STEC clinical isolates induce neutrophil transmigration across intestinal epithelial cells

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IEC harderfunction or by inhibiting the ability of growth factors to modulateepithelialfunction/ growth. Polarized HT29/cI.19A human IEC were grown on filters and collagen-coatedslide chambers. Confluent IEC were infected with trypsin-activated simian rotavirus (SA11) at a multiplicity of infection of 10, or treated with medium alone. At various times (1-24hrs) after infection (a.i.), lEO were tested for chloride secretion in modified Ussing chambers, and for transapithelial resistance (TER) using a voltohmeter. At the same time points, fixed IEC on slides were assayedby immunofluorescencn (IFA) for actin, tubulin, and occludin and ZO-1, and cell lysates were tested by immunoprecipitation (IP)-western blotting (WB) for actin, TJ proteins, and polyubiquitin-EGF-R.At 6hrs a.i., rotavirus infected IEC had increasedbaseline (6---2 vs 0/.~A/cm2: n =4, p60% by 12 hrs in infected IEC (n=8,p40% vs controls at 12 hrs a.i.; n=3, p
Heat Shock Protein, Hsp72, Reduces C.Di#icile Toxin A-mediated Effects on Colonic Caco2 Cells by Inhibiting Toxin-Induced Mitochondrial Damage Mark W. Musch, Tom S. Liu, Univ of Chicago, Chicago, IL; CharalabosPothoulakis, J Thomas Lamnnt, Harvard Univ, Boston, MA; Eugene B. Chang, Univ of Chicago, Chicago, IL Backgroundand Aims C. difficile toxin A (TxA) alters epithelialcell functions through a number of pathways,including UDP-glucosylationof the GTP-bindingprotein RhoAand mitochondrial damage.We previously showedthat endogenousexpressionof the heat shock protein, hsp72, in human colonic Caco2bbe (C2) cells conferred protection to the injurious effects of TxA, i.e. anti-senseinhibition of hsp72 was associatedwith increasedsensitivity to the toxin's effects on cell viability and transepithelialelectricalresistance(TER). In this study, we investigatedtwo potential mechanisms underlying hsp72 protection, protection of RhoA and mitochondrial function. MethodsC2 cells, which havehigh endogenoushsp72 protein, were stably transfected with hsp72 anti-senseconstruct. Two clonesexhibiting reducedendogenoushsp72 expression were selectedand their sensitivity to purified TxA determined.Cellsampleswerethen harvested at varying times following exposure to vehicle or TxA and analyzedfor RhoA glucosylation, RhoA-hsp72 binding, and mitochondrial function. Potential binding of hsp72 with RhoA was assessed by co-immunoprecipitation using anti-hsp72 antibody. RhoA glucosylation was determined by an in vitro assay using purified C3 exoenzymeand [32P]-NAD. Mitochonddal function was assessed by cell ATP levels and measurement of mitochondrial and cytosolic cytochrome C oxidase. Results Confirming previous results, RhoA co-immunoprecipitated with hsp72 in samplesfrom control and TxA-treatedcells, However,there were no significant differences at any time point in RhoA glucosylation induced by TxA in vector alone- or hsp72 anti-sense transfncted cells, the latter having little or no endogenous hsp72 expression. In contrast, TxA (30ng/ml) caused a more rapid decline in cell ATP in the hsp72 anti-sense clones. This effect was observedas early as 2 h and was nearly maximalby 12 h. Mitochondrial damage, assessed by cytochrome release, was also apparent in the hsp72 anti-sense cells at 2 hours and was maximal between 8 and 12 hours. Vector-transfectedcells demonstrated far less damageat all but later timepoints. Conclusions: Hsp72 reduces the injurious effects of TxA on colonic epithelial cells by decreasing mitochondria damage, but not through preventing RhoAglucosylation.By decreasingoxidativedamage,cellularviability and functions such as TER may be protected.

