Activation of NF-kappa B in intestinal epithelial cells by enteropathogenic Escherichia coli

Suzana D. Savkovic, Athanasia Koutsouris, and Gail Hecht

Section of Digestive and Liver Diseases, Department of Medicine, University of Illinois, Chicago, Illinois 60612

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The initial response to infection is recruitment of acute inflammatory cells to the involved site. Interleukin (IL)-8 is the prototypical effector molecule for this process. Transcription of the IL-8 gene is primarily governed by the nuclear transcription factor (NF)-kappa B. Intestinal epithelial cells produce IL-8 in response to infection by enteric pathogens yet remain quiescent in a milieu where they are literally bathed in normal bacterial flora. We therefore sought to investigate NF-kappa B activation in response to enteropathogenic Escherichia coli (EPEC), nonpathogenic E. coli, and bacterial lipopolysaccharide in an intestinal epithelial cell (T84) model and to determine whether EPEC-induced activation of NF-kappa B factor is causally linked to IL-8 production. We report herein that NF-kappa B is activated by EPEC, yet such a response is not extended to nonpathogenic organisms or purified E. coli lipopolysaccharide. Transcription factor decoys significantly diminished IL-8 production in response to EPEC, demonstrating a causal relationship. Furthermore, deletion of specific EPEC virulence genes abrogates the NF-kappa B-activating property of this pathogen, suggesting that specific bacterial factors are crucial for inducing this response. These studies show for the first time that infection of intestinal epithelial cells with EPEC activates NF-kappa B, which in turn initiates IL-8 transcription, and highlight the differential response of these cells to bacterial pathogens vs. nonpathogens.

interleukin-8; inflammation; infectious diarrhea; epithelial immune response; nuclear factor-kappa B

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

ONE OF THE INITIAL host responses to infection by various pathogens is recruitment of acute inflammatory cells, primarily polymorphonuclear leukocytes (PMN), to the site. Several studies employing various animal models and in vitro systems have identified interleukin (IL)-8 to be one of the principal chemoattractants responsible for summoning PMN to the site of infection or tissue injury (11). Intestinal epithelial cells have been shown to respond to enteric infectious agents by producing a variety of proinflammatory cytokines, including IL-8, monocyte chemotactic protein-1, granulocyte/monocyte colony-stimulating factor, and tumor necrosis factor-alpha (TNF-alpha ) (16). A previous study from our laboratory (31) showed that IL-8 antibodies abrogated ~50% of the chemotactic activity for PMN present in medium from cultured intestinal epithelial cell (T84) monolayers infected with enteropathogenic Escherichia coli (EPEC).

The regulation of IL-8 gene expression is beginning to be elucidated. The promoter region of the IL-8 gene contains binding sequences for several transcription factors, including nuclear factor (NF)-IL-6, NF-kappa B, AP-1, AP-3, and octamer binding proteins (25). Previous studies have suggested that these transcriptional factors do not have equivalent effects on IL-8 gene activation, and in fact NF-kappa B and NF-IL-6 appear to synergistically activate IL-8 gene transcription (21, 22, 24, 25). Although cooperation with another transcription factor, preferentially NF-IL-6 (25) but also AP-1 (22, 25, 33), is necessary, NF-kappa B has been demonstrated to be the most crucial factor for initiation of IL-8 gene transcription (24).

A number of factors activate NF-kappa B, including various cytokines, phorbol esters, and several viruses (1). In addition, expression of a great variety of genes is controlled by NF-kappa B (1). Activated NF-kappa B is a dimeric protein complex, either homo- or heterodimeric. Several protein subunits compose the NF-kappa B family, including p50, p52, p65, c-Rel, and Rel B. It is believed that the variability in the composition of NF-kappa B dimers may contribute to the specificity of gene regulation, as a particular NF-kappa B sequence may bind certain NF-kappa B complexes but not all of them (18, 24, 27).

