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,
*
Department of Molecular Microbiology,
Department of Pathology and Immunology, and
Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Urinary tract infections (UTIs)3 are some of the most common infectious diseases, with greater than seven million cases reported annually in the United States (3). Escherichia coli is the causative organism in 70–90% of UTIs (3). Nearly all uropathogenic E. coli (UPEC) express surface-adhesive organelles known as type 1 pili. Type 1 pili are composite structures comprised of a 7-nm thick rod joined to a thin 3-nm tip fibrillum containing the adhesin, FimH (4, 5). FimH mediates binding to mannosylated glycoproteins and has been shown to be critical in the ability of uropathogenic E. coli to establish bladder infections (6, 7). In addition, Fim host receptor interactions trigger the invasion of E. coli into bladder epithelial cells, an event that has been proposed to facilitate bacterial persistence in the urinary tract (8, 9). However, type 1-piliated E. coli are also known to stimulate host defenses, such as epithelial cell apoptosis and cytokine production (8, 10). It has been proposed that type 1 pili can directly activate bladder epithelial cells leading to the production of IL-6 and IL-8 (11).
LPS is a critical factor for the induction of inflammatory cytokines in
macrophages. The recognition of LPS by macrophages involves the
LPS-binding protein or soluble CD14, which facilitates the transfer of
LPS to membrane-bound CD14 (mCD14), a GPI-linked receptor (12, 13). The CD14-LPS complex is thought to associate with Toll-like
receptor 4 (TLR4), which leads to the activation of NF-
B and
subsequently the production of proinflammatory cytokines (12, 14). In contrast, the recognition of LPS by epithelial cells is
unclear. The majority of epithelial cells do not respond as well as
macrophages, if at all, to soluble LPS (1, 10, 12, 15, 16, 17, 18). Therefore, it has been assumed that LPS is not the
primary molecule involved in epithelial recognition of Gram-negative
pathogens. In this report, we now describe the mechanism by which type
1 pili augment the activation of bladder epithelial cell cytokine
production and define the role of LPS in this response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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AAEC185 (19), AAEC185/put2002, AAEC185/pSH2, and UTI 89 were grown static in Luria broth for 48 h at 37°C to induce the expression of type 1 pili. UTI 89 is a type 1-piliated clinical cystitis isolate kindly provided by Dr. Sol Langermann from Medimmune (M. A. Mulvey, J. D. Schilling, and S. J. Hultgren, manuscript in progress). Before all experiments, the expression of type 1 pili was confirmed by mannose-inhibitable yeast agglutination.
Cell culture
5637 epithelial cells (derived from a human bladder carcinoma, HTB-9; American Type Culture Collection, Manassas, VA) were cultured in RPMI 1640 medium (BioWhittaker, Walkersville, MD) plus 10% FBS (Sigma, St. Louis, MO) at 37°C in a water-saturated atmosphere of 95% air and 5% CO2.
IL-6 stimulation assay
5637 epithelial cells were seeded into 24-well plates at
0.3 x 105 to 1 x
105 cells per well and grown to confluency over 2
days. Forty-eight-hour bacterial cultures were pelleted by
centrifugation, resuspended in PBS, and diluted to the indicated
concentrations in RPMI 1640 medium plus 10% FBS plus the growth
inhibiting antibiotic trimetrioprim-sulfamethoxazole (TMP-SMZ)
(Alpharma) at concentrations of 54 µg/ml and 270 µg/ml,
respectively. TMP-SMZ was used to allow the accurate determination of
dose-response relationships. Bacterial suspensions were titered at the
onset of the infection to determine the number of live bacteria in
every experiment. One milliliter of the bacterial suspension or 20
ng/ml of human rIL-1
(positive control for cytokine induction) (R&D
Systems, Minneapolis, MN) was added per well of epithelial cells. The
bacteria were spun onto the cells by low-speed centrifugation and
incubated at 37°C for the indicated times. Supernatants were
collected, cell debris and bacteria were removed by centrifugation, and
samples were frozen at -80°C until assayed using an IL-6 ELISA (R&D
Systems).
Incubation with FimCH-coated beads
FimCH-coated beads or BSA-coated beads were prepared as previously described (9). The beads were removed from storage buffer (10 mg/ml BSA, 0.1% sodium azide, 5% glycerol, in PBS (pH 7.4)), washed three times with PBS, and resuspended in the original volume of PBS. For experiments, 1 µl of FimCH-coated beads or BSA-coated beads was added to 5637 cells (approximately five beads/epithelial cell). The beads were spun onto the cells by low-speed centrifugation and incubated at 37°C for 6 h. The supernatants were analyzed as described above.
