| IL-22, due to its limited primary structure similarity to the well-known anti-inflammatory and immunosuppressive cytokine IL-10, was named IL-TIF for "IL-10-related T cell-derived Inducible Factor" and belongs to the family of IL-10. IL-22 was produced by a variant of Thl7 cells which also co-expressed cytokine IL-17. Studies suggested that IL-17 was involved in asthma pathogenesis and Thl7 cells were also related to asthma. Thus, IL-22 was predicted to involve in asthma pathogenesis. Some reports showed the level of IL-17 increased in the sera of allergic asthma patients, however, there is no data showed the expression of IL-22 in the sera of allergic asthma patients.IL-22 receptor complex was composed of IL-22R1 and IL-10R2. IL-10R2 had been shown to be ubiquitously expressed, which was a part of several cytokine receptors, Therefore IL-22R1 was the specific receptor chain and the expression of the IL-22R1 chain should determine whether a cell was IL-22 target or not. No IL-22R1-expression was detected in a variety of resting or activated primary cells. It was only expressed by non-hematopoietic cells such as hepatocytes, keratinocytes, lung and intestinal epithelial cells. In respiratory system, the expression of IL-22R1 was detected in human airway epithelial cells and human alveolar epithelial cells. There was no report about the expression of IL-22 in human airway smooth muscle cells and human airway fibroblast cells.There were many activated cytokines in the serum of asthma patients. The effect of asthma serum on the expression of IL-22R1 in human airway epithelial cells (HECs), human airway smooth muscle cells (HSMCs) and human fibroblast cells (HFs) had not been reported yet. The role of IL-22 in cell proliferation, apoptosis and death of these three kinds of cells had not clarified yet. Cell proliferation and death of human airway epithelial cells, human airway smooth muscle cells and human fibroblast cells stimulated by IL-22 of different concentration are observed by MTT method and FACS in this study.IFN-y could dramatically increase the expression of IL-22R1 and IL-10R2 in keratinocyte depended of concentration and duration. This suggested that the susceptibility to IL-22 increased in keratinocyte with Thl cells. Many scientists reported TGF-βis essential for Thl7 differentiation. TGF-βwas one of the most pleiotropic molecules which had been shown to be intricately involved in basic cellular activities. In the lung, TGF-P not only influenced the synthesis of ECM components such as fibronectin and collagen from bronchial epithelial cells, fibroblasts, and airway smooth muscle cells, but also controled the expression of matrix-degrading proteases and the levels of antiproteases. IL-4 was produced by Th2 cells which was believed to play a role in allergic bronchial asthma, and has been suggested to cause hyperresponsiveness of airway smooth muscle. The influence of IFN-y, IL-4 and TGF-βon the expression of IL-22R1 in HECs, HFs, and HSMCs had not been reported yet. Understanding the role of those cytokines on the expression of IL-22R1 of HECs, HFs, and HSMCs and the effect of IL-22 with different concentration on cell proliferation and death of these three kinds of cells, may lead to a better understanding of effect of IL-22 on human airway cells and led to a novel targets to treat allergic asthma.Objectives1. To detect the level of IL-22 in the sera of asthma patients and healthy controls and measure the lung function of asthma patients. To compare the level of serum IL-22 between bronchial dilation test positive group and bronchial provocation positive group of asthma patients. 2. To detect the expression of IL-22R1 in HECs, HFs, and HSMCs.3. To compare the expression of IL-22R1mRNA before and after sensitizing by asthma sera. To compare the IL-22R1 of HECs, HFs, and HSMCs stimulated by IFN-γ, IL-4 and TGF-β.4. To detect the effect of IL-22 with different concentration on cell proliferation and death of HECs, HFs, and HSMCs.Methods1. Collect sera of 41 asthma patients and 20 healthy controls and detect the level of IL-22 and IL-17 in these sera by ELIS A methods. Measure lung function of asthma patients and divide hose asthma patients into two groups according to the result of FEV1%:bronchial dilation test positive group (FEV1%≤70), bronchial provocation positive group (FEV1%<70%) and compare the level of IL-17 and IL-22 in those two groups.2. Measure the expression of IL-22RlmRNA in HECs, HFs, and HSMCs by real-time PCR assay and measure the protein expression of IL-22R1 in those three kinds of cells by Western blot.3. Collect sera of severe asthma patients without using glucocorticoid recently (IgE>1000U/ml). Detect the expression of IL-22R1mRNA in HECs, HFs, and HSMCs stimulated by severe asthma sera by real time PCR assay. Measure OD value of those three kinds of cells which were dealt with IL-22 with different concentration by MTT assay and cell apoptosis and necrosis per cent by FACS.4. Measure the expression of IL-22R1mRNA in HFs, and HSMCs stimulated by IFN-γ, IL-4 and TGF-βfor 24h. Measure protein expression of IL-22R1 in those two kinds of cells stimulated by IFN-γ, IL-4 and TGFβ.Results:1. There is no significant difference of the level of IL-22 in sera of asthma patient and healthy control(the value of P is 0.07 and 0.293, separately); while the level of serum IL-17 in asthma patients is significantly higher than healthy control. Serum level of IL-17 and IL-22 in bronchial dilation test positive group is significantly higher than bronchial provocation positive group.2. The expression of L-22Rlwere detected in HECs, HFs, and HSMCs by real-time PCR, Western blot and immunofluorescence assay.3. The expression of IL-22R1mRNA decreased to 9% of the control cells in HECs sensitized by asthma serum while in HSMCs, the expression of IL-22R1mRNA increased to 345 times of the control cells. There is no change in the expression of IL-22R1mRNA in HFs.4. IL-22mRNA and protein increased in HSMCs after stimulated by IFN-y, IL-4 and TGF-βfor 24h. For HFs, IL-22mRNA and protein decreased in IFN-y, IL-4 group while increased in TGF-βgroup.5. Proliferation of HECs decreased by a IL-22 concentration-depended way. OD value significantly decreased in 100ng/ml and 1000ng/ml IL-22 groups after stimulated for 12h (P<0.01) and in 1000ng/ml group for 24h (P<0.05). OD value showed a decreasing trend depended on IL-22 increasing. 1000ng/ml IL-22 significantly reduced OD value of HSMCs (P<0.01); IL-22 with 100ng/ml can change cell proliferation that OD value added after stimulated for 6h and 12h while suppressed after 24h. In HFs, OD value significantly decreased in 100ng/ml and 1000ng/ml groups stimulated by IL-22 for 12h(P<0.05); OD value showed a decreasing trend depended on IL-22 increasing.6. Apoptosis decrease in HECs stimulated by IL-22 and at the same time, normal cells increased(P<0.01);10ng/ml IL-22 significantly added cell necrosis. Cell necrosis showed an increasing trend depended on IL-22 increasing. There is no significant difference of normal cells rate, cell apoptosis and cell necrosis in HSMCs. There is no significant difference of normal cells rate and cell apoptosis in HFs. However, IL-22 significantly reduced those cells'necrosis.Conclusion1. IL-22 may involve in asthma pathogenesis, and HECs, HSMCs and HFs are the targets of IL-22 in airway. 2. IL-22 plays a dual role on bronchial epithelial cells and asthma which may be related to the course of the disease and the status of HECs. In HSMCs, the role of IL-22 maybe depend on its concentration or the expression of IL-22R1. In HFs, IL-22 looks like a protective cytokine and the role is very limited in established asthma.3. IFN-γ, IL-4 and TGF-βcan have an effect on IL-22 role by regulating IL-22R1 expression in HECs, HSMCs and HFs. |
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