Induction of human colonic intestinal epithelial cell death by interferon-γ

April 1998 allowing its members to be identified by PCR using degenerate primers in combination with library screening. Based on sequence homology among the rat cNT1, rat SPNT and other bacterial nucleoside transporters, degenerate oligonucleotides flanking the conserved regions: (PYLADMT) [5'-primer CCNTAYYTNGCNGAYATGAC (N=A/G/C/T, Y=C/T), 512 fold of degeneracy] and (NEFVAYQ) [3'-primer TGRTANGCNAC RAAYTCRTT (N=A/C/G/T, R=A/G, Y=C/T), 256 fold of degeneracy] were designed and were used to amplify cDNAs from a rabbit ileal library. Using low stringency amplification of an annealing temperature of 37°C and 30 cycles of amplification, these paired primers generated a predicted 546bp PCR product that was subsequently subcloned into PCRII vector. Colonies were selected, and cDNAs were sequenced. Sequences revealed that a mixture of two partial cDNAs was isolated. These two cDNAs were subsequently used to screen a rabbit ileal library to obtain their respective full length clones. Clone 10 is 2.3kb long and encodes a protein of 651 aa with a predicted size of 71.6kD. This protein exhibits 80% amino acid identity with rat cNT1 and is presumably the rabbit cNT1. Clone 5 is 2.9kb long and encodes a protein of 658 aa with a predicted size of 72.2kD, and has 82% amino acid identity with the rat SPNT and presumably is the the rabbit SPNT. Both rabbit cNT1 and SPNT contain 6 putative C-kinase sites. Three of 6 C-kinase sites are conserved between rabbit and rat cNT1, and 4 out of 6 of these sequences are also conserved between rabbit and rat SPNT. Northern analysis with rabbit ileal mRNA showed that rabbit cNT1 and rabbit SPNT hybridized to transcripts of 3.2kb and 3.4kb, respectively. To further biochemically characterize cNT1 and SPNT, rabbit cNT1 and SPNT were epitope tagged with the 11 amino acid VSVG epitope (vesicular stomatitis viral glycoprotein). Rabbit cNTI/VSVG and rabbit SPNT/VSVG were then transiently transfected into Cos-7 ceils. Western blot analysis of crude lysates of cNTI/VSVG/Cos-7 cells and SPNT/VSVG/Cos-7 cells showed that both rabbit cNT1/VSVG and rabbit SPNT/VSVG have a Mr of 54kD. Thus, we have isolated cDNAs encoding full-length rabbit cNT1 and SPNT and transiently expressed these two proteins in Cos-7 cells. In conclusion: 1) we have cloned two members of the rabbit Na dependent nucleoside transporter gene family, cNT1 and SPNT; 2) rabbit cNT1 and SPNT exhibit 65% in amino acid identity and are 54kD proteins when expressed in Cos-7 cells; 3) the presence of conserved putative C-kinase phosphorylation sites in cNT1 and SPNT suggests that these two proteins may be regulated by protein kinase C. • G1732 INDUCTION OF HUMAN COLONIC INTESTINAL EPITHELIAL CELL DEATH BY INTERFERON-'/. N. Tsuji, D. Frederick, H. Takahashi. Gastrointestinal Unit, Harvard Medical School and Massachusetts General Hospital, Boston, MA. Background/Purpose: Colonic epithelial cell injury is a common manifestation of inflammatory bowel diseases (IBD). The inflammatory reaction is presumably initiated and propagated by the local release of cytokines. Interferon (IFN)-T is an inflammatory cytokine which plays a critical role in host defense and the manifestation of inflammatory response. Activated T lymphocytes which produce IFN-'/were detected in the lamina propria of the patients with Crohn's disease, and the mucosal production of IFN-'/increased during exacerbation of the disease. Interestingly, anti-IFN-'/ monoclonal antibody (Mab) prevented colitis in murine models of IBD which display a highly polarized Thj phenotype. Thus, IFN-'/has been shown to be an important pathogenic factor in the induction of colitis, but IFN-'/alone is not cytotoxic to colonic epithelial ceils. Since TNF-tx and FasL play a pivotal role in lymphocytotoxicity, we studied the effect of IFN-'/on Fas- and TNFa-mediated cytotoxicity to human intestinal epithelial cells. In addition, we investigated if the effect of endotoxin on the growth of intestinal epithelial Cells is modulated by IFN-T, since bacterial endotoxin may be important for the disruption of epithelial cell barrier in IBD. Methods: Human colonic epithelial cell line, HT-29 was cultured with agonistric anti-human Fas Mab (CH11) (100 ng/ml) or TNF-tx (10 ng/ml) in the presence of a various concentrations of IFN-'t (0.1-1,000 U/ml). Cytotoxicity was determined by MTT assay. Apoptosis was assessed by the increase of hypodiploid peak in nucleus DNA stained by propidium iodide using flow cytometry. Since we found that CH11 Mab was contaminated with endotoxin and modulated the growth of HT-29 cells, we removed endotoxin from all reagents using endotoxin neutralization affinity ligand (Endo-X endotoxin neutralization resin). Results: Neither IFN-'/, agonistic anti-Fas Mab nor TNF-ct alone was cytotoxic to HT-29 cells. IFN-'/was found to sensitize this epithelial cells to Fas- and TNFR-mediated cytotoxicity in a dose-dependent manner. Endotoxin increased the growth of HT-29 cells at a concentration of 1-100 ~tg/ml. Maximum effect was observed at 100 lag/ml. Interestingly, preincubation of HT-29 cells With IFN-T (1-1,000 U/ml) for 24 hours reversed the proliferative effect of endotoxin (100 lag/ml) to cytotoxic effect on epithelial ceils in a dose dependent manner. Conclusion: IFN-'/ sensitized human colonic epithelial cells to Fas-, TNF-, and endotoxin-mediated Cytotoxicity. Since IFN-'/is mainly produced by Th~ helper T cells, it may play an integral role in the induction of colonic epithelial cell death and the disruption of epithelial cell barriers in IBD with a highly polarized Th I phenotype.

