Takeshi Shibahara

Migration of intestinal intraepithelial lymphocytes into a polarized epithelial monolayer

Intraepithelial lymphocytes (IEL) are a phenotypically distinct population of lymphocytes that reside in mucosal epithelia, below the intercellular tight junctions. Although adhesive functions of this population have been previously studied, relatively little is known about IEL migration from the microvasculature into the epithelium. We demonstrated that cultured human IEL were capable of migration into polarized epithelial cells in vitro, where they assumed a subjunctional position, identical to that observed in vivo. The migration was rapid and efficient and was directionally polarized, such that IEL migrated into epithelial monolayers from the basolateral, but not the apical, aspect. After a 4-h period of residence, up to one-half of the IEL then exited the monolayer basolaterally. Migration was partially inhibited by pertussis toxin, suggesting a potential mechanism for IEL migration by chemokine receptor-mediated signaling. The conditions and ligand pairs used in IEL migration were different from those for neutrophils, another cell type known to migrate through epithelia. This system may serve as a model for microenvironmental homing of IEL into the epithelium.Intraepithelial lymphocytes (IEL) are a phenotypically distinct population of lymphocytes that reside in mucosal epithelia, below the intercellular tight junctions. Although adhesive functions of this population have been previously studied, relatively little is known about IEL migration from the microvasculature into the epithelium. We demonstrated that cultured human IEL were capable of migration into polarized epithelial cells in vitro, where they assumed a subjunctional position, identical to that observed in vivo. The migration was rapid and efficient and was directionally polarized, such that IEL migrated into epithelial monolayers from the basolateral, but not the apical, aspect. After a 4-h period of residence, up to one-half of the IEL then exited the monolayer basolaterally. Migration was partially inhibited by pertussis toxin, suggesting a potential mechanism for IEL migration by chemokine receptor-mediated signaling. The conditions and ligand pairs used in IEL migration were different from those for neutrophils, another cell type known to migrate through epithelia. This system may serve as a model for microenvironmental homing of IEL into the epithelium.

Adhesion molecules expressed on homing lymphocytes in model intestinal epithelia

BACKGROUND & AIMS The development of intestinal intraepithelial lymphocytes (IELs) requires the movement of lymphocytes into the epithelial compartment (i.e., IEL homing). The rules governing and the biologic consequences of IEL homing are poorly understood. The aims of this study were to examine the adhesion molecules involved in IEL homing and the phenotypic alteration of lymphocytes as a consequence of homing. METHODS We previously developed an in vitro IEL homing model consisting of human IEL cell lines and a polarized monolayer of human intestinal epithelial T84 cells. Homing capacity of lymphocytes was assessed by measuring their migration into epithelial monolayers, and phenotypic analysis was performed by flow cytometry. RESULTS In this model, approximately 30% of lymphocytes moved into the epithelial monolayer, regardless of the lymphocyte concentration. Flow cytometric screening of adhesion molecules revealed that homed lymphocytes expressed high levels of integrin alphaXbeta2 and alphaEbeta7 and low levels of alpha4beta7 compared with non-homed lymphocytes. In addition, subpopulations sorted as alphaXbeta2(high) or alphaEbeta7(high) independently showed greater homing capacities. After homing, alphaEbeta7 and intercellular adhesion molecule 1 (ICAM-1) on homed lymphocytes were significantly up-regulated, which was consistent with their high expression observed on freshly isolated human IELs. The up-regulation of alphaEbeta7 (but not ICAM-1) was completely dependent on epithelial-derived transforming growth factor beta1 (TGF-beta1). The expression of alphaXbeta2 was observed on a small population of freshly isolated human IELs, and was markedly induced by stimulation. Also, epithelial-derived TGF-beta1 down-regulated the alphaXbeta2 expression (an event likely to occur after homing). CONCLUSIONS Our findings indicate a relationship between IEL alphaXbeta2 and alphaEbeta7 expression and homing into intestinal epithelia. We also show that phenotypic alteration of IELs is induced by close interaction with intestinal epithelia as a consequence of homing.

Efficacy of endoscopic nasobiliary drainage for the prevention of pancreatitis after papillary balloon dilatation: a pilot study.