Invasion assays and cytochalasin D treatment
5637 cells were pretreated with 0.1 µg/ml of cytochalasin D in RPMI 1640 medium plus 10% FBS plus TMP-SMZ for 30 min at 37°C. After the preincubation, 10 µl of 1 x 108 CFU/ml bacteria in PBS was added to the cells. Bacteria were spun onto cells and incubated at 37°C for 6 h. At the 6-h time point, supernatants were either collected for ELISA or incubated with 100 µg/ml of gentamicin to kill any extracellular bacteria. After 2 h in the presence of gentamicin, the wells were washed, lysed with 0.1% Triton X-100, and titered for viable counts of intracellular bacteria.
To determine whether intracellular E. coli could
continuously stimulate epithelial IL-6 production, cells were left
uninfected or infected with 20 µl of AAEC185/pSH2
(OD600 0.5) for 1 h (sufficient time for
significant invasion to occur (9)). Cells were then washed
with PBS and incubated for 2 h with media containing 100 µg/ml
of gentamicin to kill any extracellular bacteria. Subsequently, the
cells were washed five times with PBS and fresh medium containing 15
µg/ml of gentamicin was added to the wells. Also, at this time,
uninfected wells were left unstimulated or stimulated with 20 ng/ml of
IL-1. Supernatants were collected at 2, 6, and 24 h after the
addition of the low-concentration gentamicin solution and were analyzed
as described above. Intracellular titers of AAEC185/pSH2 were
determined at each time point as described above.
Inhibition of LPS-mediated responses
Log increments of E.coli LPS 055:B5 (Sigma) ranging from 0.5 ng/ml to 50 µg/ml were preincubated with or without 1 µg/ml of polymyxin B sulfate (Sigma) for 15 min. 5637 cells were subsequently stimulated with the LPS preparations and the IL-6 concentration of the supernatants was determined 6 h poststimulation as described above. To analyze the inhibitory properties of detoxified LPS, 5637 cells were preincubated with or without 100 µg/ml detoxified LPS derived from LPS 055:B5 (Sigma) for 30 min at 37°C. The cells were subsequently stimulated with LPS 055:B5 at concentrations ranging from 0.5 ng/ml to 50 µg/ml for 6 h, after which the IL-6 concentration of the supernatants was determined as described above. Bacterial suspensions were prepared as described above and left untreated, treated with polymyxin B (1 µg/ml) (17), or treated with gentamicin (100 µg/ml). Cytokine stimulation assays were performed as described above. To block epithelial recognition of LPS, 5637 cells were preincubated with 100 µg/ml detoxified LPS for 30 min at 37°C. After the pretreatment, 10 µl of 1–3 x 108 CFU/ml (multiplicity of infection 1–3) bacteria in PBS were added to the cells. Bacteria were spun down onto cells and incubated at 37°C for 6 h. The supernatants were analyzed as described above.
RT-PCR assay
THP-1 cells (TIB-202; American Type Culture Collection) were grown in RPMI 1640 medium (Life Technologies, Rockville, MD) containing 2 mM L-glutamine, 1.5g/l sodium bicarbonate, 4.5 g/l glucose, 1.0 mM sodium pyruvate, 0.05 mM 2-ME, and 10% fetal bovine serum (Sigma). Approximately 24 h before RNA isolation, 5 x 106 THP-1 cells were differentiated with 10 ng/ml PMA (Sigma). 5637 cells were grown to confluence as described above.
RNA was extracted from 
5 x 106 cells
using TRIzol reagent (Life Technologies) according to manufacturer’s
protocols. Subsequently, the RNA was treated with DNase I,
Amplification Grade (Life Technologies) according to manufacturer’s
protocols. RNA samples were stored at -80°C.
RT-PCR was performed using the Enhanced Avian RT-PCR Kit (Sigma) according to manufacturer’s protocols for the one-step method. Approximately 18 ng of RNA template was added to each reaction. Primers for TLR4 (506 bp) (20) and TLR2 (346 bp) (20) have been previously described. The products of the reverse transcriptase reaction were amplified by PCR for 35 cycles, with an annealing temperature of 52°C. Initial extention time was 45 s and every 5 cycles the extension time was increased by 30 s. PCR products were analyzed on a 1.5% agarose gel.