38O2 Modulation of Tight Junction (TJ) Structure by Enteroinvasive E.coli (EIEC) Is Prevented by H202Scavengers and Protein Kinaae C(PKC) Inhibitors. Silvia C. Resta-Lenert,Kim E. Barrett, UCSD, San Diego, CA We have shown that EIEC increases levels of nitric oxide (NO) and peroxide (H202)produced by infected intestinal epithelial cells (IEC). The rise in NO correlated with increasedchloride secretion after infection, and NOS inhibitors reversedthis effect. Subsequently,barrier integrity was severelycompromised in infected cells. We hypothesizedthat colonizationof lEG by EIEC requires altered TJ and that this effect is mediated by oxides and peroxides. Moreover, the effect of EIECon barrier integrity may involve cooperationwith other cells, such as neutrophils (PMNs). Studies were conducted using polarizedHT29/cI.19Acells, a human lEG.Thesecells were exposedto EIEC(029:NM) or probiotics, S.thermophilus(ST) and L.acidophilus(LA), on the apical side and cocuitivatedwith isolated PMNs on the basolateralside of the monolayer. NO and H202levels were measured in supernatantsat 0-24 hrs after infection (a.i.), by Griess and xylnnol orange reactions, respectively.Infected IECand controls were harvestedat various times for immunofluorescent (IFA) staining of intact cells and protein analysis of cell lysates in the presenceor absenceof TEMPONEor ascorbic acid (Asc), a H202spin trap and scavenger, respectively, or H-7, a PKC inhibitor. Levels of NO (80_+3 vs 2.5-+0.5 vs 7.5_+2/~M/L, EIEC vs control vs ST/LA at 12hrs a.i.;n = 6,p<0.001) and H202 (42 _+10 vs 2 + I vs 3-+ I /~M at 18hrs a.i.;n=4,p60% inhibition of reduction in phosphorylation for occludin and ZO-1 compared to untreated infected lEG; n=3, p
3805 RELM-Beta Is a Novel Intestine-Specific Secreted Protein that Is Expressed in the Colonic Crypt Epithelium. Xiaoming Wen, Univ of PennsylvaniaSch of Medicine, Philadelphia,PA; Scott T. Cooper, Weimian He, Claire M. Steppan, Mitchell A. Lazar, Gary D. Wu, Univ of Pennsylvania, Philadelphia, PA Various epithelial cell lineagesin the intestine are capableof secreting bioactive proteins that play important roles in the physiology of the alimentarycanal.We describethe characterization of RELM-Beta, a novel intestine-specific gone with a unique pattern of expression. RELMBeta (Resistin-Like Molecule) is a member of a new gone family of secreted proteins that contain a highly conserved cysteinn rich C-terminus and a signal pnptide sequence at its amino-terminal end. The founding member of this gene family, Resistin, is a novel hormone expressed by adipocytes that is regulated by ligands for the PPARgammanuclear hormone receptor and plays a role in the pathogenesisof adult onset diabetes (Steppan et al. Nature 2000, in press). RELM-Betawas identified by a search of the NCBI mouse EST databasefor genes related to Resistin. RELMs were not detected in the nearly completed genomes of either C. nlngans or D. melanogaster, suggesting that RELMs may be specific to higher organisms. The human and mouse RELM-Beta proteins are highly conserved, especially in the cysteine-dch C-terminus that is most similar to Resistin. Transfection of 293T cells with flag-tagged RELM-beta confirmed that this protein is secreted. Northern blots and in situ hybridization showed RELM-Betato be expressedspecifically in the intestinal tract with the highest level of expression localized to the proximal colonic crypt epithelium. Interestingly, although the expression of the colonic ion transport gone, DRA, on the surface epithelium and c-Myc in colonic crypt cells are unchanged in germ-free mice, the mRNA for RELM-beta is decreasedby approximately3- fold. RELM-Betaprotein was undetectablein germ-free mice by immunoblot. This suggests that the RELM-betagone expression is specifically regulated by colonization of the colon with normal enteric bacteria. Characterizationof the RELM-Beta gene by 5'-RACE PCR and homology comparison shows that this gene contains 4 exons, a high consensus TATA box, and a conserved core promoter of 250 bp. Ultimately, further investigation of RELM-Beta may provide insights into biological properties of this secreted protein as well as how genes are targeted to the colonic crypt epithelium in response to bacterial colonization.

