Acute induction of human IL-8 production by intestinal epithelium triggers neutrophil infiltration without mucosal injury.

AIM: Neutrophil migration in the intestine depends on chemotaxis of neutrophils to CXC chemokines produced by epithelial cells. The goal of this project was to determine if acute induction of a CXC chemokine gradient originating from intestinal epithelial cells is sufficient to induce neutrophil influx into intact intestinal tissue. METHODS AND RESULTS: The authors developed a double transgenic mouse model with doxycycline induced human IL-8 expression restricted to intestinal epithelial cells. Doxycycline treatment of double transgenic mice for three days resulted in a 50-fold increase in the caecal IL-8 concentration and influx of neutrophils into the lamina propria. Although neutrophils entered the paracellular space between epithelial cells, complete transepithelial migration was not observed. Doxycycline treatment also increased the water content of the caecal and colonic stool, indicating dysfunctional water transport. However, the transmural electrical resistance was not decreased. Neutrophils recruited to the intestinal epithelium did not show evidence of degranulation and the epithelium remained intact as judged by histology. CONCLUSIONS: This conditional transgenic model of chemokine expression provides evidence that acute induction of IL-8 in the intestinal epithelium is sufficient to trigger neutrophil recruitment to the lamina propria, but additional activation signals are needed for full activation and degranulation of neutrophils, mucosal injury, and complete transepithelial migration.

CD98-mediated links between amino acid transport and beta 1 integrin distribution in polarized columnar epithelia.

In non-polarized cells, CD98 has been shown to both influence beta(1) integrins and heterodimerize with LAT-2, which confers amino acid transport capability on the LAT-2/CD98 heterodimer. Since LAT-2 is most heavily expressed in intestine and CD98 associates with the beta(1) integrin splice form selectively found in such epithelia, we investigated the relationship and polarity of these proteins using the intestinal epithelial model Caco2-BBE. CD98 was found to selectively coimmunoprecipitate with both LAT-2 and beta(1) integrin, and, logically, all three proteins were polarized to the same (basolateral) domain. Furthermore, expression of CD98 in polarized epithelia lacking human CD98 (MDCK cells) disrupted beta(1) integrin surface distribution and cytoskeletal architecture, suggesting that CD98 can influence integrin function. Expression of a CD98 mutant lacking the specific residues conferring LAT-2 binding similarly affected cells, confirming that the latter effect was not due to LAT-2 sequestration. Use of CD98 truncation mutants suggest that a 10-amino acid domain located at the putative cytoplasmic tail/transmembrane domain interface was necessary and sufficient to induce the phenotype change. We conclude that the CD98/LAT-2 amino acid transporter is polarized to the same domain on which beta(1) integrin resides. CD98 appears to associate with beta(1) integrin and, in doing so, may influence its function as revealed by disruption of the outside-in signaling that confers cytoskeletal organization. Furthermore, such findings suggest a link between classic transport events and a critical element of barrier function: integrin-mediated influences on cytoskeletal organization.

Cutting edge: bacterial flagellin activates basolaterally expressed TLR5 to induce epithelial proinflammatory gene expression.

Flagellin, the structural component of bacterial flagella, is secreted by pathogenic and commensal bacteria. Flagellin activates proinflammatory gene expression in intestinal epithelia. However, only flagellin that contacts basolateral epithelial surfaces is proinflammatory; apical flagellin has no effect. Pathogenic Salmonella, but not commensal Escherichia coli, translocate flagellin across epithelia, thus activating epithelial proinflammatory gene expression. Investigating how epithelia detect flagellin revealed that cell surface expression of Toll-like receptor 5 (TLR5) conferred NF-kappaB gene expression in response to flagellin. The response depended on both extracellular leucine-rich repeats and intracellular Toll/IL-1R homology region of TLR5 as well as the adaptor protein MyD88. Furthermore, immunolocalization and cell surface-selective biotinylation revealed that TLR5 is expressed exclusively on the basolateral surface of intestinal epithelia, thus providing a molecular basis for the polarity of this innate immune response. Thus, detection of flagellin by basolateral TLR5 mediates epithelial-driven inflammatory responses to Salmonella.

The role of CD47 in neutrophil transmigration. Increased rate of migration correlates with increased cell surface expression of CD47.

