Research on The Effect of Intraepitherial Lymphocytes on Intestinal Mucosal Permeability

• Project/Area Number: 12671278
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Digestive surgery
• Research Institution: Osaka University (2001-2002); Kinki University (2000)
• Principal Investigator: USUI Noriaki Osaka University, Graduate School of Medicine, Assistant Professor, 医学系研究科, 助手 (30273626)
• Co-Investigator(Kenkyū-buntansha): KUBOTA Akio Osaka Medical Center and Research Institute for Maternal and Child Health, Department of Pediatric Surgery, Department Director, 部長(研究職) (10161671) ; 大柳 治正 近畿大学, 医学部, 教授 (00030958)
• Project Period (FY): 2000 – 2002
• Project Status: Completed (Fiscal Year 2002)
• Budget Amount: ¥2,900,000 (Direct Cost: ¥2,900,000); Fiscal Year 2002: ¥500,000 (Direct Cost: ¥500,000); Fiscal Year 2001: ¥1,000,000 (Direct Cost: ¥1,000,000); Fiscal Year 2000: ¥1,400,000 (Direct Cost: ¥1,400,000)
• Keywords: Mucosal Permeability / Gut Associated Lymphoid Tissue / Intestinal Mucosal Immunit / Intraepithelial Lymphocytes / Co-culture / Caco-2 / TEER / 腸管免疫 / CaCo-2 / co-culture

Research Abstract

Purpose : The aim of this study is to evaluate the influence of gut associated lymphoid tissue (GALT) on intestinal mucosal permeability by using a co-culture system of Caco-2 human enterocytes monolayer with lymphocytes harvested from normal and inflammatory SD rats
Materials and methods : Human Caco-2 enterocytes were seeded onto the lower aspect of porous membranes of a two-chamber system. "They were cultured for 10 14 days and allowed to form a polarized monolayer. After the measwement of transepithelial electric resistance (TEER) by using an epithelial voltohmmeter, Raji cells (a human lymphoid cell line with predominant B cell features), lymphocytes harvested from rat intestinal Peyer's patches (PPL), lamina propria lymphocytes (LPL) and intraepithelial lymphocytes (IEL) were added to the upper surface of the membranes and allowed to migrate through the pores in between the Caco-2 cell monolayers. After incorporation of Lymphocytes, TEERs were monitored for 3 days. PPL, LPL and IE L were also harvested from the bowel of indometacin induced inflammatory SD rats, in order to investigate the influence of bowel inflammation on these lymphocytes
Results : TEERs were elevated to 200-300 ohm.cm^2 14 days after seeding 1 x10^6 of Caco-2 cells per well, implying the integrity of tight junction. Co-culture with Raji cells for 3 days caused a decrease in TEERs, in a dose-dependent, manner. Permeability to dextran blue was 0.0t±0.0%, 0.0±0.0%, 0.0±0.0%, 0.6±0.3% and 15.411.5% at the Raji cell numbers of 0, 5x10^5 1x10^6, 2x10^6 and 4x10^6, respectively. Co-culture with 1x10^6 of PPL, 1x10^6 of LPL and 1x10^6 of IEL have not decreased TEERs of Caco-2 cell monolayeis. The intestine of SD rats that have been given Indometacin showed a thinner wall and fraglity, indicating an existence of bowel influnmation. Co-cultwe with PPL, LPL and IEL harvested from Indometacin induced inflammatory bowel also have not decreased TEERs of Caco-2 cell monolayers
Conclusion : GULT, such as PPL, LPL and IEL, deliverd from normal rats and inflammatory rats indicated no influence on the permeability of polarized human Caco-2 enterocyte monolayers. Further investigation may be required to clarify the mechanism of cell-cellular interaction between GALT and enterocytes