalbumin 1 mg (containing aluminum hydroxide 5 mg) subcutaneously at day 7 and day 14 after birth respectively. The rats were sacrificed 3 days or 6 days after allergen exposure. The mRNA expressions of Foxp3 and CD28 in thymus and spleen were measured by Real-time PCR.The results showed that the mRNA expressions of Foxp3 and CD28 in thymus at day 7 group were increased simultaneously compared with control group (7.1±1.7 vs 4.0±1.6, 224.6±78.8 vs 125.6±63.5, P<0.05). But day 14 group had a decrease of Foxp3 mRNA (3.9±1.5 vs 6.8±2.6, P<0.05) and an increase of CD28 mRNA in spleen (33.2±5.7 vs 13.5±1.6, P<0.01).Conclusions:1. There is a"time-window"phenomenon in the protective role of the early allergen exposure for preventing the development of asthma. The"time-window"occurs about within 2 week after birth in rats.2. Early exposure to allergen inhibits asthma in adulthood probably by means of inducing the regulatory T cells."Time-window"maybe depends on whether the regulatory T cells are able to be induced by exposure to allergen at different time.3. Exposure to allergen within the"time-window"might induce the generation of regulatory T cells by increased expression of CD28 signal pathway. However, exposure to allergen out of"time-window"probably activates T cells and inhibits the role of regulatory T cells.