CD47, a cell surface glycoprotein, plays an important role in modulating neutrophil (PMN) migration across endothelial and epithelial monolayers. Here we show that anti-CD47 monoclonal antibodies (mAbs) delay PMN migration across collagen-coated filters or T84 epithelial monolayers toward the chemoattractant formylmethionylleucylphenylalanine (fMLP). Despite delayed transmigration by anti-CD47 mAbs, the numbers of PMN migrating across in either condition were the same as in the presence of control non-inhibitory mAbs. Cell surface labeling and immunoprecipitation demonstrated upregulation of CD47 to the PMN cell surface with kinetics similar to those of the transmigration response. Subcellular fractionation studies revealed redistribution of CD47 from intracellular compartments that co-sediment with secondary granules to plasma membrane-containing fractions after fMLP stimulation. Experiments performed to investigate potential signaling pathways revealed that inhibition of tyrosine phosphorylation with genistein reversed the anti-CD47-mediated PMN migration delay, whereas inhibition of phosphatidylinositol 3-kinase only partially reversed anti-CD47 effects that correlated with a rapid increase in PMN cell surface CD47. Analysis of the contribution of epithelial-expressed CD47 to PMN transmigration revealed that PMN migration across CD47-deficient epithelial monolayers (CaCO2) was significantly increased after stable transfection with CD47. These results suggest that cell surface CD47 and downstream tyrosine phosphorylation signaling events regulate, in part, the rate of PMN migration during the inflammatory response.

Colonic epithelial hPepT1 expression occurs in inflammatory bowel disease: transport of bacterial peptides influences expression of MHC class 1 molecules.

BACKGROUND & AIMS: hPepT1 is an intestinal epithelial apical membrane transporter responsible for uptake of di/tripeptides (including bacterial derived proinflammatory n-formyl peptides). hPepT1 expression normally has a strict axial gradient-highest in the proximal small intestine with no expression in the colon. METHODS: Small intestinal-like cells (Caco2-BBE), and colonic-like cells (HT29-Cl.19A), and colonic mucosa from diseased and control patients were used in the present study. RESULTS: hPepT1 expression occurs aberrantly in the colon with chronic ulcerative colitis (6 patients) and Crohn's disease (4 patients), but not in normal colon (4 patients) or colon with microscopic colitis (4 patients). To model expression of hPepT1 by colonic-like cells in inflamed states, we stably transfected HT29-Cl.19A cells with a modified hPepT1 tagged on the N-terminus with green fluorescence protein. Analysis of transfected cells revealed that: GFP-hPepT1 protein, like the natural protein, is targeted to the apical plasma membrane. In addition, the tagged protein retains the capability of di/tripeptide absorption, and the expression of the tagged protein by HT29-Cl.19A cells permits absorption of N-formyl-methionyl-leucyl-phenylalanine (fMLP), as occurs in hPepT1 expressing Caco2-BBE cells. fMLP uptake by colonic cells expressing GFP-hPepT1 specifically enhances major histocompatibility complex class I surface expression. CONCLUSIONS: These data collectively indicate that, in some states of chronic inflammation, hPepT1 may be anomolously expressed in the colon. Further, transport of fMLP by hPepT1 potentially stimulates expression of key accessory immune molecule, MHC-1.

Neutrophil-epithelial crosstalk at the intestinal lumenal surface mediated by reciprocal secretion of adenosine and IL-6.

Adenosine is formed in the intestinal lumen during active inflammation from neutrophil-derived 5' AMP. Using intestinal epithelial cell line T84, we studied the effect of adenosine on the secretion of IL-6, a proinflammatory cytokine involved in neutrophil degranulation and lymphocyte differentiation. Stimulation of T84 monolayers with either apical or basolateral adenosine induces A2b receptor-mediated increase in IL-6 secretion, which is polarized to the apical (luminal) compartment. In addition, Salmonella typhimurium, TNF-alpha, and forskolin, known inducers of IL-6 secretion in intestinal epithelial cells, also stimulate IL-6 secretion into the apical compartment. We show that IL6 promoter induction by adenosine occurs through cAMP-mediated activation of nuclear cAMP-responsive element-binding protein (CREB). We also show that IL-6 released in the luminal (apical) compartment achieves a sufficient concentration to activate neutrophils (from which the adenosine signal originates), since such IL-6 is found to induce an intracellular [Ca(++)] flux in neutrophils. We conclude that adenosine released in the intestinal lumen during active inflammation may induce IL-6 secretion, which is mediated by cAMP/CREB activation and occurs in an apically polarized fashion. This would allow sequential activation of neutrophil degranulation in the lumen -- a flow of events that would, in an epithelium-dependent fashion, enhance microbicidal activity of neutrophils as they arrive in the intestinal lumen.

