- 學位論文
金黃色葡萄球菌超級抗原造成異位性皮膚炎皮膚過敏性發炎反應之持續與惡化的分子機轉
Molecular Mechanisms that Staphylococcal Superantigens Contribute to the Persistence and Exacerbation of Allergic Skin Inflammation in Atopic Dermatitis
摘要
過去文獻顯示，高達95% 的異位性皮膚炎患者的皮膚上有金黃色葡萄球菌的增生與感染。此菌會分泌許多外毒素，其中，以金黃色葡萄球菌腸毒素B為異位性皮膚炎患者皮膚上最常見的金黃色葡萄球菌外毒素。這些金黃色葡萄球菌的外毒素是超級抗原，它們可以穿透皮膚，刺激帶有某些特定的T細胞受體beta鏈變異區 (variable region of beta chain of T cell receptor，TCRVbeta) 的T細胞，造成異位性皮膚炎皮膚過敏性發炎反應的持續與惡化。 本研究的目標，是要探討金黃色葡萄球菌超級抗原造成異位性皮膚炎皮膚過敏性發炎反應之持續與惡化的分子機轉。其特定目的包括 : 1) 比較異位性皮膚炎病人、無異位性皮膚炎的呼吸道過敏病病人、及健康人的血清中金黃色葡萄球菌腸毒素A之特異性IgE抗體與金黃色葡萄球菌腸毒素B之特異性IgE抗體的濃度 ; 2) 探討血清中金黃色葡萄球菌腸毒素A之特異性IgE抗體或金黃色葡萄球菌腸毒素B之特異性IgE抗體的濃度和異位性皮膚炎的臨床嚴重程度或皮膚感染病史之間的相關性 ; 3) 比較異位性皮膚炎病人與健康人的金黃色葡萄球菌腸毒素B反應型 (TCRVbeta3陽性或TCRVbeta12陽性或TCRVbeta17陽性) CD4陽性T細胞，在受到金黃色葡萄球菌腸毒素B的刺激之後，所引起的活化、分裂增殖、細胞激素的製造、趨化激素受體的表現、細胞凋亡、caspase-3的活化、以及抗凋亡蛋白Bcl-2及Bcl-2 mRNA濃度的變化 ; 4) 探討外加介白質-4對健康人的金黃色葡萄球菌腸毒素B反應型CD4陽性T細胞之金黃色葡萄球菌腸毒素B引起的細胞凋亡、caspase-3的活化、以及Bcl-2及Bcl-2 mRNA濃度的變化的影響 ; 5) 探討藉由加入抗介白質-4中和抗體來抑制本身製造的介白質-4對異位性皮膚炎病人的金黃色葡萄球菌腸毒素B反應型CD4陽性T細胞之金黃色葡萄球菌腸毒素B引起的細胞凋亡、caspase-3的活化、以及Bcl-2及Bcl-2 mRNA濃度的變化的影響。 我們以酵素免疫分析法來測定血清中金黃色葡萄球菌腸毒素A之特異性IgE抗體與金黃色葡萄球菌腸毒素B之特異性IgE抗體的濃度。此外，我們以免疫螢光染色法及Annexin V染色法倂流式細胞儀分析與同步定量聚合酶連鎖反應，來分析在有或沒有外加重組介白質-4或抗介白質-4中和抗體的情況下分別給予金黃色葡萄球菌腸毒素B刺激的周邊血單核細胞。 結果發現，和無異位性皮膚炎的呼吸道過敏病病人或健康人比較起來，異位性皮膚炎病人有較高的血清中金黃色葡萄球菌超級抗原之特異性IgE抗體的濃度。血清中金黃色葡萄球菌超級抗原之特異性IgE抗體的濃度和異位性皮膚炎的臨床嚴重程度之間有正相關。這些結果暗示，金黃色葡萄球菌超級抗原是一種過敏原，其會引起異位性皮膚炎病人的B細胞製造功能上有意義的金黃色葡萄球菌超級抗原之特異性IgE抗體。然而，血清中金黃色葡萄球菌超級抗原之特異性IgE抗體的濃度和皮膚感染病史之間並無相關。在CD4陽性T細胞受到金黃色葡萄球菌超級抗原的刺激之後所產生的一連串的反應方面，我們發現，CD4陽性T細胞之活化與分裂增殖的程度，在異位性皮膚炎病人與健康人身上是相似的。然而，金黃色葡萄球菌超級抗原的刺激，在健康人的CD4陽性T細胞，主要是引起干擾素-gamma (第一型T輔助細胞細胞激素) 的製造與趨化激素受體CXCR3陽性細胞的增加，而在異位性皮膚炎病人的CD4陽性T細胞，主要是引起介白質-4 (第二型T輔助細胞細胞激素) 的製造與趨化激素受體CCR4陽性細胞的增加。和健康人比較起來，異位性皮膚炎病人的CD4陽性T細胞比較不容易發生金黃色葡萄球菌超級抗原引起的Bcl-2的減少、caspase-3的活化、及細胞凋亡。此外，外加介白質-4可以抑制健康人的CD4陽性T細胞之金黃色葡萄球菌超級抗原引起的Bcl-2的減少、caspase-3的活化、及細胞凋亡。抑制本身製造的介白質-4可以增加異位性皮膚炎病人的CD4陽性T細胞之金黃色葡萄球菌超級抗原引起的Bcl-2的減少、caspase-3的活化、及細胞凋亡。這些結果暗示，在受到金黃色葡萄球菌超級抗原的刺激之後，異位性皮膚炎病人的CD4陽性T細胞會製造介白質-4，而這些介白質-4可以透過抑制金黃色葡萄球菌超級抗原引起的Bcl-2的減少，來抑制CD4陽性T細胞之金黃色葡萄球菌超級抗原引起的caspase-3的活化與細胞凋亡。這會導致被金黃色葡萄球菌超級抗原活化的T細胞無法被清除，皮膚的過敏性發炎反應也就無法終止。 本研究的結果，可以釐清金黃色葡萄球菌超級抗原造成異位性皮膚炎皮膚過敏性發炎反應之持續與惡化的分子機轉，以助於發展對異位性皮膚炎特定的預防與治療之道。在臨床上的應用，本研究暗示，測定血清中金黃色葡萄球菌超級抗原之特異性IgE抗體的濃度，可以提供重要的訊息，以助於異位性皮膚炎病人的追蹤治療。當異位性皮膚炎的臨床病情惡化或併發皮膚感染時，早期積極地使用抗生素來抑制金黃色葡萄球菌，以避免皮膚的T細胞繼續受到金黃色葡萄球菌超級抗原的刺激，對於異位性皮膚炎病情的控制會很有幫助。
並列摘要