A number of cell types produce IL-8 in response to various stimuli (4, 11, 23), including other cytokines, such as IL-1 or TNF-alpha , and infectious agents, such as viruses and bacterial products like lipopolysaccharide (LPS). The involvement of NF-kappa B activation in IL-8 production in response to infectious agents, such as viruses and bacterial LPS, has been demonstrated recently. For example, infection of respiratory epithelium with respiratory syncytial virus has been shown to stimulate the production of IL-8 via a mechanism that involves activation of both NF-kappa B and NF-IL-6 (21). In monocytes, bacterial LPS is a potent stimulus of NF-kappa B activation and IL-8 production (12, 22). The commonality of the cell types used in the studies cited above is that they exist in a sterile environment. Contact with any infectious agent, therefore, is perceived as a threat, and a rapid defensive response is crucial for host survival. Intestinal epithelial cells are different, however. Colonic cells, in particular, exist peacefully in an environment heavily colonized by bacteria and yet evoke an inflammatory response on contact with pathogens (23). Because the molecular mechanisms by which IL-8 production by intestinal epithelia is increased in response to bacterial infection have not been studied, we used an in vitro model of enteric infection, cultured intestinal epithelial T84 cells and EPEC, to investigate these events.

Specifically, the aims of this study were to examine the effect of a bacterial enteric pathogen, EPEC, on NF-kappa B activation in intestinal epithelial cells, to determine whether EPEC-activated NF-kappa B is linked to IL-8 expression in response to infection, to investigate whether intestinal epithelial cells discriminate between pathogens and nonpathogens with regard to NF-kappa B activation, and to determine whether specific EPEC factors are required to induce NF-kappa B activation in intestinal epithelial cells.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell culture. The T84 cells were a generous gift from Dr. Kim Barrett (University of California, San Diego, CA). Passages 40-55 were used for these studies and were grown in a 1:1 (vol/vol) mixture of Dulbecco-Vogt modified Eagle's medium and Ham's F-12 medium with 7% fetal calf serum (20).

Bacterial strains and infection of host cells. The EPEC strain (E2348/69) used in these studies is a wild-type strain that demonstrates localized adherence to HEp-2 cells (26) and T84 cells (32). JPN-15, derived from strain E2348/69, has spontaneously lost the pMAR2 plasmid that encodes the bundle-forming pilus required for initial, or nonintimate, attachment. This strain therefore adheres minimally, if at all, to T84 cells (32). CVD206 is a derivative of the wild-type strain in which the eaeA gene has been deleted (15). The eaeA gene encodes the outer membrane protein intimin (14), which is important for the formation of the attaching and effacing lesion characteristic of EPEC. UMD864 has a deletion of the espB gene (6), whose product, espB, is essential for activation of signal transduction (8). Both CVD206 and UMD864 are incapable of intimate attachment (6). E2348/69, JPN-15, and CVD206 were generous gifts from Dr. James Kaper (Center for Vaccine Development, University of Maryland, Baltimore, MD). Strain UMD864 was kindly provided by Dr. Michael Donnenberg (Infectious Diseases, University of Maryland, Baltimore, MD). Bacterial cultures grown overnight in Luria-Bertani broth were diluted (1:33) in serum- and antibiotic-free T84 medium containing 0.5% mannose and grown to mid log growth phase. The bacterial suspension was pelleted and then resuspended and layered onto T84 cell monolayers, as described previously (31). In addition to the EPEC strains, five human E. coli commensals were isolated by the Clinical Microbiology Laboratory (University of Illinois, Chicago, IL) for use in these studies. A lab strain of E. coli, JM109, was also used.

Transmigration assay. Inverted monolayers were constructed as originally described by Parkos et al. (28). Monolayers were incubated in T84 serum- and antibiotic-free medium for 24 h before infection with EPEC. Monolayer integrity was assessed by measuring transepithelial electrical resistance (20). The PMN transepithelial migration assay has been described in detail by others (28) and with specific adaptations for EPEC by our lab (31). The infected T84 monolayers were transferred, apical side down, into a 24-well tissue culture tray containing Hanks' balanced salt solution with Ca2+ and Mg2+. Isolated PMN (106) were added to the basolateral side (top reservoir) of each monolayer and incubated for 2 h at 37°C. To determine the role of IL-8 in EPEC-associated PMN transmigration, neutralizing antibody to IL-8 (30 µg/ml; Genzyme, Cambridge, MA) was added 20 min before the addition of PMN. Positive controls were represented by PMN transmigration in response to 1 µM formyl-Met-Leu-Phe (fMLP). The number of PMN that had transmigrated was quantitated by myeloperoxidase assay as previously described (28, 31).

