Ruemmele F M, Beaulieu J F, Dionne S, Levy E, Seidman E G, Cerf-Bensussan N, Lentze M J
Laboratory of Gastrointestinal Immunology, Division of Gastroenterology, Department of Paediatrics, Children's Hospital Medical Centre, University of Bonn, Bonn, Germany.
Gut. 2002 Dec;51(6):842-8. doi: 10.1136/gut.51.6.842.
Circulating levels of endotoxin (or lipopolysaccharide (LPS)) and anti-endotoxin antibodies are increased in patients with inflammatory bowel disease, supporting the hypothesis of a role for endogenous bacterial products in the pathogenesis of these disorders.
The aim of this study was to analyse the direct effects of LPS on intestinal epithelial cell turnover.
LPS significantly inhibited growth of the human non-transformed immature crypt cell line (HIEC), whereas IEC-6 cell proliferation was stimulated by LPS. As LPS is a physiological inducer of tumour necrosis factor alpha (TNFalpha) in various cell systems and this cytokine exerted similar anti-proliferative (HIEC) or growth stimulatory (IEC-6 cells) effects, the study thus tested the hypothesis that endogenously produced TNFalpha in response to LPS mediates this growth modulatory effect in an autoparacrine/paracrine way. Therefore, during LPS stimulation, the biological activity of TNFalpha was blocked using neutralising anti-TNFalpha antibodies, as well as inhibitory, antagonistic antibodies directed against the p55 TNF receptor, signalling the antimitotic TNFalpha effect in HIEC. Both experimental approaches completely abolished the growth modulatory effects of LPS in HIEC/IEC-6 cells. Production and secretion of TNFalpha by HIEC/IEC-6 cells in response to LPS was confirmed on mRNA and protein level by reverse transcription polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay. LPS signalling was independent of CD14 in HIEC, as these cells lack this receptor. However, HIEC expressed TLR4 and MD2 resulting in a fully functional signalling complex as demonstrated by RT-PCR, western blot, and immunofluorescence analyses.
These results support the hypothesis that LPS induced changes of intestinal epithelial cell turnover may directly contribute to the pathogenesis of inflammatory epithelial cell lesions by endogenous TNFalpha production by enterocytes.
炎症性肠病患者体内内毒素（或脂多糖（LPS））和抗内毒素抗体的循环水平升高，支持内源性细菌产物在这些疾病发病机制中起作用的假说。
本研究旨在分析LPS对肠上皮细胞更新的直接影响。
LPS显著抑制人未转化的未成熟隐窝细胞系（HIEC）的生长，而LPS刺激IEC-6细胞增殖。由于LPS是多种细胞系统中肿瘤坏死因子α（TNFα）的生理诱导剂，且该细胞因子具有类似的抗增殖（HIEC）或生长刺激（IEC-6细胞）作用，因此本研究检验了以下假说：内源性产生的TNFα对LPS的反应以自分泌/旁分泌方式介导这种生长调节作用。因此，在LPS刺激期间，使用中和抗TNFα抗体以及针对p55 TNF受体的抑制性、拮抗性抗体阻断TNFα的生物活性，这些抗体可传导HIEC中的抗有丝分裂TNFα效应。两种实验方法均完全消除了LPS在HIEC/IEC-6细胞中的生长调节作用。通过逆转录聚合酶链反应（RT-PCR）和酶联免疫吸附测定在mRNA和蛋白质水平证实了HIEC/IEC-6细胞对LPS的反应产生并分泌TNFα。HIEC中的LPS信号传导不依赖于CD14，因为这些细胞缺乏该受体。然而，HIEC表达TLR4和MD2，如RT-PCR、蛋白质印迹和免疫荧光分析所示，形成了功能完全的信号复合物。
这些结果支持以下假说：LPS诱导的肠上皮细胞更新变化可能通过肠细胞内源性产生TNFα直接促成炎症性上皮细胞病变的发病机制。