Neutrophil transepithelial migration: regulation at the apical epithelial surface by Fc-mediated events.

Neutrophil (PMN) transepithelial migration is a major effector of epithelial defense in inflammatory diseases involving mucosal surfaces. However, major receptor-ligand interactions between epithelial cells and PMN remain incompletely characterized. To better define the molecular events involved in PMN interactions with epithelial cells, we produced a monoclonal antibody called g82 that inhibited PMN transepithelial migration in the physiological basolateral-to-apical direction. The g82 antigen localized to the apical surface of human colonic epithelium and was significantly upregulated under inflammatory conditions. Immunoprecipitation revealed two polypeptides of M(r) 207 and 32 kDa. F(ab')(2) fragments from g82 IgG had no effect on transmigration, suggesting Fc dependence. Further experiments confirmed dependence on the PMN Fc receptor CD32A and that the observed effects were secondary to a failure of PMN to detach from the apical epithelial surface. These Fc-mediated events were epitope specific since binding, isotype-matched antibodies did not affect detachment. These results identify a new mechanism for retention of PMN at the apical epithelial surface following transepithelial migration. This pathway may be important in pathogen clearance and mucosal pathophysiology associated with autoimmunity.

Clostridium difficile toxins disrupt epithelial barrier function by altering membrane microdomain localization of tight junction proteins.

The anaerobic bacterium Clostridium difficile is the etiologic agent of pseudomembranous colitis. C. difficile toxins TcdA and TcdB are UDP-glucosyltransferases that monoglucosylate and thereby inactivate the Rho family of GTPases (W. P. Ciesla, Jr., and D. A. Bobak, J. Biol. Chem. 273:16021-16026, 1998). We utilized purified reference toxins of C. difficile, TcdA-10463 (TcdA) and TcdB-10463 (TcdB), and a model intestinal epithelial cell line to characterize their influence on tight-junction (TJ) organization and hence to analyze the mechanisms by which they contribute to the enhanced paracellular permeability and disease pathophysiology of pseudomembranous colitis. The increase in paracellular permeability induced by TcdA and TcdB was associated with disorganization of apical and basal F-actin. F-actin restructuring was paralleled by dissociation of occludin, ZO-1, and ZO-2 from the lateral TJ membrane without influencing the subjacent adherens junction protein, E-cadherin. In addition, we observed decreased association of actin with the TJ cytoplasmic plaque protein ZO-1. Differential detergent extraction and fractionation in sucrose density gradients revealed TcdB-induced redistribution of occludin and ZO-1 from detergent-insoluble fractions constituting "raft-like" membrane microdomains, suggesting an important role of Rho proteins in maintaining the association of TJ proteins with such microdomains. These toxin-mediated effects on actin and TJ structure provide a mechanism for early events in the pathophysiology of pseudomembranous colitis.

Cryptdin-3 induces novel apical conductance(s) in Cl- secretory, including cystic fibrosis, epithelia.

