Sawai T, Goldstone N, Drongowski R A, Coran A G, Harmon C M
Section of Pediatric Surgery, University of Michigan, Mott Children's Hospital, Ann Arbor, MI 48109-0245, USA.
Pediatr Surg Int. 2001 May;17(4):275-9. doi: 10.1007/s003830100593.
Intestinal secretory immunoglobulin A (sIgA) plays an important role in gut mucosal immunity in vivo; however, in-vitro enterocyte models for studying the mechanisms of these effects are lacking. This study utilizes a cell-culture model to investigate the effect of sIgA on bacterial translocation (BT) across human enterocytes co-cultured with human lymphoid cells (Raji cells). This model is intended to mimic in-vivo enterocyte/lymphocyte interactions found in intestinal follicle-associated epithelia. Human Caco-2 enterocytes were grown to confluence on porous filters in the apical chamber of a two-chamber cell-culture system. After differentiation, human B lymphoid cells (Raji cells) were added to the basolateral surface of Caco-2 monolayers for 3 days' co-culture, followed by washing away of unincorporated Raji cells. Transepithelial electrical resistance (TEER) was used to measure tight-junction permeability. Monolayers were treated with or without sIgA, IgG (negative control), or mannose (positive control). BT across the cell monolayer was determined 1.5 h after addition of Escherichia coli. Statistical analysis was by the Kruskal-Wallis test, P below 0.05 considered significant. In co-culture monolayers treated with sIgA, IgG, or mannose, there was no significant effect on TEER; however, the magnitude of BT across cells treated with sIgA (1.3 +/- 0.4 log10CFU/ml) and mannose (1.6 +/- 1.1 log10CFU/ml) was significantly decreased compared to PBS (3.9 +/- 0.4 log10CFU/ml) and IgG (2.9 +/- 0.6 log10CFU/ml) controls (P < 0.05). sIgA BT inhibition was dose-dependent. BT inhibition by sIgA and mannose was additive (0.5 +/- 1 log10CFU/ml). Inhibition of BT was negated when sIgA and mannose were removed by washing prior to E. Coli addition (3.6 +/- 0.5 log10CFU/ml), suggesting that both inhibitors act through bacterial binding.
肠道分泌型免疫球蛋白A（sIgA）在体内肠道黏膜免疫中发挥着重要作用；然而，目前缺乏用于研究这些作用机制的体外肠上皮细胞模型。本研究利用细胞培养模型来研究sIgA对与人类淋巴细胞（Raji细胞）共培养的人肠上皮细胞的细菌移位（BT）的影响。该模型旨在模拟在肠道滤泡相关上皮中发现的体内肠上皮细胞/淋巴细胞相互作用。将人Caco-2肠上皮细胞在双室细胞培养系统顶室的多孔滤膜上培养至汇合。分化后，将人B淋巴细胞（Raji细胞）添加到Caco-2单层细胞的基底外侧表面进行3天的共培养，然后洗去未结合的Raji细胞。使用跨上皮电阻（TEER）来测量紧密连接通透性。单层细胞分别用或不用sIgA、IgG（阴性对照）或甘露糖（阳性对照）处理。在添加大肠杆菌1.5小时后测定跨细胞单层的细菌移位。采用Kruskal-Wallis检验进行统计分析，P值低于0.05认为具有统计学意义。在用sIgA、IgG或甘露糖处理的共培养单层细胞中，对TEER没有显著影响；然而，与磷酸盐缓冲液（PBS，3.9±0.4 log10CFU/ml）和IgG（2.9±0.6 log10CFU/ml）对照相比，用sIgA（1.3±0.4 log10CFU/ml）和甘露糖（1.6±1.1 log10CFU/ml）处理的细胞的细菌移位量显著降低（P<0.05）。sIgA对细菌移位的抑制作用呈剂量依赖性。sIgA和甘露糖对细菌移位的抑制作用具有相加性（0.5±1 log10CFU/ml）。在添加大肠杆菌之前通过洗涤去除sIgA和甘露糖后，细菌移位的抑制作用消失（3.6±0.5 log10CFU/ml），这表明两种抑制剂均通过细菌结合起作用。