Intestinal Disorders A425

G1733

H. PYLORI-EXTRACT AND AMMONIA SUPPRESS ANGIOGENESIS IN VITRO: POSSIBLE IMPLICATIONS IN SPRANCHNIC VASCULARTURE. S, Tsuii, S. Kawano, E.S. Gunawan, W-H. Sun, H. Sawaoka, M. Tsujii, N. Kawai, M. Hod. Dept. of Med., Osaka Univ. School of Medicine, Suita, Japan Baekvronnd: Intestinal epithelia consume glutamine to release ammonia to spranchnic circulation. H. pylori also produces ammonia by urea/urease reaction. Therefore splanchnic tissues are exposed to several ten vM(portal blood) - 10 raM(gastric lumen) of ammonia. The present studies were designed to clarify influences of H. pylori-extract and ammonia on angiogenesis. Materials and Methods: Human umbilical vein endothelial cells, lxl04 cells/ml were seeded onto 96-well plates, They were incubated for 24 hours with H. pylori-extract (diluted to 20-2,000 fold) and 0.1-10 mM urea, or 0.01-100 mM ammonia/ammonium chloride (NH3/NH4C1; pH7.4), and subjected to SH-thymidine uptake and cell viability assays. Their increase in cell number, cell migration to the denuded area, and tube-formation on reconstituted extraceUular matrix(Matrigel) were also assessed. Results: H. pylori-extract and urea suppressed 3H-thymidine uptake of HUVEC in a dose-dependent manner to both of the substances. H. pyloriextract with 10 mM urea lowered cell-replication by 96% of the control. However, endothelial cell viability was not decreased after exposure to H. pylori-extract and urea. NH3/NH4CI also suppressed endothelial cell proliferation at concentrations more than 0.1 mM in a dose-dependent manner. Furthermore, they inhibited increase in cell number, cell migration and tube-formation of HUVEC. Conclusion: Endothelial cell-replication, migration and tube-formation are critical steps for angiogenesis. We found that H. pylori-extract and ammonia suppressed in-vitro angiogenesis at concentrations which could be found in spranchnic organ. These results indicate an important role of ammonia in spranchnic angiogenesis including H. pylori-infected peptic ulcer subjects. G1734 I N H I B I T I O N O F INTESTINAL EPITHELIAL Na+-H ÷ EXCHANGE

