Although intestinal epithelial cells (IECs) are known as an important source for IL-6, it is not known whether mechanical forces affect IL-6 production. We investigated how transmural pressure modulates IL-6 synthesis and activation of transcription factors in IECs. Pressure was loaded onto IEC-18 cells by introducing compressed helium gas into the cell culture flask for 1-48 h. IL-6 release into the culture media was determined by cell proliferation bioassay using an IL-6-dependent mouse hybridoma cell line (7TD1). Exposure to pressure (80 mmHg) significantly enhanced IL-6 release into the culture media from IEC-18 cells at 12 h. Under control conditions, IL-6 secretion was directed to the basolateral side, but after exposure to pressure IL-6 secretion was increased in both the apical and basolateral sides. A nuclear factor kappa B (NF-kappaB) decoy reversed completely the pressure-induced increase of IL-6 secretion by IEC-18 cells. Pressure treatment enhanced IL-6 mRNA expression in IECs within 6 h. Pressure loading significantly enhanced the activation of both NF-kappaB and NF-IL-6 from 1h in the nuclear protein of IEC-18 cells as assessed by the electrophoretic mobility shift assay using FITC-conjugated specific primers. Increased phosphorylation of I-kappa B was also demonstrated in the cytosol of IEC cells within 1h by Western blot analysis. These results suggest a possible role for pressure loading in immune modulation of the intestinal mucosa by the stimulation of IL-6 release from intestinal epithelial cells.

Intestinal mucosal immunity is modulated by cytokine release from intestinal cells, but little is known about the relation between nutrient absorption and cytokine release. In this study, we examined how exposure to fatty acids affects the production of growth-regulated oncogene/cytokine-induced neutrophil chemoattractant-1 (GRO/CINC-1) and interleukin (IL)-6 in rat intestinal epithelial cells (IEC). The long-chain fatty acids, oleic, linoleic and arachidonic acids, and the middle-chain fatty acid octanoic acid were administered to subconfluent cultures of IEC-6 cells alone, or in combination with IL-1beta and transforming growth factor (TGF)-beta. The GRO/CINC-1 and IL-6 concentrations in culture media were determined by sandwich enzyme immunoassay. In epithelial cells, GRO/CINC-1 and IL-6 mRNA expression were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and mitogen-activated protein kinase (MAPK) activities determined by immunoblotting. Administration of long-chain fatty acids significantly increased the GRO/CINC-1 and IL-6 secretion into culture media, and this secretion was markedly increased (P < 0.05) in the presence of IL-1beta or TGF-beta. Octanoic acid had no effect on GRO/CINC-1 or IL-6 production. Furthermore, treatment with long-chain fatty acids significantly enhanced the GRO/CINC-1 and IL-6 expression that was induced by IL-1beta or TGF-beta. MAPK activity was significantly enhanced by treatment with long-chain fatty acids. Inhibitors of phospholipase C, protein kinase C or MAPK significantly reduced the fatty acid-induced increase in GRO/CINC-1 secretion, whereas a calcium/calmodulin inhibitor did not attenuate the secretion. These results suggest that long-chain fatty acids enhance cytokine release under conditions of inflammatory stimulation in the intestinal mucosa.

BACKGROUND & AIMS:The basic mechanisms of food allergies are still unknown. The aims of this study were to investigate whether endothelins (ETs) in the intestinal mucosa are involved in the pathogenesis of intestinal anaphylaxis.METHODS:Sprague-Dawley rats were sensitized to chicken egg albumin (EA) by intraperitoneal injection. Fourteen days after sensitization, EA was administered in the jejunal segments to induce intestinal anaphylaxis. Net water outflux and histamine release into loops and serum concentrations of rat mast cell protease II (RMCP-II) were determined. ET-1 and ET-3 concentrations in the jejunal mucosa were determined, and expression of the corresponding messenger RNAs was examined by competitive polymerase chain reaction.RESULTS:In sensitized animals, challenge with intraluminal antigen caused a significant increase in net water outflux and histamine release together with an elevation of serum RMCP-II concentrations. Mucosal concentrations of ET-1 and ET-3 and expression of their messenger RNAs were significantly increased in sensitized animals after EA challenge. Treatment with an ETA-receptor antagonist, but not an ETB-receptor antagonist, attenuated the increase in net water outflux, histamine release, and serum RMCP-II concentrations in rats with EA-induced intestinal anaphylaxis.CONCLUSIONS:Release of ETs in the intestinal mucosa increased in sensitized animals after EA challenge. ETs may play a significant role in the development of intestinal anaphylaxis via an ETA receptor.