Objective: Endoscopic papillary balloon dilatation (EPBD) has been reported to increase the risk of post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis (4%-11%). Based on the hypothesis that performing endoscopic nasobiliary drainage (ENBD) could prevent this complication, we performed EPBD combined with ENBD (EPBD/ENBD) and analyzed the risk of pancreatitis. Methods: Thirty-four patients underwent EPBD followed by ENBD for common bile duct stone(s). Serum amylase levels the following morning and incidence of pancreatitis were compared with those previously reported and with complications of simple diagnostic ERCP performed in our institution. Results: After EPBD/ENBD, amylase levels the following morning were 214.5 ± 152.9 U/L, and no cases developed pancreatitis or hyperamylasemia (>3 times normal). These outcomes were favorable compared with previous EPBD reports. Furthermore, despite the stress of EPBD/ENBD after ERCP, these outcomes were better, even compared with simple ERCP performed at our institution [amylase levels: 318.7 ± 475.2 U/L; hyperamylasemia: 16.5% (P = 0.006); pancreatitis: 7.1%]. Conclusion: Although EPBD has been regarded as a risk factor for post-ERCP pancreatitis, our results suggest the possibility that application of ENBD after EPBD decreases the incidence of pancreatitis and should be studied further. We speculate that ENBD itself prevents pancreatic duct obstruction by residual stones or papillary edema.

Daily intake of sulforaphane-rich broccoli sprouts prevents progression of high salt diet-induced gastric atrophy in H. pylori-infected C57/BL6 mice in vivo

5170 Background: Sulforaphane, shown to be abundantly present in broccoli sprouts, possesses anticarcinogenic activity, since it strongly induces nrf-2 (NF-E2 p45-related factor-2) gene-dependant antioxidant (or phase 2) enzymes, such as glutathione-S- transferase (GSH) and NADPH:quinone reductase(QR), which exert as free radical scavengers, thereby protecting cells from oxidative injury (PNAS, 94:10367, 1997). It has been reported that nrf-2 is strongly expressed in gastric mucosa (Cancer Res 61:3299,2001). Recent studies have also shown that sulforaphane has strong bactericidal activity against Helicobacter pylori (Hp) in vitro (PNAS 99:7610, 2002). We have previously shown that high salt diet accelerates progression of gastric atrophy in Hp-infected mice in vivo by enhancing oxidative stress and by exacerbating Hp-induced gastritis. (Gastroenterology 122: A1, 2002). Based on these backgrounds, we examined if 1) sulforaphane inhibits Hp colonization in vivo , and if 2) sulforaphane prevents progression of high salt diet-induced gastric atrophy in Hp-infected gastric mucosa via activation of nrf-2 dependent antioxidant ezymes. Methods: C57BL/6 wild type (nrf-2+/+) or nrf-2 gene kockout (nrf-2-/-) female mice were inoculated with Hp Sydney Strain; SS1. Mice were daily fed with high salt diet (7.5% NaCl), supplemented with or without 2.5 mmol sulforaphane or homogenate of broccoli sprouts, which contains 2.5 mmol sulforaphane. Mice were sacrificed at 8 wks after starting the treatment. Degree of gastritis and atrophy was evaluated by updated Sydney system. DNA damage was estimated by measuring mucosal level of 8-OHdG. Expressions of TNF-a, IL-1b, MIP-2, and COX-2 were analyzed by real time PCR. Results: 1. Treatment with either sulforaphane or broccoli sprouts enhanced activities of GSH and quinone reductase in gastric mucosa in nrf2+/+, but not in nrf-2-/- mice in vivo . 2. Treatment with sulforaphane or broccoli sprouts attenuated mucosal expressions of TNF-a, IL-1b, MIP-2, and COX-2, mitigated gastritis and DNA damage, and prevented progression of high salt diet-induced gastric atrophy in nrf2+/+, but not in nrf-2-/- mice. 3. Treatment with either sulforaphane or broccoli sprouts decreased Hp colonization in gastric mucosa, but failed to induce complete eradication of Hp in both nrf2+/+ nrf-2-/- mice in vivo .Conclusions: Daily intake of sulforaphane-rich broccoli-sprouts attenuates gastritis and prevents progression of gastric atrophy in Hp-infected mice fed with high salt diet, effects appear to be mediated in part by enhancing activities of nrf-2 gene dependent anti-oxidant enzymes against oxidative stress induced by Hp infection, and in part by suppressing Hp colonization.