RESULTS IN INCREASED TRANSEPITHELIAL RESISTANCE AND DECREASED MYOSIN LIGHT CHAIN PHOSPHORYLATION. J.R. Turner1, E.D. Black 1, J,M. Angle 1, H. Alli ], and J.L. Madara 2, IDepartment of Pathology, Wayne State University and Harper Hospital, Detroit, MI and 2Department of Pathology, Emory University, Atlanta, GA. We have previously shown that activation of Na÷-glucose cotransport results in increased tight junction (TJ) permeability, both in isolated intestinal mucosae and monolayers of Caco-2 cells. This Na+-glucose cotransportinduced TJ regulation is associated with myosin light chain (MLC) phosphorylation, a biochemical marker of cytoskeletal tension, and can be prevented by inhibition of myosin light chain kinase (Turner et al., 1997. Amer. J. Physiol.). Activation of Na+-glucose cotransport is accompanied by rapid cytoplasmic acidification and subsequent alkalinization (MacLeod et al., 1996. J. Biol. Chem.). This cytoplasmic alkalinization is required for regulatory volume decrease (RVD), and inhibition of Na+-H÷ exchange (NHE) prevents both alkalinization and RVD. Since tight junction regulation, cytoplasmic alkalinization, and RVD all follow activation of Na÷-glucose cotransport, we hypothesized that cytoplasmic alkalinization might also be required for TJ regulation. METHODS: Caco-2 cell monolayers with active physiological Na+-glucose cotransport were grown on semipermeable supports. Electrophysiology, and 32p radiolabelling were assessed by standard methods. RESULTS: Inhibition of Na÷-H+ exchange (NHE) in Caco-2 cell monolayers results in a rapid increase in transepithelial resistance (TER). Inhibition of NHE with lmM amiloride, 200uM dimethyl amiloride (DMA), or 50uM methyl-isobutyl amiloride (MIA) results in 49%_+6%, 23%+0%, and 15% -+ 1% increases in TER, respectively, within 15 minutes. This increase is dose-dependent, with rank potency of MIA > DMA > amiloride. Inclusion of 5mM NH4C1 to bypass NHE inhibition and allow cytoplasmic alkalinization prevents TER elevations that follow inhibition of NHE. Increases in TER of 34% _+9% and 23 -+ 12%, for DMA and MIA, respectively, were reduced to 10% +_8% and 9% -+ 12% when NHaC1 was added. TER of monolayers treated with NH4C1 alone was 6% _+9% greater than control monolayers. MLC phosphorylation was decreased in monolayers exposed to NHE inhibitors by 28%, 17%, and 43% for amiloride, DMA, and MIA, respectively. CONCLUSIONS: These data demonstrate that (1) cytoplasmic acidification, via inhibition of NHE, results in increased TER, (2) that this increase in TER may be prevented by preventing the acidification, and (3) that inhibition of NHE also results in decreased MLC phosphorylation. Thus, activation of NHE after Na÷-glucose cotransport may be part of the intracellular signalling cascade which results in increased MLC phosphorylation and decreased TER after Na+-glucose cotransport. Supported by the NIH/NIDDK DK-K08-02503 (J.R.T.) and DK-R37-35932 (LL.M.) and the F.M.R.E. of Wayne State University.