Quantitation of IL-8. T84 cells were grown in 24-well plates containing 1 ml of tissue culture medium/well. Medium from cells infected with EPEC was collected after 2, 4, 6, and 24 h. IL-8 was quantitated using a dual-antibody enzyme-linked immunosorbent assay (ELISA) kit from R&D Systems (Minneapolis, MN), following the manufacturer's protocol.

Electrophoretic mobility shift assay. T84 cells were infected with EPEC as described above. At specified times after infection, cells were trypsinized, pelleted, and resuspended in phosphate-buffered saline. All subsequent steps were performed at 4°C. The cells were incubated for 15 min in 400 µl of a hyposmotic buffer [in mM: 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; pH 7.8), 10 KCl, 2 MgCl2, 0.1 EDTA, 3 phenylmethylsulfonyl fluoride (PMSF), and 3 1,4-dithiothreitol (DTT)]. Cell membranes were broken using a Dounce homogenizer, and the nuclei were pelleted by centrifugation for 5 min in a Microfuge. The pelleted nuclei were resuspended in a high-salt buffer [in mM: 50 HEPES (pH 7.4), 50 KCl, 200 NaCl, 0.1 EDTA, 3 PMSF, and 3 DTT, as well as 10% glycerol] to solubilize DNA binding proteins and then gently shaken for 30 min at 4°C. Extracts were spun in a Microfuge for 10 min, and aliquots of the supernatants containing nuclear proteins were stored at -70°C. Protein concentrations were determined by the Bradford assay. Binding reactions were performed at room temperature for 30 min using 5 µg of nuclear proteins and 0.5 ng [25,000 counts/min (cpm)] of labeled oligonucleotide in 15 µl of binding buffer containing (in mM) 10 tris(hydroxymethyl)aminomethane (Tris) · HCl (pH 7.5), 50 NaCl, 50 KCl, 1 MgCl2, 1 EDTA, and 5 DTT, as well as 5% glycerol and 1 µg poly(dI-dC).

The sequences of the oligomers used for the electrophoretic mobility shift assays (EMSAs) were as follows: NF-kappa B, 5' TCG AGC GGC AGG GGA ATT CCC CTC TCC 3'; AP-1, 5' AGC TTA AAG CAT GAG TCA GAC ACCT 3' (30); and -80 to -71 base pairs (bp) from promoter region of IL-8 gene (putative NF-kappa B binding sequence), 3× GGAATTTCCT (12) (GIBCO-BRL). Annealed oligomers were radiolabeled with delta -32P (DuPont NEN, Boston, MA), using T4 polynucleotide kinase (Promega, Madison, WI) to yield a specific activity of >108 cpm/µg. Resolution was accomplished by electrophoresis of the reaction solution on vertical 5% nondenaturing polyacrylamide gels using 0.25× TBE buffer [1× TBE buffer contains (in mM) 22.3 Tris · HCl, 22.3 boric acid, and 0.25 EDTA (pH 8.0)]. Protein binding was assessed via autoradiography. All radiographic images were reproduced by scanning, using Desk Scan software (Hewlett-Packard), and then transferred into Power Point (Microsoft) for labeling.

Supershift EMSA. Supershift assays were used to determine which specific members of the NF-kappa B family were activated by EPEC infection. In these studies, EMSAs were performed as described above except that rabbit antibodies (1 µg/reaction) against the NF-kappa B proteins p50, p52, p65, c-Rel, and Rel B (Santa Cruz Biotechnology, Santa Cruz, CA) were added during the binding reaction period.

