人類心臟瓣膜纖維母細胞受草綠色鏈球菌葡萄糖傳遞酶活化引發白血球趨化作用之研究

Activation of human valvular fibroblasts by glucosyltransferases of viridans streptococci to induce chemotaxis of leukocytes

張家榮(Chia-Jung Chang)

指導教授 ： 賈景山

國立臺灣大學/醫學院/口腔生物科學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.01579

摘要

轉糖鏈球菌(Streptococcus mutans)是屬於草綠色鏈球菌的一群，並是容易造成亞急性心內膜炎的伺機性病原菌。心內膜炎的致病過程主要是由於血液中的病原菌聚集到受傷瓣膜表面，並分泌細菌毒力因子造成瓣膜慢性發炎反應的形成。草綠色鏈球菌葡萄糖傳遞酶(glucosyltransferase)是引起心臟瓣膜發炎一個相當重要的細菌性調節素，其具有刺激人類周邊血液單核細胞及人類臍帶靜脈內皮細胞產生大量IL-6的能力。由於心臟瓣膜主要由外層的內皮細胞及內層的纖維母細胞構成；因此，本實驗利用純化的葡萄糖傳遞酶刺激心臟移植病人瓣膜所培養的纖維母細胞，探討在人類瓣膜初級培養的細胞是否會受到葡萄糖傳遞酶刺激而引起發炎反應，藉以找尋草綠色鏈球菌造成感染性心內膜炎的致病機轉。
葡萄糖傳遞酶具有刺激人類瓣膜纖維母細胞產生IL-6、IL-8、及MCP-1，並可以造成細胞內AKT、MAPKs及IκB-α的磷酸化，及NF-κB的細胞核轉位。利用PI3K、MAPK及NF-κB的抑制劑可有效抑制葡萄糖傳遞酶所誘導之IL-6、IL-8及MCP-1的產生。顯示其可透過活化人類瓣膜纖維母細胞的PI3K-AKT-MAPK-NF-κB訊息路徑，促進發炎反應的發生。此外葡萄糖傳遞酶可以促使人類單核球細胞株(THP-1及U937)進行趨化性移動(Chemotaxis)，並藉由活化人類瓣膜纖維母細胞表現ICAM-1，促進趨化的單核球黏附；同時藉由產生大量的CXCR4專一性配體，導致大量人類初級T淋巴球及T淋巴球細胞株Jurkat的趨化性移動趨化。
由於葡萄糖傳遞酶也會活化人類單核白血球產生高量的IL-1β，因此，當活化的單核白血球浸潤至瓣膜組織中，其分泌的IL-1β可能會加劇發炎反應的發生。因此本研究中利用IL-1β刺激人類瓣膜纖維母細胞發現的確會促使IL-6、IL-8、及MCP-1的產生，並活化人類瓣膜纖維母細胞表現ICAM-1；同時會造成大量人類單核球細胞株U937進行趨化性移動及黏附。
經由以上實驗結果推論，在草綠色鏈球菌引起的感染性心內膜炎中，葡萄糖傳遞酶會刺激人類瓣膜纖維母細胞產生大量的細胞激素和趨化激素，誘發白血球的浸潤；受活化的浸潤單核白血球會分泌IL-1β，促進瓣膜纖維母細胞更進一步的活化，導致心臟瓣膜慢性且持續性發炎反應的發生。

關鍵字

轉糖鏈球菌 ； 葡萄糖傳遞酶人類瓣膜纖維母細胞 ； 趨化作用

並列摘要

Streptococcus mutans is a member of viridans group streptococci and one of the opportunistic pathogens causing subacute infective endocarditis (IE). The major process in the pathogenesis of IE is initiated when pathogens circulating in the bloodstream colonize to damaged valves and then secrete virulence factors to induce a chronic inflammatory response. Glucosyltransferases (GTFs) of viridans streptococci are the important bacterial modulins and potent inducers of interleukin-6 (IL-6) synthesis and release from human PBMC and HUVEC. Because the heart valves were constituted primary by meshes of fibroblasts, we investigated the primary culture of valvular fibroblasts from heart transplant patients to study the immune response stimulated by GTFs and to define the mechanism of IE triggered by the viridans streptococci.
We reported that GTFs attached to human primary valvular fibroblasts triggered the release of IL-6, IL-8 and MCP-1, and enhanced the phosphorylation of AKT, MAPKs and IκB-α and the nuclear translocation of NF-κB. The increase in IL-6, IL-8 and MCP-1 was abrogated by addition of PI3K, MAPKs, and NF-κB inhibitors, suggesting that the up-regulation of IL-6, IL-8 and MCP-1 was mediated through PI3K, MAPKs, and NF-κB signaling pathways. We also demonstrated that valvular fibroblasts treated by GTFs promoted the chemotaxis and adhesion of monocytic cell lines. In addition, GTFs-stimulated valvular fibroblasts could induce the release of CXCR4-specific ligand to enhance the recruitment of T lymphocyte.
Because GTFs could stimulate human mononuclear cells to produce interleukin-1β(IL-1β), we hypothesized that the infiltration of GTFs-activated mononuclear cells could play a role in enhancing inflammatory response. We found that exogenous IL-1β could activate valvular fibroblasts, trigger the release of IL-6, IL-8 and MCP-1, and up-regulate the expression of ICAM-1. In addition, IL-1β-stimulated fibroblasts could promote the chemotaxis and adhesion of monocytic cell lines.
Taken together, these results indicate that GTFs could induce the synthesis and release of inflammatory cytokine and chemokine to trigger the infiltration of leukocytes a characteristic in the pathogenesis of IE caused by viridans group streptococci. Furthermore, GTFs-activated mononuclear cells could secrete IL-1β to further stimulate valvular fibroblasts and sustain the formation of chronic endocardial inflammation.

並列關鍵字

Streptococcus mutans ； glucosyltransferase ； human valvular fibroblasts ； chemotaxis

參考文獻

吳淑惠. 2005. 篩選體內表現基因載體的建立與心內膜炎免疫反應之特徵. 碩士 國立臺灣大學醫學院口腔生物科學研究所碩士論文

葉秋月. 2006. 草綠色鏈球菌葡萄糖傳遞酶與內皮細胞的交互作用參與感染性心內膜炎的免疫致病機制. 博士 國立臺灣大學醫學院微生物學研究所博士論文

Ajdic, D., McShan, W. M., McLaughlin, R. E., Savic, G., Chang, J., Carson, M. B. et al. (2002) Genome sequence of Streptococcus mutans UA159, a cariogenic dental pathogen. Proc Natl Acad Sci U S A 99: 14434-14439.

Al-Okla, S., Chatenay-Rivauday, C., Klein, J. P., and Wachsmann, D. (1999) Involvement of α5β1 integrins in interleukin-8 production induced by oral viridans streptococcal protein I/IIf in cultured endothelial cells. Cell Microbiol 1: 157-168.

Alter, P., Hoeschen, J., Ritter, M., and Maisch, B. (2002) Usefulness of cytokines interleukin-6 and interleukin-2R concentrations in diagnosing active infective endocarditis involving native valves. Am J Cardiol 89: 1400-1404.