Opening of anion-conductive pathways in apical membranes of secretory cells lining mucosal surfaces is a critical step in salt and water secretion and, thus, hydration of sites including airway and intestine. In intestine, Paneth cells are positioned at the base of the secretory gland (crypt) and release defensin peptide, in mice termed cryptdins, into the crypt lumen. Because at least some defensins have been shown to form anion-conductive channels in phospholipid bilayers, we tested whether these endogenous antimicrobial peptides could act as soluble inducers of channel-like activity when applied to apical membranes. To directly evaluate the possibility of cryptdin-3-mediated apical anion conductance (G(ap)), we have utilized amphotericin B to selectively permeabilize basolateral membranes of electrically tight monolayers of polarized human intestinal secretory epithelia (T84 cells), thus isolating the apical membrane for study. Cryptdin-3 induces G(ap) that is voltage independent (deltaG(ap) = 1.90 +/- 0.60 mS/cm2) and exhibits ion selectivity contrasting to that elicited by forskolin or thapsigargin (for cryptdin-3, Cl- = gluconate; for forskolin and thapsigargin, Cl- >> gluconate). We cannot exclude the possibility that the macroscopic current induced by cryptdin could be the sum of cation and Cl- currents. Cryptdin-3 induces a current in basolaterally permeabilized epithelial monolayers derived from airway cells harboring the deltaF508 mutation of cystic fibrosis (CF; deltaG(ap) = 0.80 +/- 0.06 mS/cm2), demonstrating that cryptdin-3 restores anion secretion in CF cells; this occurs independently of the CF transmembrane conductance regulator channel. These results support the idea that cryptdin-3 may associate with apical membranes of Cl--secreting epithelia and self-assemble into conducting channels capable of mediating a physiological response.

Characterization of epithelial chemoattractants for human intestinal intraepithelial lymphocytes.

BACKGROUND & AIMS: Although homing of intraepithelial lymphocytes (IEL) into intestinal epithelia seems to be guided by signals from epithelia, little is known concerning functional epithelial-derived chemoattractants for IEL. METHODS: Epithelial chemoattractants for IEL were analyzed using chemotaxis chamber system, enzyme-linked immunosorbent assay, and in situ hybridization using human epithelial lines and IEL lines. RESULTS: Epithelial-conditioned media induced IEL chemotaxis, and this activity was markedly enhanced by prestimulation of epithelia with interferon-(IFN)-gamma. This chemotaxis (stimulation +) was significantly inhibited by neutralizing antibodies to IFN-gamma inducible protein-10 (IP-10) or to monokine induced by IFN-gamma (MIG). Furthermore, while high amounts of IP-10 and MIG were detected in epithelial-conditioned media after IFN-gamma stimulation, equivalent concentrations of recombinant IP-10 and MIG reproduced IEL chemotaxis. Production of IP-10 and MIG in fresh epithelial cells was supported by in situ hybridization and enzyme-linked immunosorbent assay. Lastly, fresh human IEL constitutively expressed CXCR-3 (the common receptor for IP-10 and MIG), and fresh IEL also exhibited chemotaxis to by rIP-10, rMIG, and epithelial-conditioned media. CONCLUSIONS: Epithelial cells produce chemoattractants for IEL, and such chemokine production is regulated by proinflammatory cytokines such as IFN-gamma. IP-10 and MIG may serve as potentially important epithelial chemokines for IEL, especially under inflammatory conditions.

Negative regulation of epithelium-neutrophil interactions via activation of CD44.

Polymorphonuclear neutrophil (PMN) migration across epithelia is a common feature of active inflammation. Given the suggested role of carbohydrates in this process, we examined the receptor CD44. The standard CD44 isoform was expressed at the cell surface of PMN. PMN migration across model polarized intestinal epithelia was reduced (by 60%) if the CD44 receptor was activated by either a specific antibody (clone IM7) or the natural soluble ligand, hyaluronic acid. This inhibitory effect following receptor activation occurred with both basolateral-to-apical- and apical-to-basolateral-directed migration. The anti-CD44 antibody similarly reduced PMN migration through filters in the absence of epithelia, while preincubation of the antibody with the epithelium did not alter subsequent PMN transepithelial migration. These data suggest that PMN, rather than epithelial, CD44 is responsible for these effects. A similar inhibitory effect of anti-CD44 antibody was also observed on migration of intraepithelial lymphocytes. The molecular mechanism involved in such negative signaling following CD44 activation may include modulation of outside-in cell signaling. While neither the anti-CD44 antibody nor CD44 ligand affected PMN mobilization of intracellular Ca(2+), both led to increased adenylate cyclase activity, an inhibitory signal for PMN migration. Together, these results suggest that CD44 of PMN may potentially serve as a negative regulator of leukocyte migration across biological surfaces such as columnar epithelia.

Salmonella typhimurium translocates flagellin across intestinal epithelia, inducing a proinflammatory response.

