Transcytosis of Murine-Adapted Bovine Spongiform Encephalopathy Agents in an In Vitro Bovine M Cell Model

Transmissible spongiform encephalopathies (TSE), including bovine spongiform encephalopathy (BSE), are fatal neurodegenerative disorders in humans and animals. BSE appears to have spread to cattle through the consumption of feed contaminated with BSE/scrapie agents. In the case of an oral infection, the agents have to cross the gut-epithelial barrier. We recently established a bovine intestinal epithelial cell line (BIE cells) that can differentiate into the M cell type in vitro after lymphocytic stimulation (K. Miyazawa, T. Hondo, T. Kanaya, S. Tanaka, I. Takakura, W. Itani, M. T. Rose, H. Kitazawa, T. Yamaguchi, and H. Aso, Histochem. Cell Biol. 133:125-134, 2010). In this study, we evaluated the role of M cells in the intestinal invasion of the murine-adapted BSE (mBSE) agent using our in vitro bovine intestinal epithelial model. We demonstrate here that M cell-differentiated BIE cells are able to transport the mBSE agent without inactivation at least 30-fold more efficiently than undifferentiated BIE cells in our in vitro model. As M cells in the follicle-associated epithelium are known to have a high ability to transport a variety of macromolecules, viruses, and bacteria from gut lumen to mucosal immune cells, our results indicate the possibility that bovine M cells are able to deliver agents of TSE, not just the mBSE agent.Transmissible spongiform encephalopathies (TSE) or prion diseases, including human Creutzfeldt-Jakob disease (CJD) and endemic sheep scrapie, are fatal neurodegenerative diseases. The host cellular prion protein (PrP^C), which is thought to have neuroprotective function, is expressed in both humans and a range of other animal species (36), and PrP^C expression is essential for TSE disease susceptibility (7). The prion hypothesis suggests that infectious abnormally folded prion protein (PrP^Sc) is the primary or sole composition of the infectious agent of TSE (known as the prion). However, the molecular composition of PrP^Sc remains speculative and unclear. It is well known that the detergent-insoluble and relatively proteinase K (PK)-resistant prion protein (PrP-res) is detectable in many kinds of TSE-infected tissues, including the brain. Although some studies have revealed that PrP-res does not correlate with infectivity levels in animal tissues as well as in subcellular fractions (37, 40), PrP-res is a useful surrogate marker for TSE infection.Bovine spongiform encephalopathy (BSE) is a TSE of cattle. The first case of BSE in the world was found in the United Kingdom in 1986 (41), and it spread to continental Europe, North America, and Japan. At present, BSE is a threat to human health because of the appearance of BSE-linked variant Creutzfeldt-Jakob disease (vCJD). The cattle BSE agent appears to spread to the cattle population through the consumption of rendered meat and bone meal contaminated with BSE-infected brain or spinal cord (32). Likewise, the transmission of vCJD to humans is likely to have occurred following the consumption of BSE-contaminated food (6, 13, 45). In cases of oral transmissions such as BSE and vCJD, TSE agents first have to cross the gut epithelium, but the exact mechanisms for intestinal invasion still are unknown.Intestinal epithelial cells are bound to each other by tight junctions. This close-packed structure forms a highly selective barrier for macromolecules and limits the access of pathogenic bacteria to the underlying host tissues (43). Gut epithelia are composed of two different epithelial types. One is the villous epithelium, and the other is the follicle-associated epithelium (FAE), which overlies gut-associated lymphoid tissues (GALTs) such as Peyer''s patches. The FAE is considerably different from the surrounding villous epithelium, in that it contains membranous (M) cells. Because M cells have a high capacity for the transcytosis of a wide range of macromolecules, viruses, and microorganisms, they are specialized epithelial cells and act as an antigen sampling system from the gut lumen (28). M cells are, however, exploited by some pathogenic microorganisms and viruses as the entry site to invade the body (20, 29). In fact, some experiments have proposed that M cells transport TSE agents (12) and that Peyer''s patches including the FAE are associated with TSE disease susceptibility (35). In contrast, some authors have suggested the M cell-independent pathway as the main transport route of TSE agents across the intestinal epithelium (16, 23, 27). The intestinal cell types involved in the transport of TSE agents therefore are still a matter of controversy at this stage.Recently, we succeeded in the establishment of a bovine intestinal epithelial cell line (BIE cells) and the development of an in vitro bovine M cell model by coculture with murine intestinal lymphocytes or the supernatant of bovine peripheral blood mononuclear cells (PBMC) stimulated by interleukin 2 (IL-2) (25). In this study, we investigate whether M cells can transport the murine-adapted BSE (mBSE) agent using BIE cells. We demonstrate here that M cell-differentiated BIE cells are able to deliver mBSE agents at least 30-fold more efficiently than undifferentiated BIE cells, although a small number of the mBSE agents pass through undifferentiated BIE cells. Our findings thus provide an insight into the uptake mechanisms of TSE agents, including the cattle BSE agent from the gut lumen.