Section of Digestive and Liver Diseases, Department of Medicine, University of Illinois, Chicago, Illinois 60612
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ABSTRACT |
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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)-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-
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-
B factor is causally linked to IL-8 production. We
report herein that NF-
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-
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-
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-B
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INTRODUCTION |
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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- (TNF-
) (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-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-
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-
B has been demonstrated to be the most
crucial factor for initiation of IL-8 gene transcription (24).
A number of factors activate NF-B, including various cytokines,
phorbol esters, and several viruses (1). In addition, expression of a
great variety of genes is controlled by NF-
B (1). Activated NF-
B
is a dimeric protein complex, either homo- or heterodimeric. Several
protein subunits compose the NF-
B family, including p50, p52, p65,
c-Rel, and Rel B. It is believed that the variability in the
composition of NF-
B dimers may contribute to the specificity of gene
regulation, as a particular NF-
B sequence may bind certain NF-
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-, and
infectious agents, such as viruses and bacterial products like
lipopolysaccharide (LPS). The involvement of NF-
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-
B and NF-IL-6 (21). In monocytes, bacterial
LPS is a potent stimulus of NF-
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-B activation in intestinal
epithelial cells, to determine whether EPEC-activated NF-
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-
B activation, and to determine
whether specific EPEC factors are required to induce NF-
B activation
in intestinal epithelial cells.
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MATERIALS AND METHODS |
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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).
Supershift EMSA.
Supershift assays were used to determine which specific members of the
NF-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-
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-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-
B binding motif (5' G GGG ACT TTC CGC TGG GGA CTT TCC AGG GGG ACT TTC C 3')
or a mutant NF-
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-
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.
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RESULTS |
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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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EPEC activates NF-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-
B is most important in initiating transcription (25). To
investigate whether EPEC infection of intestinal T84 cells activated
NF-
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-
B binding sequence (see
MATERIALS AND METHODS). Figure
2A
compares EMSAs from uninfected control and EPEC-infected T84 cells. As
shown, EPEC activates NF-
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-
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-
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-
B complexes of higher molecular weights than those
seen on induction with EPEC and TPA (Fig.
2A). That these constitutively
present complexes represent NF-
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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Effect of nonpathogenic E. coli and E. coli LPS on
NF-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-
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-
B activation was
assessed by EMSA, as shown in Fig. 3.
Neither nonpathogenic E. coli (strain JM109) nor pure E. coli LPS activated
NF-
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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EPEC-activated NF-B binds to the promoter region of
the IL-8 gene.
In the above experiments, NF-
B activation by EPEC was detected using
oligonucleotides consisting of a common binding sequence for NF-
B
(30). The promoter region of the IL-8 gene, however, contains a
specific NF-
B binding site (24). To assess whether the particular
NF-
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-
B binding site) from the promoter region of
the IL-8 gene. As shown in Fig. 4, the
specific NF-
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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TFD experiments link EPEC-induced NF-B activation
and IL-8 expression.
Because NF-
B regulates the expression of numerous genes, TFD
experiments were performed to determine whether EPEC-activated NF-
B
was directly related to EPEC-induced IL-8 production. Oligonucleotides consisting of either the wild-type or mutant NF-
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-
B (2), hence inhibiting NF-
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-
B activation and IL-8 production.
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Identification of specific NF-B subunits activated
in T84 cells infected with EPEC.
NF-
B exists as dimeric complexes, either homo- or heterodimers. To
identify the specific NF-
B subunits that comprise the NF-
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-
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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The product of the EPEC espB gene is key
for activation of NF-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-
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-
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-
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-
B. Instead, stimulation of signal transduction pathways in the
host cells by the espB gene product is
crucial.
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DISCUSSION |
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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-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-
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-
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-
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-B. Nevertheless, that specific bacterial
virulence factors are required to activate NF-
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-
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-
B and the subsequent events
resulting in PMN transmigration. Alternatively, rearrangement of host
cytoskeletal proteins by EPEC may be responsible for NF-
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-
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-
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-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-
B and the
inflammatory response in intestinal epithelia will aid in elucidating
the regulation of this process.
In other cell systems, NF-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-
B proteins, including p65, c-Rel,
and Rel B. The specific NF-
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-
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-B. Although similar models have been used to investigate the effects of virus-induced NF-
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.
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ACKNOWLEDGEMENTS |
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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).
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FOOTNOTES |
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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.
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