Transcription factor decoy experiments. Transcription factor decoy (TFD) experiments have been reported by others to be an effective and specific method for blocking NF-kappa B-dependent events (7, 19). Double-stranded phosphorothioate oligonucleotides are efficiently taken up into the cytoplasm of cells, where they bind activated transcription factors and prevent translocation to the nucleus and subsequent DNA binding (2). For these studies, T84 monolayers were incubated for 20 h with 40 µM oligonucleotide consisting of either the NF-kappa B binding motif (5' G GGG ACT TTC CGC TGG GGA CTT TCC AGG GGG ACT TTC C 3') or a mutant NF-kappa B binding sequence (5' GTC TAC TTT CCG CTG TCT ACT TTC CAC GGT CTA CTT TCC 3'). The mutant sequence has been shown previously to not bind NF-kappa B (19). Monolayers were then washed and infected with EPEC. After 6 h, medium was collected and IL-8 was measured as described above.

Statistical methods. Comparisons of data between groups were made using the Student's t-test. Differences were considered significant when P < 0.05.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Quantitation of IL-8 production by T84 monolayers in response to EPEC infection. In a previous report using the inverted T84 model described here, we demonstrated that EPEC infection induced the transepithelial migration of PMN (31). Data from these studies showed that ~50% of the chemotactic activity in medium collected from EPEC-infected monolayers could be inhibited by IL-8-neutralizing antibodies. To better define the effect of EPEC infection on IL-8 production by host intestinal epithelial cells, IL-8 was quantitated by ELISA. As shown in Fig. 1, IL-8 production by uninfected control monolayers and monolayers incubated with EPEC for 2 h was negligible (<20 pg/ml). By 4 h, however, IL-8 was present at a concentration of 90 ± 6 pg/ml and by 6 h at a concentration of 110 ± 6 pg/ml. IL-8 production in response to five human commensal E. coli strains was also determined and was found to be negligible (16.5 ± 6.8 pg/ml).


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Fig. 1.   Interleukin (IL)-8 production in response to enteropathogenic Escherichia coli (EPEC) infection. EPEC infection of T84 monolayers stimulates production of IL-8 in a time-dependent manner. T84 monolayers were infected with EPEC, and medium was collected for quantitation of IL-8 at 2, 4, 6, and 24 h. Values are means ± SE.

To define the extent to which IL-8 was responsible for EPEC-induced PMN transmigration across intact intestinal epithelial monolayers, transmigration assays were performed using the inverted monolayer system in the presence and absence of IL-8 neutralizing antibodies. In these studies, the addition of IL-8 neutralizing antibodies decreased PMN transmigration in response to EPEC infection by 60% (14 × 104 vs. 6 × 104 transmigrating PMN in the absence and presence of IL-8 antibody, respectively), indicating that, early in the course of infection (at least up to 3 h), IL-8 is responsible for the majority of PMN chemotaxis. In contrast, antibody to IL-8 had no effect on fMLP-driven transmigration (26.7 × 104 vs. 33.9 × 104 transmigrating PMN in the absence and presence of IL-8 antibody, respectively).

EPEC activates NF-kappa B transcription factors in intestinal epithelial T84 cells. The above data suggest that EPEC attachment to host intestinal epithelial cells in some way regulates expression of the IL-8 gene. Previous studies have suggested that of the several transcription factor binding sites present in the promoter region of the IL-8 gene, NF-kappa B is most important in initiating transcription (25). To investigate whether EPEC infection of intestinal T84 cells activated NF-kappa B, EMSAs were performed. Extracts of nuclear proteins from control and EPEC-infected T84 cells were incubated with 32P-labeled oligonucleotides consisting of a consensus NF-kappa B binding sequence (see MATERIALS AND METHODS). Figure 2A compares EMSAs from uninfected control and EPEC-infected T84 cells. As shown, EPEC activates NF-kappa B transcription factors in intestinal epithelial T84 cells. Time course studies showed that the intense signal seen at 1 h postinfection was significantly diminished after 3 h of incubation with EPEC. As a positive control, T84 cells were treated with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA; 100 ng/ml for 1 h), a known inducer of NF-kappa B activity (1). To confirm the specificity of this signal, competitor studies using 100-fold excess cold oligonucleotide were performed. These experiments demonstrated that the NF-kappa B binding activity was inhibited by such competition (Fig. 2B). In contrast, competition using an unrelated binding sequence (AP-1) failed to eliminate the signal (data not shown). Interestingly, EMSAs from uninfected T84 cells demonstrated NF-kappa B complexes of higher molecular weights than those seen on induction with EPEC and TPA (Fig. 2A). That these constitutively present complexes represent NF-kappa B signals was confirmed by competition experiments (not shown). Interestingly, these bands disappear when cells are infected with EPEC or treated with TPA.


