Cholera toxin elicits epithelial TNF production; Potential role in adjuvant activity

AI02 AGA ABSTRACTS

680 PROBIOTICS, MONONUCLEAR CELLS, AND EPITHELIAL CELLS: AN ANTI-INFLAMMATORY NETWORK. Liam O'Mahony, Maria Feeney, John MacSharry, Barry Kiely, Jane MeCarthy, Gerald C. O'Sullivan, Fergus Lj Shanahan, John K. Collins, Nui, Cork, Ireland. Background & Aim: We previously reported the selection criteria of Lactobacillus salivarius (subsp. salivarius, strain UCCI18) as a probiotic bacterium, and described the microbiologic and immunologic outcomes of feeding trials in healthy human volunteers. We also confirmed the antiinflammatory therapeutic efficacy of L. salivarius UCC118 in the IL-1O knockout murine model of enterocolitis. The aim of the present study was to elucidate the mechanisms underlying the modulation of mucosal inflammation using an in vitro model with human cells. Methods: A transwell system was used, involving two compartments separated by a porous membrane enabling fluid phase exchange but no cell-cell contact; the upper compartment contained a monolayer of CaCo-2 epithelial cells on the membrane, with peripheral blood mononuclear cells (PBMCs) in the lower compartment. Intracellular and secreted cytokines (TNF-a, IL-6, IL-IRA, soluble IL-6R and IFNy) were assayed by flow cytometry and ELISA respectively, after the probiotic was added to either compartment. In addition, gene array technology was used to screen cytokine and related molecular responses. Results: TNF-aproduction by PBMCs in all three assay systems was consistently inhibited by the epithelium, and the inhibitory effect was significantly enhanced by the presence of the probiotic. This was found to be a strain-dependent effect, when IS other lactobacillus strains, which elicited variable to no responses, were screened. In addition, production of IL-ll3and IFNyby PBMCs in the presence of the probiotic was also reduced by the epithelial monolayer, while IL-5, IL-6 and TGFf3were increased. Thus, the L. Salivarius UCCI18 probiotic is capable of inducing a Th2 cytokine pattern, while suppressing the production of inflammatory cytokines. Conclusion: (1) the results indicate a tricellular signalling network involving downregulation of lymphoid cells by the epithelium that is enhanced by the probiotic; (2) optimal anti-inflammatory effects of probiotics may depend, in part, on the integrity of the epithelium; (3) the data strengthen the rationale for a probiotic therapeutic strategy in mucosal inflammation.

681 CHOLERA TOXIN ELICITS EPITHELIAL TNF PRODUCTION; POTENTIAL ROLE IN ADJUVANT ACTIVITY. Richard S. Pitman, Cormac T. Taylor, Richard S. Blumberg, Sean P. Colgan, Brigham & Women's Hosp, Boston, MA. Background: Cholera toxin (CT) is a highly potent mucosal adjuvant of potential therapeutic importance in the design of successful methods of oral vaccine delivery. The toxin consists of a single A subunit (CTA) and 5 identical B subunits (CTB). Cell binding and internalization of CT is achieved by the interaction of CTB with cell membrane-associated glycolipids. Following uptake, CTA mediates its toxic activity through the activation of Gs proteins, resulting in elevated levels of intracellular cyclic adenosine monophosphate (cAMP). The mechanisms by which CT promotes immune responses within the intestinal mucosa remain to be elucidated. In this study we have investigated the impact of CT on intestinal epithelial phenotype using the colonic epithelial cell line T84. Methods: T84 cells were grown to confluence and exposed apically to cholera toxin (20nM; 0-24 hours). Messenger RNA and protein expression were determined by semi-quantitative PCR and Western blotting respectively. TNF levels were measured by sandwich ELISA. Activation of NFKB was assessed by determining the presence of p65 within isolated nuclear extracts. Results: Exposure of T84 monolayers to CT resulted in a timedependent induction of TNF mRNA expression (max. levels were seen at 8 hours). TNF protein expression was induced by CT with maximum levels detected at 24 hours (19.1:t2.83 pg/monolayer vs. untreated cells 3.7:t0.326 pg/ monolayer). The B subunit alone did not stimulate the production of TNF indicating a requirement for the A subunit in mediating CT-induced TNF release. Inhibition of the cAMP-dependent protein kinase, PKA with the inhibitor H-89 (lO/LM) had no affect on the levels of TNF induced by CT (93.2:t7.5% of control), indicating that CT is likely to elevate levels of TNF in a cAMP-independent manner. CT did however induce the activation of the transcription factor nuclear factor kappa B (NFkB), an activity known to promote TNF gene transcription. Conclusions: CT stimulates intestinal epithelial TNF release. This effect is dependent upon the presence of the A subunit of the toxin and yet appears to be independent of toxin-induced increases in cellular cAMP. The induction of NFKB activity by CT represents a potential mechanism by which the toxin induces TNF production. In conclusion, CT-induced epithelial TNF may contribute to the immunomodulatory qualities of the toxin.