The skin of atopic dermatitis (AD) patients exhibits a striking susceptibility to colonization and infection with Staphylococcus aureus, which can secrete various exotoxins including staphylococcal enterotoxin B (SEB) being the most common. These exotoxins are superantigens. They may penetrate the skin barrier, stimulate T cells bearing the specific variable region of beta chain of T cell receptor (TCRVbeta), and contribute to the persistence and exacerbation of allergic skin inflammation in AD. The objective of this study was to clarify the molecular mechanisms that staphylococcal superantigens (SsAgs) contribute to the persistence and exacerbation of allergic skin inflammation in AD. The specific aims were to 1) compare serum staphylococcal enterotoxin A (SEA)-specific IgE and SEB-specific IgE levels among AD patients, patients with respiratory allergy without AD, and healthy subjects; 2) investigate the correlation between serum SEA-specific IgE or SEB-specific IgE levels and AD severity or previous skin infections; 3) determine whether there are differences between AD patients and healthy subjects in SEB-induced activation, proliferation, cytokine production, chemokine receptor expression, apoptosis, caspase-3 activation, and changes of anti-apoptotic protein Bcl-2 and Bcl-2 mRNA levels of SEB-reactive (TCRVbeta3+ or Vbeta12+ or Vbeta17+) CD4+ T cells; 4) investigate the effect of exogenously added interleukin-4 (IL-4) on SEB-induced apoptosis, caspase-3 activation, and changes of Bcl-2 and Bcl-2 mRNA levels in SEB-reactive CD4+ T cells from healthy subjects; 5) investigate the effect of inhibition of endogenous IL-4 by using anti-IL-4 neutralizing antibodies (Abs) on SEB-induced apoptosis, caspase-3 activation, and changes of Bcl-2 and Bcl-2 mRNA levels in SEB-reactive CD4+ T cells from AD patients. Serum SEA-specific IgE and SEB-specific IgE levels were determined by enzyme immunoassay. By using immunofluorescence and Annexin V staining followed by flow cytometric analysis and real-time polymerase chain reaction, we analyzed peripheral blood mononuclear cells with or without SEB stimulation in the presence or absence of recombinant IL-4 or anti-IL-4 neutralizing Abs. We found that AD patients had higher serum SsAg-specific IgE levels than patients with respiratory allergy without AD and healthy subjects. There were positive correlations between serum SsAg-specific IgE levels and AD severity. These findings suggest that SsAgs play the role of allergens and thus may induce the production of functionally-relevant SsAg-specific IgE Abs in AD patients. However, there was no correlation between serum SsAg-specific IgE levels and previous skin infections. About the sequential responses of CD4+ T cells induced by SsAg stimulation, both activation and proliferation of CD4+ T cells were similar in AD patients and healthy subjects. However, SsAgs induced production of interferon-gamma (type 1 T helper cell (Th1) cytokine) in CD4+ T cells from healthy subjects and IL-4 (type 2 T helper cell (Th2) cytokine) in those from AD patients. SsAgs induced up-regulation of chemokine receptor CXCR3+ cells in CD4+ T cells from healthy subjects and CCR4+ cells in those from AD patients. CD4+ T cells from AD patients were more resistant to SsAg-induced decrease of Bcl-2, caspase-3 activation, and apoptosis than those from healthy subjects. Moreover, exogenously added IL-4 inhibited SsAg-induced decrease of Bcl-2, caspase-3 activation, and apoptosis in CD4+ T cells from healthy subjects. Inhibition of endogenous IL-4 increased SsAg-induced decrease of Bcl-2, caspase-3 activation, and apoptosis in CD4+ T cells from AD patients. These findings suggest that following SsAg stimulation, IL-4 produced by CD4+ T cells in AD patients can inhibit SsAg-induced caspase-3 activation and apoptosis of CD4+ T cells through inhibiting SsAg-induced decrease of Bcl-2. This may impair deletion of SsAg-activated T cells and resolution of allergic skin inflammation. The results of this study may clarify the molecular mechanisms that SsAgs contribute to the persistence and exacerbation of allergic skin inflammation in AD, which is important for exploring the specific therapy and prevention of AD. In clinical application, this study suggests that the determination of serum SsAg-specific IgE levels may provide important information in the follow-up and therapy of AD patients. Early and aggressive antibiotic treatment of skin infection or exacerbated AD to prevent SsAg exposure of skin T cells is helpful to disease control of AD.
參考文獻
延伸閱讀
- 徐世達（2009）。異位性皮膚炎與金黃色葡萄球菌超級抗原。台灣氣喘衛教學會會刊，(16)，12-18。https://doi.org/10.6669/TAAE.2009.16.12
- 蔡紋如（2005）。異位性皮膚炎和金黃色葡萄球菌感染。臺灣兒童過敏氣喘及免疫學會學會通訊，6(2)，2-3。https://doi.org/10.7025/HEJ.200506.0002
- Wang, J. T., Lin, S. F., Chiu, H. L., Wang, L. C., Tai, H. M., Jiang, C. F., Chang, S. C., & Chu, S. H. (2004). 一教學醫院院內methicillin抗藥性金黃色葡萄球菌感染之分子流行病學與控制. Journal of the Formosan Medical Association, 103(1), 32-36. https://doi.org/10.29828/JFMA.200401.0005
- Souza, J. A. C. D., Nogueira, A. V. B., Souza, P. P. C. D., Kim, Y. J., Lobo, C. S., Oliveira, G. J. P. L. D., Cirelli, J. A., Garlet, G. P., & Jr., C. R. (2013). SOCS3 Expression Correlates with Severity of Inflammation, Expression of Proinflammatory Cytokines, and Activation of STAT3 and p38 MAPK in LPS-Induced Inflammation In Vivo. Mediators of Inflammation, 2013(), 269-278-231. https://doi.org/10.1155/2013/650812
- Oyama, N., & Kaneko, F. (2011). Cell-Type-Specific Differentiation and Molecular Profiles in Skin Transplantation: Implication of Medical Approach for Genetic Skin Diseases. Journal of Transplantation, 2011(), 221-226. https://doi.org/10.1155/2011/501857