3803 TLR-Mediated Innate Immune Responsesto Bacterial Products (PAMPs) Are Dynamically Regulated via Tyrosine-Phosphorylated Multireceptor Huterocomplex Formation in the Intestinal Epithelium Elkn Carlo, Daniel K. Podolsky, Gastrointestinal Unit, MGH, CSIBD, Harvard Medical Sch, Boston, MA Background: We have recently demonstrated that Toll-like receptors (TLRs) may mediate innate immune responsesto LPS and other bacterialcell wall componentsin intestinalepithelial cells (lEG)to initiatethe production of proinflammatorychemokinesand cytokinns (J. Immunol. 2000 164:966; Infect. Immun. 2000 68:7010). The aim of this study was to assess the role of TLR complex formation and phosphoryletion in mediating cell responses to pathogenassociated molecular patterns (PAMPs). Material and Methods: Crosslinking studies with DSS and subsequentimmunoprecipitationwere carried out using extracts of two human intestinal epithelial cell lines (Caco-2 and T84) and colonic biopsies (obtained from healthy volunteers) with various specific antibodies against TLR2, TLR3 and TLR4 and protein A-agarosa. To ascertain specificity of the reactions, non-immune sera was used as negativecontrol. Results: TLR2, TLR3 and TLR4 are constitutivelytyrosine-phosphorylated.Receptorphosphorylationis differentially regulatedby LPS and PGN.TLR2,TLR3 and TLR4 form heteromedcmultireceptor complexesat approx. 188kDain-vitro and in-vivo, suggestingthat TLRs may interactwith each other in intestinal epithelial cells. LPS-/PGN-stimulationslead to significant downregulationof TLR3-TLR4 or TLR2-TLR3 complex formation, respectively.All TLR-complexformations are constitutivelytyrosine-phosphorylated.Treatmentwith alkalinephosphataseleadsto disruption of the TLR3-TLR4complex, but not of TLR2-TLR3, suggestingspecific modulation of interactions by protein dephosphorylation.In contrast, MEK inhibition promotesTLR3-TLR4complex formation. Conclusions: This is the first report that TLRs may functionally interact in a multireceptor complex which may be differentially modulatedby ligands and phosphorylation.

3806 STEC Clinical Isolates induce Neutrophil Transmigration across Intestinal Epithelial Cells Bryan P. Hurley, New England Medical Ctr, Tufts Univ, Boston, MA; Cheleste M. Thorpe, New England Medical Ctr, Boston, MA; Mireia Guerau, Tufts Univ, Boston, MA; David W K Acheson, New England Medical Ctr, Tufts Univ, Boston, MA Background: Shiga toxin producing E. coil (STEC)causes diarrhea that can range in severity from wateryto bloody.STECinfection has also beenassociatedwith serioussystemiccomplications such as hemorrhagiccolitis (HC) and hemolytic-uremicsyndrome (HUS).STECcolonizes the gut and produces Shiga toxins (Stxs) which cross the intestinal barrier and target the endothelia of susceptibletissue in multiple sites resulting in HC and HUS. Our hypothesis is that the amount of Stx absorbed from the gut lumen may be crucial in determining disease severity. Here we addressedthe role of acute inflammation in the intestine on Stx uptake by investigating whether nnutrophil (PMN) transmigration across the intestinal epithelial cells (IECs) had an effect on Stx translocation in vitro. We also determined the effect of STECon IL-8 secretion and their ability to induce PMN transmigration across lEGs. Methods: IECs

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(T84) were grown on permeable filters forming polarized barriers. PMNs were added to the basolateral surface and stimulated to transmigrate with fMLP, Stx was added to the apical surface and the amount that crossed in the absence or presence of PMN transmigration was measured.A group of 10 STECclinical isolates characterizedby multiplex PCRfor the presence of the virulence factors hlyA, eae, stxl, and stx2 were assessed for ability to induce PMN transmigration across lEGs. Transmigrated PMNs were quantified by MPO activity. Results: PMN transmigration across the IEC barrier enhanced Stx translocation and the amount correlated with the number of PMNs migrating. STEC strains were capable of inducing PMNs to transmigrate. There were reproducible differences in the degreeof PMN transmigration elicited by different strains. Several eae(-) strains induced significantly more PMNs to transmigrate than the eae(+ ) strains. Furthermore these eae(-) strains induced IECs to secrete more IL8 than the eae(+) strains. No differences in adhesion were observed between eae(+) and eae(-) strains. Conclusion: This study implicates the inflammatory response to STEC infection as a potential means to enhance Stx movement to the underlying tissue. We have shown that various STEC are capable of inducing IL-8 secretion and PMN transmigration. It was surprising to observethat eae(-) STECstrains induced significantly more PMNs to transmigrate and caused higher IL-8 secretion when compared with eae(+) strains. Future work will address bacterial factors in eae(-) strains responsiblefor inducing these inflammatory readouts.