682 CYTOTOXIC EFFECTS ON CULTURED CELLS OF ORGANIC ACIDS PRODUCED BY INTESTINAL ANAEROBIC BACTERIA. Toshiaki Sakurazawa, Toshifumi Ohkusa, Koichiro Ariake, Ken-ichi Ishii, Ichizen Takashimizu, Tokyo Med and Dental Univ, Tokyo, Japan. Aim:Spontaneous colitis does not appear in HLA-B27 transgenic, interleukin-2 knockout, IL-1Oknockout, or T-cell-receptor-a knockout rodents when they are germfree, so bacteria normally present in the intestine are important in the development of such colitis. We reported earlier that there

GASTROENTEROLOGY Vol. 118, No.4

are more individuals in the genus Bacteroides among the intestinal flora in mice with colitis caused by dextran sulfate sodium than in untreated controls (Gastroenterology 1990; 98: 694). Anaerobic bacteria, including Bacteroides species, are normally more abundant than aerobic bacteria in the intestine, where they produce organic acids. Butyric acid, one such acid, causes apoptosis in cell lines. In this study, we identified the concentration needed for cultured cells to be killed by organic acids produced by intestinal anaerobic bacteria. Methods:One of five organic acids (acetic, propionic, butyric, lactic, or succinic) was added to the culture medium of five cell lines: Vero, HeLa, HEp-2, DLD-I (colon cancer), and NIH 3T3. We calculated the lowest concentration that killed >99% of cells by counting nonadherent cells under a microscopy, and checked the results by counting apoptotic cells (those that were stained with propidium iodide) by flow cytometry. Cells not treated with an organic acid were used as controls. Results:The lowest concentrations that killed cells are shown below. The mean (for the five cell lines) concentration needed was lowest for butyric acid, and increased in the order of succinic, propionic, lactic, and acetic acid. Conciusion:Five organic acids produced by anaerobic bacteria killed cells by causing apoptosis. The minimum concentration that killed the cells was no higher than the concentration likely to be present in the colon. This cytotoxicity may contribute to the pathogenesis of colonic ulcers. Cell Lines Vero Hela HEp·2 DU)·1 NIH 313

Acetic

Propionic

Butyric

Lactic

Succinic

17 15 15 20 20

50 11 10 10 7.0

1.5 4.5 5.5 4.0 4.5

12 13 13 13 7.0

6.5 5.5 6.5 85 3.0

683 ENTEROPATHOGENIC E. COLI ACTIVATES NF-KB, BUT DOES NOT INCREASE PARACELLULAR PERMEABILITY, BY A CAL· CIUM·INDEPENDENTIMAP KINASE-DEPENDENT PATHWAY. Suzana D. Savkovic, Akila Ramaswamy, Athanasia Koutsouris, Gail Hecht, Univ of Illinois at Chicago, Chicago, IL; Univ of Illinois, Chicago, IL. We have shown previously that EPEC infection of host intestinal epithelia results in two important functional alterations: initiation of the inflammatory cascade by activating transcription factor NF-KB and an increase in paracellular permeability. The increase in paracellular permeability by EPEC is Ca++dependent. Gerwitz, et al (Gastro 116:A882, 1999) reported that Cat+is required for NF-KB activation by Salmonella. The aim of this study was to determine the signaling pathways by which EPEC activates NF-KB and to investigate if these pathways are divergent from those that alter intestinal permeability. Phosphorylation and proteosomal degradation of the inhibitory molecule IKB precede NF-KB activation. T84 or Caco-2 cells were infected with EPEC and assessed for IKBaphosphorylation and degradation by immunoblotting. IKBaphosphorylation was seen by 0.5 h and degradation by I h post-infection. These events were cytokine independent as neither TNF-anor IL-I f3receptor antibodies prevented their occurrence. A role for Cat+ was examined by chelating with BAPTA-AM and determining the impact on EPEe-induced IKBadegradation and/or IL-8 production. In contrast to what has been reported for Salmonella, BAPTA-AM had no effect on EPEC-induced IKBadegradation or IL-8 expression. Instead, MAP kinase inhibitors prevented both EPEC-induced IKBadegradation and IL-8 production (EPEC alone 307:t22 pg/crrr': EPEC + 20/LM apigenin 34:t 16; EPEC + 5/LMPD98059 {ERK specific} 37:t 13; EPEC + 50/LMPD98059 14:t4; EPEC + 20/LM SB203580 {p38 specific} 24:t 10). Immunoblots using phosphospecific Abs to ERKI/2, JNKI/2, and p38 showed that by IS min post-infection, each of these enzymes was phosphorylated suggesting activation. In contrast, MAP kinase inhibitors had no effect on EPEC-induced alterations in paracellular permeability as assessed by % change in transepithelial resistance after 6 h of infection (uninfected control -4:t6; EPEC -35:t5; EPEC + apigenen -49:t6; EPEC + 50/LM PD98059 -32:t5; EPEC + 20/LM SB203580 -36:t5). We conclude that EPEC activation of NF-KB: I) is an early event that is cytokine independent; 2) utilizes Ca++ independent pathways thus differentiating it from Salmonella; 3) is dependent on MAP kinases; and 4) employs signaling pathways that are distinct from those that alter paracellular permeability. This is the first time that specific EPEC-induced signaling cascades have been demonstrated to have independent functional consequences.

684 INCREASED NITRIC OXIDE (NO) CONCENTRATIONS IN THE INTESTINAL MUCOSA OF PATIENTS WITH INFLAMMATORY BOWEL DISEASE rrsr» AS DETECTED BY A NOVEL CHEMI· LUMINESCENCE METHOD. Ali Banan, Richard Hutte, Yang Zhang, Sandeep Choudhary, Todd M. Murphy, Ali Keshavarzian, Rush Univ, Chicago, IL; Seivers, Inc, Boulder, CO. NO has been implicated in the underlying pathophysiology of IBD. Hence, measurements of mucosal NO concentration is of interest for studying pathophysiological alterations in this disorder. However, to date there are neither any reported studies on the actual measurements of NO in the