Background—Increased production of reactive metabolites of oxygen and nitrogen has been implicated in chronic inflammation of the gut. The object of this study was to examine the magnitude and location of nitric oxide synthase (NOS) activity and peroxynitrite formation in the colonic mucosa of patients with ulcerative colitis in relation to the degree of inflammation.Subjects—Thirty three patients with active ulcerative colitis (17 with mild or moderate inflammation, 16 with severe inflammation).Methods—Inducible NOS activity was determined in the colonic mucosa by measuring the conversion ofl-arginine to citrulline in the absence of calcium. The localisation of NOS and nitrotyrosine immunoreactivity was assessed immunohistochemically using the labelled streptavidin biotin method.Results—Inducible NOS activity increased in parallell with the degree of inflammation of the mucosa. Expression of inducible NOS was found not only in the lamina propria, but also in the surface of the epithelium. Peroxynitrite formation as assessed by nitrotyrosine staining was frequently observed in the lamina propria of actively inflamed mucosa.Conclusions—Nitric oxide and peroxynitrite formation may play an important role in causing irreversible cellular injury to the colonic mucosa in patients with active ulcerative colitis.

Although it has been speculated that active oxidants and mitochondrial membrane damages play roles in ethanol-induced gastric mucosal damages, its detail remains unknown. The present study was designed to investigate whether ethanol induces oxidative stress and mitochondrial permeability transition (MPT) before cell death of gastric mucosal cells. Rat gastric mucosal cells (RGM-1) were kept in serum-free Dulbecco's modified Eagle's medium before addition of various concentrations of ethanol. Nuclear morphological aftemations and membrane barrier dysfunction of RGM-1 cells were assessed by staining with Hoechst 33342 and propidium iodide, respectively. To assess the contribution of oxygen-derived free radicals and intracellular glutathione, scavenger of hydrogen peroxide and the hydroxyl radical, N,N-dimethylthiourea, glutathione precursor, N-acetyl-L-cysteine, and an inhibitor of alcohol dehydrogenase, 4-methylpyrazole were added before treatment with ethanol. To investigate MPT, calcein and tetramethylrhodamine methyl ester were loaded before addition of ethanol, and the changes of fluorescence intensity were monitored using a laser scanning confocal microscope. Ethanol (>5% v/v) dose-dependently increased the number of propidium iodide-positive cells, suggesting a diminished barrier function of cell membrane. After addition of ethanol, mitochondria were filled quickly with calcein indicating MPT, which was accompanied by mitochondrial depolarization, as shown by loss of tetramethylrodamine methyl ester before cell death. Ethanol-induced cell death was significantly attenuated by simultaneous incubation with either N,N-dimethylthiourea or N-acetyl-L-cysteine, suggesting the importance of intracellular redox states in inducing cellular damage, whereas such change was not attenuated by 4-methylpyrazole. Present results suggest that ethanol treatment induces intracellular oxidative stress and produces MPT and mitochondrial depolarization, which are preceding cell death in gastric mucosal cells. Intracellular antioxidants, such as glutathione, may have a significant protective action against ethanol in gastric mucosal cells.

association between NHE3 and villin is not mediated through actin.In summary: (i) NHE3 and villin co-immunoprecipitate from rabbit ileal BBM; (ii) villin associates with fusion proteins made to the COOH-terminal of NHE3; (iii) carbachol does not change the association of villin with NHE3; (iv) there is no actin present in this complex of villin and NHE3.In conclusion: these studies demonstrate association of NHE3 in the brush border with a cytoskeletal protein, villin and suggest a role for the microvillar cytoskeletal protein villin other than in brush border morphogenesis.Perhaps villin functions in the restriction of NHE3 to the apical membrane or in the endocytic retrieval of NHE3, which we have previously shown is a mechanism for the short-term protein kinase regulation of NHE3.