This study investigated whether soluble paracrine factors mediated Salmonella-induced IL-8 expression in polarized model intestinal epithelia. We found that the basolateral media of model epithelia that had been apically infected with Salmonella typhimurium for a short period (10 minutes) could activate IL-8 secretion in virgin model epithelia, demonstrating that a proinflammatory factor (PIF) was indeed present. Initial characterization found that PIF was a heat-stable protein with a molecular mass of about 50 kDa that acts on the basolateral, but not apical, surface of model intestinal epithelia to elicit IL-8 secretion. PIF was not present in the media of model epithelia stimulated with other inducers of IL-8 secretion (TNF-alpha or carbachol) but was present in S. typhimurium supernatants, indicating PIF is of bacterial origin. PIF was purified from bacterial culture supernatants by anion/cation exchange chromatography and SDS-PAGE and found by using microsequencing to be the protein flagellin. In support of this finding, flagellin-deficient S. typhimurium mutants did not secrete detectable levels of PIF (i.e., a bioactivity that induced IL-8 secretion when placed basolaterally on model epithelia). Furthermore, viable flagellin-deficient mutant organisms (fliC/fljB and flhD) failed to elicit IL-8 secretion when added apically to model intestinal epithelia. These findings indicate that translocation of flagellin across epithelia, subsequent to apical epithelial-S. typhimurium interaction, is likely a major means of activating a mucosal inflammatory response.

Transepithelial resistance can be regulated by the intestinal brush-border Na(+)/H(+) exchanger NHE3.

Initiation of intestinal Na(+)-glucose cotransport results in transient cell swelling and sustained increases in tight junction permeability. Since Na(+)/H(+) exchange has been implicated in volume regulation after physiological cell swelling, we hypothesized that Na(+)/H(+) exchange might also be required for Na(+)-glucose cotransport-dependent tight junction regulation. In Caco-2 monolayers with active Na(+)-glucose cotransport, inhibition of Na(+)/H(+) exchange with 200 microM 5-(N,N-dimethyl)- amiloride induced 36 +/- 2% increases in transepithelial resistance (TER). Evaluation using multiple Na(+)/H(+) exchange inhibitors showed that inhibition of the Na(+)/H(+) exchanger 3 (NHE3) isoform was most closely related to TER increases. TER increases due to NHE3 inhibition were related to cytoplasmic acidification because cytoplasmic alkalinization with 5 mM NH(4)Cl prevented both cytoplasmic acidification and TER increases. However, NHE3 inhibition did not affect TER when Na(+)-glucose cotransport was inhibited. Myosin II regulatory light chain (MLC) phosphorylation decreased up to 43 +/- 5% after inhibition of Na(+)/H(+) exchange, similar to previous studies that associate decreased MLC phosphorylation with increased TER after inhibition of Na(+)-glucose cotransport. However, NHE3 inhibitors did not diminish Na(+)-glucose cotransport. These data demonstrate that inhibition of NHE3 results in decreased MLC phosphorylation and increased TER and suggest that NHE3 may participate in the signaling pathway of Na(+)-glucose cotransport-dependent tight junction regulation.

Molecular physiology and pathophysiology of tight junctions. IV. Regulation of tight junctions by extracellular stimuli: nutrients, cytokines, and immune cells.

The epithelial lining of the gastrointestinal tract forms a regulated, selectively permeable barrier between luminal contents and the underlying tissue compartments. Permeability across the epithelium is, in part, determined by the rate-limiting barrier of the paracellular pathway-the most apical intercellular junction referred to as the tight junction (TJ). The TJ is composed of a multiprotein complex that affiliates with the underlying apical actomyosin ring. TJ structure and function, and therefore epithelial permeability, are influenced by diverse physiological and pathological stimuli; here we review examples of such stimuli that are detected at the cell surface. For example, luminal glucose induces an increase in paracellular permeability to small molecules. Similarly, but by other means, cytokines and leukocytes in the vicinity of the epithelium also regulate TJ structure and paracellular permeability by influencing the TJ protein complex and/or its association with the underlying actin cytoskeleton.

Prokaryotic regulation of epithelial responses by inhibition of IkappaB-alpha ubiquitination.