3807 Mucosal Heat Shock Protein 25 Is Rapidly induced In Small Bowel Lympllo~jtes and Epithelial Cells Following StaphylococcusEnterotoxin B (SEB)-IndocedEMerita. Mark W. Musch, Hongyu Ren, Univ of Chicago, Chicago, IL; Derek McKay, Dept Path, McMaster Univ, Intest Dis Research Prog, Hamilton Canada; Eugene B. Chang, Univ of Chicago, Chicago, IL Background and Aims: SEB is a bacterial superantigen that is a potent stimulant of T cells. Administration of SEB to mice causes rapid development of enteritis, believed to be a result of activated lamina propria lymphocytes (LPLs), which is characterized by perturbed epithelial ion transport and altered jejunal morphology. The enteritis is self-limiting and does not occur in T cell-deficient mice. We hypothesized that cytoprotective inducible heat shock proteins (hsps) of the gut mucosa might increase as a tissue response to limit cellular injury and promote wound healing. Methods: Mice were injected IP with SEB (51,g) and the intestines were harvested 4, 24, or 48 hours later. Representative colonic and jejunal sections were fixed in formalin for immunostaining and mucosa was scraped from an adjacent portion, homogenized and solubilized for Western blotting. Protein expression and localization of the inducible hsps, hsp25 and hsp72, and constitutive hsc73 were assessed using isoformspecific monoclonal antibodies. Results: Four hours after injection of SEB, LPLs of the small, but not large, intestine showed increased staining for hsp25. In some cases, increased hsp72 protein expression was also observed, but hsc73 was unchanged. In contrast, hsp25 and hsp72 expression was not detected in small intestinal epithelial cells. After 24 hours, the LPL staining was still present, but expression of hsp25 was readily detected in the epithelial cells of the small intestine, confirmed by quantitative Western blotting. However, hsp72 was not induced in the epithelium by the SEB-evokedlymphocyte activation in small or large intestines. Epithelial cell hsp25 expression increased further by 48 hours, whereas tisp72 remained unchanged. Conclusion: Activation of LPLs of the small intestine with the superanl~genSEB stimulates a time- and cell-specific heat shock protein response characterized by increased expression of the small heat shock protein, tisp25. This response is first observed in LPI_s, possibly related to the activation process. The progressive increase in hsp25 expression of the small intestinal epithelial compartment may represent a tissue response to limit the extent of immune-mediated cellular injury.