Endothelin (ET), a vasoconstrictive peptide, is known to have a variety of biological actions. Although ET is released by vascular endothelial cells, other cell populations also have been reported to synthesize and release ET. In this study, we examined whether ET is synthesized by intestinal epithelial cells and whether it affects induction of epithelial cell proliferation by interleukin-2 (IL-2). Subconfluent monolayers of intestinal epithelial cells (IEC-6 and IEC-18) were maintained in serum-free medium before addition of rat IL-2. Both IEC-6 and IEC-18 cells released ET-1 into the medium under unstimulated conditions, as determined by a sandwich ELISA. IL-2 significantly enhanced ET-1 release in a time-dependent manner. ET-3 was not detectable in the culture media of either cell line. Expression of ET-1 and ET-3 mRNA in epithelial cells was assessed by competitive PCR. Both cell lines were shown to express ET-1 mRNA, but no ET-3 mRNA was detected. IL-2 treatment enhanced ET-1 mRNA expression by both IEC-6 and IEC-18 cells. Both cell lines also expressed mRNA for ETA and ETB receptor subtypes. When cell proliferation was assessed, exogenous ET-1 induced a slight proliferative response in both types of cells that was consistent and significant at low ET-1 concentrations; cell growth was inhibited at a higher concentration (10(-7) M). IL-2 produced a significant proliferative response in both cell lines. However, the addition of ET-1 (10(-7) M) to culture media attenuated the IL-2-induced increase in cell proliferation. ETA-receptor antagonists significantly enhanced cellular proliferation, suggesting involvement of the ETA receptor in modulation of IL-2-induced intestinal epithelial cell growth.

Subserosal injection of peptidoglycan-polysaccharide (PG-PS) is known to induce chronic granulomatous enteritis. In this study, using intravital microscope we examined the dynamic process of lymphocyte postcapillary venule (PCV) interaction and the role of alpha 4-integrin in rat Peyer's patches and villus mucosa during the formation of PG-PS induced enteritis. Female Lewis rats received 20 mg rhamnose of PG-PS in ileal Peyer's patches. T lymphocytes collected from intestinal lymph were fluorescence labeled and injected into jugular vein of PG-PS-treated recipient rats. Lymphocyte behavior was observed under fluorescence microscope with SIT camera. In some experiments, T lymphocytes were preincubated with anti-alpha 4-integrin Ab before infusion. In another sets of experiments. anti-alpha 4-integrin Ab was injected at day 0, 2, 4 in PC-PS-treated rats. The intramural injection of PG-PS induced significant inflammation of Peyer's patches. Before inflammation becomes significant, rolling and sticking df T lymphocytes increased in the PCV of Peyer's patches and submucosal venules of the mucosa surrounding Peyer's patches. Antibody treatment significantly attenuated the increase of T lymphocyte rolling and sticking, and also inhibited the development of chronic inflammation with granuloma formation.

lumen, possibly associated with an increased exfoliation.Fat absorption does not directly increase the rate of DAn synthesis in the intestinal mucosa.The enzyme released by fat appears to originate from an intestinal storage pool.

Effects of intraluminal pressure on cultured intestinal epithelial cells were assessed by measuring cell proliferation and DNA synthesis after exposure to various pressures. Pressures of 40 to 120 mm Hg promoted cell proliferation and DNA synthesis. Pressure-induced DNA synthesis was further enhanced by the addition of interleukin-2, suggesting the regulation of intestinal epithelial growth by pressure in coordination with cytokines. Pretreatment with either a phospholipase C inhibitor or protein kinase C inhibitor significantly inhibited DNA synthesis promoted by pressure and interleukin-2. This study demonstrates a novel mechanism whereby pressure regulates cell growth in intestinal epithelial cells, possibly via activation of phospholipase C and protein kinase C.