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Fig. 2.   EPEC infection of intestinal epithelial T84 cells activates nuclear factor (NF)-kappa B. A: electrophoretic mobility shift assays (EMSAs) were performed by incubating nuclear proteins extracted from uninfected control monolayers (lane 1), monolayers infected with EPEC for 1 h (lane 2) and 3 h (lane 3), and 12-O-tetradecanoylphorbol 13-acetate (TPA)-treated monolayers (100 ng/ml for 1 h; lane 4) with 32P-labeled oligonucleotides consisting of NF-kappa B binding sequence. Bands labeled NF-kappa B represent NF-kappa B protein-DNA complexes whose mobility through the gel has been retarded. Unbound oligonucleotides are seen at bottom of gel (Free). B: competition studies demonstrate inhibition of NF-kappa B binding to labeled oligonucleotides by addition of 100-fold excess cold oligonucleotide, confirming specificity of binding. Lanes 1 and 2, EMSAs from EPEC-infected cells in absence and presence of competitor oligonucleotides, respectively.

Effect of nonpathogenic E. coli and E. coli LPS on NF-kappa B activity. Intestinal epithelial cells are continually exposed to bacterial flora that reside within the intestinal lumen, yet they do not constitutively express proinflammatory cytokines. We sought, therefore, to determine whether intestinal epithelial cells could discriminate between pathogenic and nonpathogenic bacteria and/or bacterial LPS, a potent activator of NF-kappa B in monocytes (12, 22). T84 monolayers were therefore incubated with pathogenic (EPEC) or nonpathogenic (JM109) E. coli or with purified E. coli LPS (serotype 0111:B4, Sigma, St. Louis, MO) at a concentration of 100 µg/monolayer, which is equivalent to the entire weight of bacteria adherent to infected cells. NF-kappa B activation was assessed by EMSA, as shown in Fig. 3. Neither nonpathogenic E. coli (strain JM109) nor pure E. coli LPS activated NF-kappa B transcription factors in T84 cells, suggesting that intestinal epithelia can differentiate pathogenic from nonpathogenic bacterial strains and tolerate high concentrations of bacterial LPS without activating the inflammatory cascade.


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Fig. 3.   Intestinal epithelial cells discriminate between pathogenic and nonpathogenic bacteria with regard to NF-kappa B activation. EMSAs using uninfected control cells are shown in lane 1. T84 cells were infected with pathogenic E. coli (EPEC; lane 2) or nonpathogenic E. coli (JM109; lane 3) or were exposed to purified E. coli lipopolysaccharide (100 µg/monolayer; lane 4). EMSAs using nuclear extracts from above groups of cells and 32P-labeled NF-kappa B oligonucleotides were then performed to determine NF-kappa B activation. TPA-treated cells served as a positive control (lane 5).

EPEC-activated NF-kappa B binds to the promoter region of the IL-8 gene. In the above experiments, NF-kappa B activation by EPEC was detected using oligonucleotides consisting of a common binding sequence for NF-kappa B (30). The promoter region of the IL-8 gene, however, contains a specific NF-kappa B binding site (24). To assess whether the particular NF-kappa B proteins activated in intestinal epithelial cells (T84) by exposure to EPEC bind to the IL-8 promoter, EMSAs were performed using oligonucleotides consisting of -80 to -71 bp (which contains the putative NF-kappa B binding site) from the promoter region of the IL-8 gene. As shown in Fig. 4, the specific NF-kappa B proteins activated by EPEC did bind to this specific region of the IL-8 promoter. Competition with 100-fold excess cold oligonucleotide completely ablated the signal (Fig. 4), demonstrating specificity of the binding.