Mouse Infections
C3H/HeN and C3H/HeJ female mice were obtained from Harlan
Sprague and Jackson Laboratory, respectively. A 48-h culture of UTI 89
was pelleted and resuspended in sterile PBS to a concentration of
2 x 109 CFU/ml. Mice were infected via
intraurethral catheterization with 50 µl of the bacterial suspension
(1 x 108 CFU). At 10 and 48 h after
infection, five mice from each group were sacrificed by cervical
dislocation and the bladders were harvested. Bladders were bisected and
either fixed in neutral buffer formalin for histological analysis, or
homogenized in sterile 0.025% Triton X-100/PBS, and titered for
surviving bacteria. The bladder tissue was embedded in paraffin and
sections were stained with hematoxylin and eosin (H&E) or with a rabbit
anti-E. coli primary Ab (BioDesign, New York, NY) and
goat anti-rabbit Ig-Cy3 secondary Ab (Jackson ImmunoResearch, West
Grove, PA). Nuclei were counterstained with 500 ng/ml Hoechst
(Sigma).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-6 is a pleiotropic cytokine that has among its functions the
amplification of neutrophil recruitment and the activation and
differentiation of T and B cells (21, 22, 23, 24, 25). The rapid
up-regulation of IL-6 during UTIs in humans and mice make this molecule
an ideal read-out for epithelial activation in response to E.
coli (26, 27). To separate the function of type 1
pili from other bacterial virulence factors, 5637 human bladder
epithelial cells were infected with K12 E. coli expressing
(AAEC185/pSH2) or not expressing (AAEC185) type 1 pili and the IL-6
concentration in the supernatants was determined. TMP-SMZ, a
bacteriostatic antibiotic frequently used in the treatment of UTIs, was
always coincubated with the bacteria to prevent growth during the assay
(3). TMP-SMZ has no affect on the induction profile of
IL-6 by type 1 and nonpiliated E. coli (unpublished data).
Inhibition of bacterial proliferation using TMP-SMZ allowed a more
detailed and accurate dissection of dose-response effects, because the
number of bacteria was constant throughout the assay. As a positive
control, 5637 cells were stimulated with IL-1.
Infection of 5637 cells with AAEC185 or AAEC185/pSH2 at doses ranging
from 103 to 108 CFU/ml
(multiplicity of infection, 0.01–100) revealed two distinct thresholds
for epithelial activation (Fig. 1
A). At 1 x
105 CFU/ml, both AAEC185/pSH2 and AAEC185 induced
5637 IL-6 production 5-fold over unstimulated cells (Fig. 1A). The threshold for type 1 pilus-specific amplification
of IL-6 expression was attained at bacterial concentrations of
1 x 106 CFU/ml. At its peak of
stimulation, AAEC185/pSH2 elicited up to 4- to 7-fold more IL-6 than
did AAEC185 (Fig. 1A). Inoculation of bladder cells with
concentrations of E. coli above 5 x
107 CFU/ml resulted in a dramatic decline of IL-6
secretion. Kinetic analysis of IL-6 production showed that from 3 to
12 h after infection, type 1-piliated E. coli induced
three times more cytokine than the isogenic nonpiliated strain at an
equivalent dose (1 x 106 CFU/ml) (Fig. 1B). The fimH- mutant strain
(AAEC185/put2002), which produces nonadhesive type 1 pili,
behaved identically to the parental strain AAEC185 with respect to IL-6
induction (Fig. 2).
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The hypothesis that FimH binding to an epithelial receptor is
sufficient to induce epithelial IL-6 was investigated by using
polystyrene latex beads coated with purified FimCH. Purification of
FimH requires that it be coexpressed with the FimC chaperone, to
prevent its misfolding and proteolytic degradation (5). In
the FimCH complex, FimH has its native receptor binding structure
(28). FimCH-coated beads efficiently bind to and are
internalized by 5637 cells via a pathway that, like that observed with
FimH-expressing bacteria, is dependent on actin polymerization,
tyrosine phosphorylation, and is mannose inhibitable (9).
When FimCH-coated beads were incubated with 5637 cells, no IL-6
induction was observed, suggesting that signals derived from FimH-host
receptor interactions are not sufficient to activate IL-6 transcription
(Fig. 2
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Cytochalasin D inhibits type 1 pilus-mediated augmentation of IL-6 production
Previous studies have demonstrated an association between
bacterial invasion and epithelial cytokine production (1, 26, 29). Cytochalasin D, a drug that inhibits internalization but
does not affect bacterial adherence (9), was used to
prevent bacterial invasion of bladder epithelial cells. Cytochalasin D
had no effect on IL-6 production when 5637 cells were incubated with
IL-1 or the noninvasive, type 1- strain
AAEC185 (Fig. 3A). However, it
reduced epithelial IL-6 induction by the type 1+
strain, AAEC185/pSH2, to levels similar to those seen after
stimulation with the noninvasive strain (Fig. 3A). At this
dose of cytochalasin D, invasion of bladder epithelial cells by
AAEC185/pSH2 was reduced by 80% over the 6-hour assay (data not
shown). Thus, the data show that FimH-mediated bacterial adherence is
not sufficient and strongly argue that subsequent invasion is required
for the augmentation of IL-6 production by epithelial cells in response
to type 1-piliated E. coli.