3809 Regulation of the Gastrointestinal Epithelial Cell Expression of CFTR Protein by Salmonella enterlca serovar Typhi, and Its Importanceto Infection and Development of Typhoid Fever. Jeffrey B. Lyczak, Gerald B. Pier, Brigham and Women's Hosp, Boston, MA Background: While widely known as a chloride channel, the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein has also been shown to be an important epithelial cell receptor mediating translocation of Salmonella enterica serovar Typhi to the gastric submucosa. The bacterial ligand for the CFTRprotein is the lipopolysacchatide (LPS) core oligosaccharide. Infection of gastrointestinal epithelium with serovar Typhi triggers a redistribution of preexisting CFTRprotein in epithelial cells from cytoplasmic stores to the plasma membrane. Since we have shown that the level of cell-surface CFTR expression is directly related to the efficiency with which serovar Typhi translocates to the submucosa, the CFTR redistribution observed likely influences the outcome of infection. Therefore, the goal of the current study is to elucidate the mechanism by which CFTR redistribution is triggered during infection. Methods: Serovar Typhi strain Ty2 and cellular extracts of this organism were tested for their ability to induce CFTR redistribution in epithelial cell lines. Redistribution was visualized by indirect immunofluorescent staining of CFTR protein on LCFSN cells and by confocal microscopic analysis of the subcellular distribution of a Green Fluorescent Protein-CFTR fusion protein present in MDCK cells. Epithelial cell uptake of serovar Typhi bacteria was measured by a standard gantamicin exclusion assay. Results: CFTRprotein levels increaseddramatically on epithelial cell membranes following exposure to either whole bacteria or cell extracts. CFTR-dependent bacterial uptake by epithelial cells was enhanced (2- to 3-fold) following redistribution of CFTR protein to the plasma membrane, demonstrating that the newly expressed cell surface CFTRprotein is accessible for binding to its bacterial ligand (LPS). The bacterial factor which triggers CFTRredistribution was sensitive to treatment with proteinase K, but not periodate, suggesting that if is protein in nature. Conclusions: Our results suggest that serovar Typhi possesses a protein factor that is capable of triggering redistribution of epithelial CFTRprotein from the cytoplasm to the cell surface, where it participates in subsequent interactions with sarovar Typhi. Identification of this bacterial factor and its epithelial cell target may provide a means of modulating the efficiency of epithelial cell invasion by serovarTyphi.

3810 16S rRNA Fluorescent/n $/tu Hybridization of Human Colon and Terminal Ileum Biopsies: A Mmms-Adlmrent Bacterial Flora Does Not Exist. Laurens A. Van de" Waaij, Hermie J. M. Harmsen, Mohsen Madjipour, Frans G. M. Kroese, Hendrik M. Van Dullemen, Nynke K. De Boer, Gjaif W. Welling, Peter L M Jansen, Acad HOSp Groningen, Groniogen The Netherlands Background: The intestinal microflora is important in the pathogenesis of inflammatory bowel diseases: a decrease in the bacterial concentration of the colon may induce a remission in Cmhn's disease. Furthermore, probiotics may prevent recurrence of pouchitis. Therefore, there is a growing interest in the intestinal microflora and its interaction with the host in health and disease. Presumably, this interaction takes place at the mucus layer. Aim: To investigate the composition of the mucus flora and its spatial relation to the host. Methods: Snapfrozen biopsies were taken of 5 colon regions and of the terminal ileum of 8 patients with a macroscopic and microscopic normal colon. Bacteria were detected in the sections by fluorescent in sltu hybridization (FISH)with probes with specificity to the major representatives (together quantitatively 70% of the hybridisable bacteria) of the anaerobicflora (Bifidobacterium spp., Bacteroides spp., Eubactedum spp., C/ostridium spp., Atopobium spp, Low G + C#2) and aerobic flora (Enterobactenaceae spp., Enterocoocus spp., Streptococcus spp., Lactobacil/us spp.). All sections were double-stained with a probe specific for all bacteria. Results: In colon and terminal ileum bacteria were only present at the luminal side of the mucus layer. No bacteria were seen within crypts, adhering to the epithelium or penetrating into the mucosa. FISH with specific probes revealed that virtually all bacteria covering the mucus layer were anaerobic. Moreover, the distribution of the different bacterial species within the thin layer covering the mucus was similar to that in faecal suspensions. Only very few microcolonies were seen. No regional differences were found. Conclusion: Our data show that bacteria are only present at the luminal side of the mucus layer. The distribution of the different bacterial species suggests that they do not colonise or adhere to the mucus but that their presence is arbitrary and temporary. We speculate that many of the beneficial and detrimental effects of the microflora and probiotics on the host are mediated by luminal bacteria derived molecules that penetrate the mucus layer,