Epithelia of the vertebrate intestinal tract characteristically maintain an inflammatory hyporesponsiveness toward the lumenal prokaryotic microflora. We report the identification of enteric organisms (nonvirulent Salmonella strains) whose direct interaction with model human epithelia attenuate synthesis of inflammatory effector molecules elicited by diverse proinflammatory stimuli. This immunosuppressive effect involves inhibition of the inhibitor kappaB/nuclear factor kappaB (IkappaB/NF-kappaB) pathway by blockade of IkappaB-alpha degradation, which prevents subsequent nuclear translocation of active NF-kappaB dimer. Although phosphorylation of IkappaB-alpha occurs, subsequent polyubiquitination necessary for regulated IkappaB-alpha degradation is completely abrogated. These data suggest that prokaryotic determinants could be responsible for the unique tolerance of the gastrointestinal mucosa to proinflammatory stimuli.

Constitutive and regulated secretion of secretory leukocyte proteinase inhibitor by human intestinal epithelial cells.

BACKGROUND & AIMS: Epithelial cells participate in immune regulation and mucosal integrity by generating a range of biologically active mediators. In the intestine, little is known about the potential endogenous anti-inflammatory molecules. Secretory leukocyte proteinase inhibitor (SLPI) is a major serine proteinase inhibitor, a potent antibiotic, and thus a potential anti-inflammatory molecule, although it is not known if it is secreted by intestinal epithelial cells. METHODS: We show, by reverse-transcription polymerase chain reaction, the presence of SLPI messenger RNA in human model intestinal epithelial cell lines (Caco2-BBE, T84, and HT29-Cl.19A) and human jejunum and colon biopsy specimens. The polymerase chain reaction product was cloned and sequenced and is identical to that of SLPI isolated previously from the human parotid gland. RESULTS: As analyzed by enzyme-linked immunosorbent assay, the constitutive secretion of SLPI occurs in a markedly polarized manner toward the apical surface and is enhanced by inflammatory mediators including tumor necrosis factor alpha and interleukin 1beta (approximately 3.5-fold increase over control value). SLPI release is also stimulated by activation of protein kinase C isoenzymes, but not by activation of adenosine 3',5'-cyclic monophosphate- or Ca(2+)-regulated signaling molecules. SLPI protein is detectable in intestinal lavage fluids collected from normal adult humans. Recombinant SLPI attenuates digestive enzyme (trypsin)- or leukocyte proteinase (elastase)-induced permeability alteration of a model epithelia in a dose-dependent manner. Moreover, SLPI exhibits an antibacterial activity against at least one major intestinal pathogen, Salmonella typhimurium. In contrast, SLPI does not influence epithelial barrier integrity as assessed by transepithelial conductance measurements or electrogenic ion transport. CONCLUSIONS: These results establish that human intestinal epithelium expresses and apically secretes SLPI, a molecule that may significantly contribute to the protection against attack from inflammatory cells and digestive enzymes, as well as against microbial infection.

Polarity of A2b adenosine receptor expression determines characteristics of receptor desensitization.

UNLABELLED: It is not known if, in polarized cells, desensitization events can be influenced by the domain on which the receptor resides. Desensitization was induced by 5'-(N-ethylcarboxamido)adenosine (NECA) and was quantitated by measurement of short-circuit current (I(sc)) in response to adenosine. NECA added to either the apical or basolateral compartments rapidly desensitized receptors on these respective domains. Although apical NECA had no effect on the basolateral receptor stimulation, basolateral NECA induced a complete desensitization of the apical receptor. We hypothesized that desensitization of apical receptor by basolateral desensitization could relate to a trafficking step in which A2b receptor is first targeted basolaterally upon synthesis and transported to the apical surface via vesicular transport/microtubules. Because desensitization is associated with downregulation of receptors, apical adenosine receptor can thus be affected by basolateral desensitization. Both low temperature and nocodazole inhibited I(sc) induced by apical and not basolateral adenosine. IN CONCLUSION: 1) a single receptor subtype, here modeled by the A2b receptor, differentially desensitizes based on the membrane domain on which it is expressed, 2) agonist exposure on one domain can result in desensitization of receptors on the opposite domain, 3) cross-domain desensitization can display strict polarity, and 4) receptor trafficking may play a role in the cross-desensitization process.