高量細菌內毒素 (LPS) 引起腸道上皮細胞凋亡及緊密連結蛋白破壞：CD14參與之機制探討

腸道屏障由單層上皮細胞及細胞之間的緊密連結 (tight junctions) 組成，可防止腸腔面細菌及抗原產物進入漿膜面循環至全身。緊密連結蛋白包括occludin、claudin及zonula occluden (ZO) 等。臨床上，有報導指出在細菌性腸炎及發炎性腸疾中均有腸道屏障缺損之現象。根據先前研究，利用高劑量革蘭氏陰性菌內毒素刺激腸道上皮細胞Caco-2之腸腔面，會引起細胞凋亡及增加細胞間通透性 (Yu et al., 2005 & 2006)。內毒素之受體複合物包括CD14、TLR-4及MD-2等在單核球及其分化細胞上可發現，然而，在腸道上皮細胞有不同的表現形式。因此，本篇研究目的為1) 探討內毒素引起緊密連結構造缺損是否為細胞凋亡途徑之下游反應，2) 檢測內毒素刺激引起腸道上皮細胞凋亡及緊密連結蛋白破壞的機制中，內毒素受體複合分子如CD14及TLR-4所扮演的角色，3) 尋找內毒素刺激引起腸道上皮細胞凋亡及緊密連結蛋白破壞所參與之細胞訊息傳遞途徑為何。實驗中將人類大腸直腸癌上皮細胞株Caco-2細胞培養達全滿，以50 μg/mL之大腸桿菌內毒素刺激腸腔面24小時後，測量細胞凋亡程度。利用Hoechst及TUNEL染色法觀察發現內毒素刺激會引起染色質濃縮及DNA片段化現象。利用流式細胞技術以DiOC6(3) 染劑亦可偵測到內毒素刺激引起粒線體膜電位下降。預先投予caspase-3抑制劑 (z-DEVD-FMK) 能阻斷內毒素刺激所引起之上皮細胞凋亡。Caco-2之控制組細胞其緊密連結蛋白如：ZO-1、occludin在免疫螢光染色下呈現鐵絲網圍欄狀。而內毒素刺激會導致緊密連結蛋白結構破壞，預先投予caspase-3抑制劑則可阻斷此現象。除此之外，預先投予polymyxin B可阻斷內毒素刺激引起之細胞凋亡及緊密連結蛋白結構改變，表示細胞凋亡反應乃因內毒素之脂質A作用所造成，而非由於其他細菌組成成份污染所致。本實驗更進一步瞭解受體複合物如：CD14及TLR-4在內毒素刺激引起腸道上皮細胞凋亡及緊密連結蛋白破壞中所參與之機制。以免疫螢光染色法及流式細胞技術可偵測到Caco-2上皮細胞中表現CD14，由共軛焦XZ連續堆疊影像顯示CD14表現在細胞頂面膜。利用三種TLR-4單株抗體 (clones HTA-125、76B357.1及lG11) 藉由免疫螢光染色法證實Caco-2細胞並無TLR-4表現，而陽性控制組T84細胞則可見TLR-4染色。在內毒素刺激下，CD14及TLR-4表現在上述細胞中並無明顯改變。此外，西方墨點法結果證實CD14 (分子量53∼55 kDa) 表現於Caco-2細胞。最後，結果顯示預先投予中和性抗CD14之抗體 (clone 134620) 能減少內毒素刺激引起之細胞凋亡及緊密連結蛋白結構之破壞，且呈現濃度依賴形式，然而，預先投予中和性抗TLR-4之抗體 (clone HTA-125) 則無作用。過去針對單核球/巨噬細胞之研究顯示，內毒素刺激會經由TLR-4引起MyD88依賴性途徑，使得轉錄因子NF-κB發生核轉移及MAPK磷酸化現象。為進一步確認或排除已知的TLR-4媒介訊息參與之可能性，利用西方墨點法檢測NF-κB、 MyD88及MAPK在Caco-2細胞之表現。結果顯示內毒素刺激Caco-2細胞不會發生NF-κB核轉移、MyD88總量改變及JNK、p38之磷酸化現象，而陽性控制組THP-1細胞則可見。綜合上述，這些結果顯示內毒素引起腸道上皮細胞Caco-2細胞凋亡會導致緊密連結蛋白結構之破壞及屏障缺損，且這些病理現象是經由CD14而非TLR-4所參與的。