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Fig. 4.   Specific NF-kappa B proteins activated by EPEC infection of intestinal epithelial cells bind to NF-kappa B binding sequence within promoter region of IL-8 gene. Previous EMSAs utilized oligonucleotides composed of a common binding sequence for NF-kappa B. For these experiments, EMSAs were performed using oligonucleotides consisting of specific sequence from promoter region of IL-8 that contains NF-kappa B binding site (-81 to -70 base pairs). EMSAs were performed with nuclear proteins from uninfected control cells in absence (lane 1) and presence (lane 2) of cold competitor oligonucleotides and from EPEC-infected cells in absence (lane 3) and presence (lane 4) of cold competitor. Lanes 5 and 6, respectively, are EMSAs performed with nuclear proteins extracted from TPA-treated cells in absence and presence of cold competitor.

TFD experiments link EPEC-induced NF-kappa B activation and IL-8 expression. Because NF-kappa B regulates the expression of numerous genes, TFD experiments were performed to determine whether EPEC-activated NF-kappa B was directly related to EPEC-induced IL-8 production. Oligonucleotides consisting of either the wild-type or mutant NF-kappa B binding sequence were taken up by cells. The presence of wild-type TFDs in the cell cytoplasm prevents nuclear translocation and DNA binding of activated NF-kappa B (2), hence inhibiting NF-kappa B-dependent events (7, 19). In these studies, wild-type TFDs significantly decreased EPEC-induced IL-8 production. Conversely, mutant TFDs had no significant effect (Fig. 5). These data demonstrate a direct connection between EPEC-induced NF-kappa B activation and IL-8 production.


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Fig. 5.   Effect of transcription factor decoys on EPEC-induced IL-8 production. T84 monolayers were incubated with oligonucleotides consisting of either wild-type NF-kappa B binding sequence (solid bar) or a mutant NF-kappa B binding sequence (hatched bar). Open bar, EPEC-infected control. Presence of wild-type sequence significantly diminished production of IL-8 in response to infection with EPEC. In contrast, mutant decoys had no impact on IL-8 expression stimulated by EPEC. Medium for IL-8 measurement was collected after 6 h of infection. Data are means ± SE; n = 3 for each group. P = 0.02 for wild-type oligonucleotides vs. infected controls, P = 0.01 for wild-type oligonucleotides vs. mutant oligonucleotides, and P = 0.09 for infected controls vs. mutant oligonucleotides.

Identification of specific NF-kappa B subunits activated in T84 cells infected with EPEC. NF-kappa B exists as dimeric complexes, either homo- or heterodimers. To identify the specific NF-kappa B subunits that comprise the NF-kappa B signal detected by EMSAs in EPEC-infected intestinal T84 cells, supershift experiments were performed. Specific antibodies to p50, p52, p65, c-Rel, and Rel B were used for these experiments. Supershift studies (Fig. 6) demonstrated that antibodies to p50 shifted nearly the entire signal and that antibodies to p52 also caused a significant shift. Anti-p65, c-Rel, and Rel B each shifted a small portion of the EPEC-induced NF-kappa B signal, as evidenced by the appearance of a higher molecular weight complex seen with each of these antibodies. This pattern differs from the one that occurs in response to treatment of T84 cells with TPA, which activates p50, p52, and p65 but not c-Rel or Rel B (data not shown).


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Fig. 6.   Identification of specific NF-kappa B proteins activated by EPEC. Supershift assays were performed using antibodies to p50, p52, p65, c-Rel, and Rel B. Antibody to p50 shifts nearly the entire NF-kappa B signal. Antibody to p52 also shifts a significant portion of the signal. Anti-p65, c-Rel, and Rel B created minor shifts, as evidenced by appearance of a new band of higher molecular weight in each lane.