3808 Cyclooxygenace-2 Activation By Noninvasive Bactada Regulates Apoptosis In Human Intestinal Primary Epithelial Cells (HIPEC) Yulan Cheng, Mahmood Akhtar, Alain P. Gobert, Jamie C. Newton, Univ of Maryland Sch of Medicine, Baltimore, MD; Asit Panja, Winthrop Univ Hosp, Mineola, NY; Keith T. Wilson, Univ of Maryland Sch of Medicine, Baltimore, MD Background: Cyclooxygenase (COX)-2 is the inducible form of the rate-limiting enzyme for prostaglandin synthesis. COX-2can downregulate apoptosis in intestinal epithelial cells (IECs). It has been reported that in transformed human IECs, only bacteria which invade can activate either COX-2 expression or apoptosis. The role of COX-2 in bacterially-induced apoptosis has not been studied in IECs.We havefound that the noninvasive pathogen Citrobacter rodentium, and to a lesser degree, nonpathogenic E. co~i, can induce COX-2 in nontransformed rat IECs. Aims: To determine if noninvasive bacteria induce COX-2 expression and apoptosis in nontransformed human intestinal primary epithelial cell (HIPEC) lines and if COX-2 regulates the apoptotic response. Methods: HIPEC lines from transverse colon and rectum were generated and maintained in long term culture (Lab invest. 80:1473-5, 2000). Cells were stimulated with nonpathogenic E. coil and enteropathogenic E. coil (EPEC) at a multiplicity of infection of 100 bacteria/cell. Bacteriawere added for 1 h or 4 h, washed away, and incubation continued in the presence of antibiotics for a total of 24 h. COX-2 mRNA levels were assessed by RTPCR, and the COX-2 product, PGE2,was assessed by enzyme immunoassay. Apoptosis was quantified by Cell Death Detection ELISA for histone-DNA fragments. Results: Both HIPEC lines had substantial basal COX-2 mRNA and PGE2levels. Results with bacterial stimulation were very similar in both cell lines. At 1 h, nonpathogenic E. coil and EPEC stimulated 1.8and 2.5-fold increases in PGE2, respectively. With 4 h stimulation, nonpathoganic E. coil induced 9-fold increases in PGE2, while EPEC induced 15-fold increases. This effect was mirrorred by significant increases in COX-2 mRNA levels with nonpathogenic E. cull (3-fold) and EPEC(5-fold). Nonpethogenic E. co/iinduced a 1.6-fold increase in apoptnsis (p < 0.05). EPEC induced a 2.1-fold increase in apoptosis (p < 0.01), and addition of the COX-2 inhibitor, NS-398 (10/~M), significantly increased EPEC-stimulatedapoptosis to 3.4-fold above control levels (p < 0.05 vs. bacteria alone). Conclusions: In nontransformed human IECs (HiPEC), noninvasive E. coil can stimulate significant increases in COX-2 expression and apoptosis. COX-2 activation regulates the level of apoptosis. COX-2 protects IECs by limiting the severity of bacterially-induced apoptosis, which may reduce inflammation, but contribute to neoplastic risk.

Ship Toxin I induces and Superinduces Multiple C-X-C Chemokinos in intestinal Epithelial Ceils Cheteste M. Thorpe, Wendy E. Smith, Bryan P. Hurley, David W. K. Acheson, New England Medical Ctr, Boston, MA Background: Exposureof humans to Shigatoxin-producing E. coil (STEC)is a risk for hemolytic uremic syndrome (HUS). Because STEC are non-invasive, the extent to which Shiga toxins (Stxs) can cross the host intestinal epithelium may determine who is at risk for HUS. The host inflammatory response may be important in Sb( uptake from the gut. We have shown that Sl~s can induce and superinduce IL-8 mRNA and protein in intestinal epithelial cells (IECs) via a ribotoxic stress response, suggesting that Stxs might influence other primary response genes, such as other chemokines associated with neutrophil migration. Methods: We used cytokine expression arrays to determine the effect of St)(1 on various C-X-Cchemokine genes in IECs. IL-8, GRO-a, and ENA-78 were further studied. Stxl effects on message stability of both IL-8 and GRO-~ were evaluated using inhibifors of transcription followed by Northem blotting. Protein expression was assessed by ELISA. Results: Six1 induces multiple C-X-C chemokine mRNAs, including GRO-cz, GRO-/3, GROw, and ENA-78. This was not observed with boiled Six1. Like IL-8, GRO-a and ENA-78mRNAs are induced and superinduced by S1~1. Furthermore, like IL-8, GRO-c~protein is expressed by IECs in response to Stxl

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