關鍵字

腸道上皮細胞；細菌內毒素；細胞凋亡；緊密連結； TLR-4 ； CD14 ；訊息傳遞途徑

並列摘要

Intestinal barrier is composed of a single layer of epithelial cells connected by tight junctions that prevents luminal bacteria and antigenic products from gaining access into the body proper. Tight junctional proteins include occludin, claudin, and zonula occluden (ZO) etc. Clinical manifestations of intestinal barrier defects were reported in bacterial enteritis and inflammatory bowel disease. Previous studies utilizing intestinal epithelial Caco-2 cell culture have demonstrated that luminal exposure to high dose of Gram (-) bacterial LPS induced cell apoptosis and increased paracellular permeability (Yu et al., 2005 & 2006). LPS receptor complexes including CD14, TLR-4 and MD-2 were identified on monocytic lineage cells. However, distinct expression patterns on intestinal epithelial cells have been reported. The aim of the current study was to 1) examine whether LPS-induced tight junctional destruction is downstream of apoptotic pathway, 2) investigate the role of LPS receptor components, i.e. CD14 and TLR-4, in the mechanism of LPS-induced intestinal epithelial apoptosis and tight junctional destruction, 3) explore the cell signaling pathway involved in the LPS-induced intestinal epithelial apoptosis and tight junctional destruction. Colorectal adenocarcinoma Caco-2 cells grown to confluence were luminally exposed to E.coli LPS at 50 μg/ml for 24 hrs and examined for cell apoptosis. Hoechst staining and TUNEL assay showed chromatin condensation and DNA fragmentation in cells following LPS exposure. Flow cytometric analysis of DiOC6(3) staining exhibited mitochondrial membrane potential collapse after LPS challenge. Pretreatment with caspase-3 inhibitor, z-DEVD-FMK, prevented the epithelial cell apoptosis induced by LPS. Immunostaining revealed chicken-wire pattern of tight junctional proteins, i.e. ZO-1 and occludin, on untreated Caco-2 cell monolayer en face. LPS caused destruction of ZO-1 and occludin in which the effect was abolished by pretreatment with caspase-3 inhibitor. In addition, polymyxin B blocked the apoptotic phenomenon and tight junctional reorganization, verifying the specificity of the lipid A moiety of LPS. The role of LPS receptor components, e.g. CD14 and TLR-4, was further assessed in the mechanism. Presence of CD14, but not TLR-4, protein expression was identified on Caco-2 epithelial cells demonstrated by immunofluorescent staining and flow cytometry. The expression of CD14 on apical membrane of cells was evidenced by confocal XZ serial imaging. The absence of TLR-4 expression on Caco-2 cells was verified using three different monoclonal antibodies (clones HTA-125, 76B357.1, and 1G11), whereas positive TLR-4 staining was showed on T84 cells. After LPS challenge, the cellular expression of CD14 and TLR-4 did not change. Moreover, western blotting results confirmed the presence of CD14 (M.W. = 53~55 kDa) on Caco-2 cells. Furthermore, our results showed that neutralizing anti-CD14 antibody (clone 134620) reduced the level of LPS-induced apoptosis and tight junctional destruction in a dose-dependent manner, whereas anti-TLR-4 (clone HTA-125) had no effect. Classical LPS/TLR-4 signaling pathway induced nuclear translocation of NF-κB and phosphorylation of MAPK by a MyD88-dependent manner. In order to confirm or rule out the involvement of TLR-4-mediated signaling pathway, western blotting was utilized to examine the expression of NF-κB, MyD88 & phosphorylation level of MAPK in intestinal epithelial Caco-2 cells. Our results showed that there are no nuclear translocation of NF-κB, phosphorylation of JNK and p38, nor augment of MyD88 in LPS challenged Caco-2 cells, in contrast to the positive control THP-1 cells. Taken together, these findings suggest that LPS-induced cell apoptosis leads to tight junctional disruption and barrier defects in intestinal epithelial Caco-2 cells. These pathological effects on enterocytes were mediated via epithelial CD14, whereas TLR-4-mediated signaling pathway did not play a role.

並列關鍵字

intestinal epithelial cells ； bacterial lipopolysaccharide (LPS) ； apoptosis ； tight junction ； TLR-4 ； CD14 ； signaling pathway

參考文獻

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2. Adibi, S.A., The oligopeptide transporter (Pept-1) in human intestine: biology and function. Gastroenterology, 1997. 113(1): p. 332-40.

3. Mackenzie, B. and J.D. Erickson, Sodium-coupled neutral amino acid (System N/A) transporters of the SLC38 gene family. Pflugers Arch, 2004. 447(5): p. 784-95.

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高量細菌內毒素 (LPS) 引起腸道上皮細胞凋亡及緊密連結蛋白破壞：CD14參與之機制探討

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