The product of the EPEC espB gene is key for activation of NF-kappa B. Because intestinal cells do not react to nonpathogenic E. coli or purified LPS, we sought to determine which EPEC factor(s) might be important in activating NF-kappa B. The genetics of EPEC have been relatively well characterized (5), and specific mutants have been created, as described in Bacterial strains and infection of host cells. To investigate which bacterial virulence gene(s) was essential for activation of NF-kappa B transcription factors, we examined the effects of several EPEC mutant strains, JPN-15, CVD206, and UMD864, on this process. As shown in Fig. 7, only the eaeA deletion mutant CVD206 activated NF-kappa B transcription factors. This mutant, although devoid of the outer membrane protein intimin (14) and unable to create attaching and effacing lesions (15), does adhere nonintimately to host cells and activate signal transduction pathways (9, 29). Interestingly, this was the only mutant that stimulated PMN transmigration, as shown in our previous publication (31). These data suggest that formation of the attaching and effacing lesion is not required for activation of NF-kappa B. Instead, stimulation of signal transduction pathways in the host cells by the espB gene product is crucial.


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Fig. 7.   Effect of EPEC mutants on NF-kappa B activity. Nuclear proteins were extracted from uninfected control cells (lane 1), cells infected with wild-type EPEC (lane 2), and mutant strains JPN-15 (lane 3), CVD206 (lane 4), and UMD864 (lane 5). EMSAs were then performed to compare NF-kappa B activation by these various mutant strains.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

It is clear that epithelia actively participate in the inflammatory process by producing several proinflammatory cytokines. The initial response to infection is recruitment of PMN. IL-8 is the prototypical epithelium-derived chemoattractant for PMN. Recent studies defining the regulation of the IL-8 gene demonstrate that nuclear transcription factors, in particular NF-kappa B, are the final messengers of a signaling process that begins extracellularly, proceeds through the cell cytoplasm and into the nucleus, and eventuates in the production and secretion of IL-8. Several activators of this pathway have been identified, including viruses, cytokines, and phorbol esters (1). In this study, we examine the ability of an enteric bacterial pathogen, EPEC, to induce the translocation of NF-kappa B from the cytoplasm to the nucleus of intestinal epithelial cells, and we investigate the specific virulence factors that stimulate this event. TFD experiments showed that EPEC-induced NF-kappa B activation and IL-8 production are directly linked. In addition, we have explored the possibility that intestinal epithelial cells can discriminate between pathogens and nonpathogens with regard to NF-kappa B activation and have determined that specific bacterial products are required to stimulate this event.

Others have previously demonstrated the failure of intestinal epithelial cells to produce cytokines in response to bacterial LPS and nonpathogenic bacteria (16). The ability of intestinal epithelial cells to differentiate between pathogens and nonpathogens and to tolerate high concentrations of LPS without activating the inflammatory cascade, although predictable, is interesting. Our studies show that this differential response occurs at the level of transcription factor activation. Should intestinal epithelia respond with the same vigor to all bacteria or bacterial components, the result would be constitutive expression of proinflammatory cytokines and uncontrolled intestinal inflammation. Such a process is descriptive of inflammatory bowel disease. In fact, aberrant regulation of inflammatory cytokines has been described in other pathological conditions, including malignancy and autoimmune disorders (17). It is interesting to speculate that inflammatory bowel disease may be attributable to deregulation of cytokine production at a molecular level.

