Sato T T, Kovacich J C, Boyle E M, Haddix T L, Weintraub A, Pohlman T H
Department of Surgery, Harborview Medical Center, University of Washington School of Medicine, Seattle 98104-9796, USA.
J Surg Res. 1998 Feb 1;74(2):103-11. doi: 10.1006/jsre.1997.5248.
We studied the capacity of isolated Bacteriodes fragilis outer membrane, B. fragilis NCTC9343 lipopolysaccharide (LPS; endotoxin), and B. fragilis NCTC9343 capsular polysaccharides to activate human umbilical vein endothelial cell (HUVEC) monolayers. To assess HUVEC activation, E-selectin expression was measured by enzyme-linked immunosorbent assay (ELISA), Northern blot analysis for E-selectin-specific mRNA, and electrophoretic gel mobility shift assay (EMSA) for NF-kappa B, a transcription factor necessary for E-selectin gene activation. Exposure of HUVECs to B. fragilis outer membrane fractions, separated from other components of the B. fragilis cell wall by isopycnic, sucrose gradient centrifugation, significantly increased surface expression of E-selectin and induced functional endothelial cell-dependent leukocyte adhesion. B. fragilis outer membranes induced translocation of NF-kappa B to HUVEC nuclei and accumulation of E-selectin mRNA in HUVEC cytoplasm. E-selectin expression induced by B. fragilis outer membranes was not blocked by polymixin B. In contrast, E-selectin expression induced by outer membrane fractions purified from E. coli was competitively inhibited by polymixin B. Neither purified B. fragilis LPS, a prominent constituent of the outer membrane, nor purified B. fragilis capsular polysaccharides induced HUVEC activation. Two different monoclonal antibodies directed against human CD14 completely inhibited B. fragilis outer membrane-induced NF-kappa B activation, E-selectin transcription, and E-selectin surface expression. We conclude that the outer membrane component of the B. fragilis cell wall contains a proinflammatory factor(s), that is not LPS, which induces human endothelial cell activation by a soluble CD14-dependent mechanism.
我们研究了脆弱拟杆菌外膜、脆弱拟杆菌NCTC9343脂多糖（LPS；内毒素）和脆弱拟杆菌NCTC9343荚膜多糖激活人脐静脉内皮细胞（HUVEC）单层的能力。为了评估HUVEC的激活情况，通过酶联免疫吸附测定（ELISA）检测E-选择素的表达，采用Northern印迹分析E-选择素特异性mRNA，并通过电泳凝胶迁移率变动分析（EMSA）检测NF-κB，NF-κB是E-选择素基因激活所必需的转录因子。将HUVEC暴露于通过等密度蔗糖梯度离心从脆弱拟杆菌细胞壁的其他成分中分离出来的脆弱拟杆菌外膜组分，可显著增加E-选择素的表面表达，并诱导功能性内皮细胞依赖性白细胞黏附。脆弱拟杆菌外膜诱导NF-κB转位至HUVEC细胞核，并使E-选择素mRNA在HUVEC细胞质中积累。脆弱拟杆菌外膜诱导的E-选择素表达不受多黏菌素B的阻断。相比之下，从大肠杆菌纯化的外膜组分诱导的E-选择素表达受到多黏菌素B的竞争性抑制。无论是纯化的脆弱拟杆菌LPS（外膜的主要成分），还是纯化的脆弱拟杆菌荚膜多糖，均未诱导HUVEC激活。两种针对人CD14的不同单克隆抗体完全抑制了脆弱拟杆菌外膜诱导的NF-κB激活以及E-选择素转录和E-选择素表面表达。我们得出结论，脆弱拟杆菌细胞壁的外膜成分含有一种促炎因子，而非LPS，该促炎因子通过可溶性CD14依赖性机制诱导人内皮细胞激活。