The present study was designed to investigate whether acute ethanol intoxication increases the production of active oxidants, and subsequently promotes apoptosis of hepatocytes. Hepatocytes were isolated from male Wistar rats, and cultured in the presence or absence of ethanol. The fluorescence in situ nick end labeling method and an enzyme-linked immunosorbent assay (ELISA) system to quantify fragmented DNA were used to estimate apoptotic change in hepatocytes. Nuclear morphological alterations and membrane barrier dysfunction of hepatocytes were assessed by staining with Hoechst 33342 and propidium iodide (PI). Intracellular glutathione level was determined as the fluorescence of monochlorobimane (MCLB), which forms conjugate with glutathione to become fluorescent. Ethanol (100 mmol/L) increased the amount of fragmented DNA and the number of apoptotic hepatocytes in vivo as well as in vitro. These ethanol-induced alterations in hepatocytes were attenuated by simultaneous incubation with either 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, or dimethylthiourea, an intracellular oxidant scavenger. Diethyl maleic acid (DMA), a glutathione depletor, enhanced the induction of apoptotic change, and decreased membrane barrier function in ethanol-treated hepatocytes, whereas ethanol per se did not increase the number of PI-positive hepatocytes. Furthermore, combination of ethanol and DMA but not ethanol alone decreased the hepatocyte MCLB fluorescence. Taken together, the present study suggests that active oxidants produced during ethanol metabolism mediate fragmentation of DNA in hepatocytes, and that intracellular antioxidants such as glutathione play a critical role in the cytoprotective mechanisms of hepatocyte against lethal cell death, ie, apoptosis, induced by ethanol.

The constant immune cell trafficking through gut-associated lymphoid tissues (GALT) is known to be critical in the maintenance of the immune barrier of the intestinal mucose. In this study, we examined how activation of T lymphocytes significantly affect lymphocyte-endothelial interaction in microcirculation of Peyer's patches and villus mucosa of intestine. T lymphocytes collected from intestinal lymph were stimulated by concanavalin A (Con A).The migration of CFSE-labeled unstimulated and Con A-treated T lymphocytes were monitored in rat intestinal microvessels using an intravital fluorescence microscope. Con A stimulation significantly increased the rolling velocity of T lymphocytes in postcapillary venules (PCV) of Peyer's patches and showed the loss of selective adherent properties to PCV as demonstrated in unstimulated T cells. By FACScan analysis, decrease in expression of L-selectin and increased expression of CD11a were observed in Con A-treated T lymphocytes. In conclusion, activation of gut-derived T lymphocytes with Con A significantly alters their migration pathway and showed loss of selective migration to Peyer's patches, possibly mediated by the changes of adhesion molecules. Copyright 1997 by Monduzzi Editore S.p.A.

Although vasoactive intestinal peptide (VIP) has been postulated to function in modulation of T cell trafficking, the exact mechanism has not been elucidated in vivo. In the present study, the effects of VIP on T lymphocyte migration were examined in rat Peyer's patches. T lymphocytes collected from intestinal lymph of rats were labeled with carboxyfluorescein diacetate succinimidyl ester and injected into the jugular vein. Peyer's patches of the recipient rats were observed with intravital fluorescence microscopy. In vivo intra-arterial infusion of or in vitro incubation with VIP did not affect the initial lymphocyte interaction with postcapillary venules of Peyer's patches. However, these treatments with VIP significantly inhibited transendothelial migration and also significantly blocked the interstitial migration of T cells and inhibited their subsequent appearance in the interfollicular lymphatics. Treatment with adenosine 3',5'-cyclic monophosphate (cAMP)-inducing agents resulted in similar inhibitory effect on T lymphocyte migration in Peyer's patches. In conclusion, VIP has significant inhibitory effects on T lymphocyte migration in Peyer's patches, possibly mediated by elevation of the intracellular cAMP concentrations.