The differential IL-8 response to a common agent, LPS, by two different cell types, monocytes and intestinal epithelial cells, suggests cell-specific regulation of cytokine production. The resistance of intestinal epithelial cells to LPS could lie at either the cell membrane or within the intracellular signaling processes that ultimately activate NF-kappa B. Nevertheless, that specific bacterial virulence factors are required to activate NF-kappa B in intestinal epithelial cells and ultimately induce transmigration of PMN (31) is supported by studies with EPEC mutants. The pathophysiological mechanisms of EPEC-induced disease are beginning to be elucidated, in part through the creation of specific EPEC mutants. Several virulence genes have been identified, including the chromosomal genes eaeA, which encodes the outer membrane attachment protein intimin (14), espB, which encodes a 37-kDa protein that stimulates signal transduction cascades in host cells (8), and sepA-sepD, which encode a protein secretory apparatus (13). In addition, a 60-megabase plasmid encodes the bundle-forming pilus (10), important for nonintimate, or the initial phase of, attachment. Of the mutants examined in our model system, only the eaeA deletion mutant, CVD206, activated NF-kappa B. Interestingly, as shown in our previous publication (31), CVD206 was also the only mutant strain that induced the transepithelial migration of PMN. These studies show that formation of the characteristic attaching and effacing lesion of EPEC is not required, since CVD206 can only attach to cells in a nonintimate fashion. Instead, our findings suggest that stimulation of host intestinal epithelial cell signaling pathways by secreted EPEC protein(s) is involved in activating NF-kappa B and the subsequent events resulting in PMN transmigration. Alternatively, rearrangement of host cytoskeletal proteins by EPEC may be responsible for NF-kappa B activation. Disruption of cytoskeletal elements, both microtubules and microfilaments, has been shown to induce gene expression (3, 30). The observation that strain JPN-15, which presumably lacks only the pMAR2 plasma, does not activate NF-kappa B or stimulate PMN transmigration (31) is likely explained by its poor attachment to T84 cells (32). Perhaps with longer exposure to JPN-15 or centrifugation of this strain onto T84 monolayers, NF-kappa B would be activated.

Regardless of the mechanisms involved, these observations support the notion that specific bacterial factors are required to stimulate the inflammatory response by intestinal epithelia. That activation of NF-kappa B in intestinal epithelia is not unique to EPEC has been demonstrated by unpublished studies from our lab. In fact, all enteric pathogens tested, including other groups of pathogenic E. coli (enterotoxigenic, enterohemorrhagic, and enteroinvasive), Salmonella, and Shigella, were found to activate this transcription factor. Further studies addressing which specific bacterial factors(s) and signaling pathways activate NF-kappa B and the inflammatory response in intestinal epithelia will aid in elucidating the regulation of this process.

In other cell systems, NF-kappa B heterodimers composed of p65 and p50 appear to be particularly potent initiators of IL-8 gene transcription. Our supershift studies show that EPEC induces primarily p50 and p52 but also to a lesser degree other NF-kappa B proteins, including p65, c-Rel, and Rel B. The specific NF-kappa B dimers that are most active in EPEC-induced transcription of the IL-8 gene in intestinal epithelial cells have not been identified. Interestingly, infection of intestinal epithelial cells by various enteric pathogens does not induce the production of equivalent amounts of IL-8. In general, invasive pathogens stimulate the production of higher concentrations than do noninvasive pathogens (16). Whether this differential response is due to variability in the specific NF-kappa B complexes, or other transcription factors involved in regulating IL-8 gene expression, that are activated by a particular pathogen is not known. Detailed comparative studies utilizing different enteric pathogens will help resolve some of the questions regarding regulation of the IL-8 gene in intestinal epithelia.

In summary, previous studies from our lab demonstrated that EPEC, a noninvasive enteric pathogen, stimulated PMN transmigration, much of which was attributable to IL-8. The present studies extend this observation and show that this bacterial pathogen influences IL-8 gene expression via activation of nuclear transcription factors, in particular, NF-kappa B. Although similar models have been used to investigate the effects of virus-induced NF-kappa B activation on IL-6 and IL-8 production by alveolar epithelial cells (22, 34), the effect of bacterial pathogens on epithelial IL-8 gene regulation had not been examined previously. Continued investigations into this area hold important clinical implications, as molecular manipulation of the inflammatory response has great potential as a future therapeutic modality.

    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-02013 and by a Veterans Affairs Merit Award (both to G. Hecht).

    FOOTNOTES

Address for reprint requests: G. Hecht, University of Illinois, Dept. of Medicine, Digestive and Liver Diseases (M/C 787), 840 S. Wood St., CSB Rm. 1115, Chicago, IL 60612.

Received 27 January 1997; accepted in final form 17 June 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Cell Physiol 273(4):C1160-C1167
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