Frey A, Giannasca K T, Weltzin R, Giannasca P J, Reggio H, Lencer W I, Neutra M R
Department of Pediatrics, Harvard Medical School, Children's Hospital, Boston, Massachusetts 02115, USA.
J Exp Med. 1996 Sep 1;184(3):1045-59. doi: 10.1084/jem.184.3.1045.
Transepithelial transport of antigens and pathogens across the epithelial barrier by M cells may be a prerequisite for induction of mucosal immunity in the intestine. Efficient transport of antigens and pathogens requires adherence to M cell apical surfaces. Coupling of antigen-containing particles to the pentameric binding subunit of cholera toxin (CTB) has been proposed as a means for increasing antigen uptake because the CTB receptor, ganglioside GM1, is a glycolipid present in apical membranes of all intestinal epithelial cells. To test the accessibility of enterocyte and M cell membrane glycolipids to ligands in the size ranges of viruses, bacteria, and particulate mucosal vaccines, we analyzed binding of CTB probes of different sizes to rabbit Peyer's patch epithelium. Soluble CTB-fluorescein isothiocyanate (diameter 6.4 nm) bound to apical membranes of all epithelial cells. CTB coupled to 14 nm colloidal gold (final diameter, 28.8 nm) failed to adhere to enterocytes but did adhere to M cells. CTB-coated, fluorescent microparticles (final diameter, 1.13 microns) failed to adhere to enterocytes or M cells in vivo or to well-differentiated Caco-2 intestinal epithelial cells in vitro. However, these particles bound specifically to GM1 on BALB/c 3T3 fibroblasts in vitro and to undifferentiated Caco-2 cells that lacked brush borders and glycocalyx. Measurements of glycocalyx thickness by electron microscopy suggested that a relatively thin (20 nm) glycocalyx was sufficient to prevent access of 1-micron microparticles to glycolipid receptors. Thus, the barrier function of the intestinal epithelial cell glycocalyx may be important in limiting microbial adherence to membrane glycolipids, and in CTB-mediated targeting of vaccines to M cells and the mucosal immune system.
M细胞介导的抗原和病原体跨上皮屏障的跨上皮转运可能是诱导肠道黏膜免疫的前提条件。抗原和病原体的有效转运需要黏附于M细胞顶端表面。有人提出将含抗原颗粒与霍乱毒素（CTB）的五聚体结合亚基偶联，作为增加抗原摄取的一种方法，因为CTB受体神经节苷脂GM1是所有肠道上皮细胞顶端膜中存在的一种糖脂。为了测试肠道上皮细胞和M细胞膜糖脂对病毒、细菌和颗粒性黏膜疫苗大小范围内配体的可及性，我们分析了不同大小的CTB探针与兔派尔集合淋巴结上皮的结合情况。可溶性异硫氰酸荧光素标记的CTB（直径6.4 nm）与所有上皮细胞的顶端膜结合。与14 nm胶体金偶联的CTB（最终直径28.8 nm）不能黏附于肠上皮细胞，但能黏附于M细胞。CTB包被的荧光微粒（最终直径1.13微米）在体内既不能黏附于肠上皮细胞或M细胞，在体外也不能黏附于分化良好的Caco-2肠道上皮细胞。然而，这些颗粒在体外能特异性结合BALB/c 3T3成纤维细胞上的GM1，以及缺乏刷状缘和糖萼的未分化Caco-2细胞。通过电子显微镜测量糖萼厚度表明，相对较薄（20 nm）的糖萼足以阻止1微米微粒接近糖脂受体。因此，肠道上皮细胞糖萼的屏障功能在限制微生物黏附于膜糖脂以及CTB介导的疫苗靶向M细胞和黏膜免疫系统方面可能很重要。