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To determine whether cytokine stimulation occurs transiently or
continuously after stimulation with type 1-piliated E. coli,
AAEC185/pSH2 was allowed to invade 5637 cells for 1 h and the
cells were then treated with gentamicin to kill extracellular bacteria.
Subsequently, the supernatant containing dead bacteria and cellular
debris was replaced with fresh medium. IL-6 production was monitored by
collecting supernatants at 2, 6, and 24 h after invasion. It
should be noted that IL-6 is stable for >24 h under these infection
conditions (unpublished data). After an initial peak between 0 and
2 h after invasion, IL-6 production by 5637 cells plateaued
between 2 and 24 h, although bacteria persisted at similar levels
intracellularly (Fig. 3B, data not shown). In contrast,
cells stimulated with IL-1 continued to produce IL-6 throughout this
time interval (Fig. 3B). These results demonstrate that the
presence of intracellular E. coli does not lead to
continuous stimulation of IL-6, suggesting that epithelial cytokine
induction by invasive bacteria is transient. Furthermore, the
observation that IL-6 levels fail to significantly increase after
2 h postinvasion demonstrates that IL-6 is poorly induced in the
absence of active bacterial internalization.
LPS is required for epithelial IL-6 production in response to type 1-piliated E. coli
The above data demonstrate that type 1 pili do not directly
activate IL-6 production, but instead mediate bacterial invasion, which
in turn amplifies the induction of IL-6. Therefore, we sought to
identify the stimulatory bacterial molecule recognized by bladder
epithelial cells. 5637 bladder epithelial cells responded to LPS in a
dose-dependent manner (Fig. 4, A and C). As expected, reagents that block LPS
recognition by host cells inhibited this response. Polymyxin B (Fig. 4A) and detoxified LPS (Fig. 4C) at
concentrations of 1 µg/ml and 100 µg/ml, respectively, shifted the
threshold of activation after stimulation with E. coli
055:B5 LPS from 5–50 ng/ml to 50–500 ng/ml. The ability of increasing
concentrations of LPS to overcome the inhibition is consistent with
these agents competitively inhibiting LPS-mediated responses in 5637
cells.
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B). In contrast,
treatment of both the piliated (AAEC185/pSH2) and nonpiliated (AAEC185)
bacteria with polymyxin B inhibited IL-6 induction by 79% and 86%,
respectively, but had no effect on IL-1 induction of IL-6 (Fig. 4B and data not shown). The invasion efficiency (no. of
internalized bacteria/no. of adhered bacteria) was not affected by
polymyxin B (data not shown).
In addition, 5637 cells were preincubated with 100 µg/ml of
detoxified E. coli LPS before bacterial stimulation.
Detoxified LPS inhibited IL-6 stimulation by type 1-piliated and
nonpiliated E. coli by 89% and 100%, respectively (Fig. 4D), but had no effect on IL-1 induction of IL-6 (data
not shown). Bacterial adherence and invasion were unaffected by the
presence of detoxified LPS (data not shown). Together these data argue
strongly that LPS is the primary bacterial factor involved in both
invasion-dependent and invasion-independent activation of IL-6
production.
5637 bladder epithelial cells express TLR4, and TLR2
TLR4 and TLR2 have been implicated in the recognition of LPS by
mammalian cells; however, recent evidence suggests that TLR4 is the
primary signal transducer involved in LPS signaling (14, 30, 31). RT-PCR was used to assess the expression of mRNAs for these
receptors in 5637 bladder epithelial cells. Differentiated THP-1 cells
(macrophage-like cells) have been shown previously to express TLR4 and
TLR2 transcripts and were used a positive control for the RT-PCR
(20). TLR4 and TLR2 mRNAs were present in the
differentiated THP-1 cells (Fig. 5).
Similarly, 5637 bladder epithelial cells clearly expressed mRNA for
TLR4 and TLR2 (Fig. 5). Thus, 5637 bladder epithelial cells express
mRNAs for receptors implicated in LPS responsiveness.
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Based on our in vitro findings, LPS-hyporesponsive mice would be predicted to have defects in epithelial responses to type 1-piliated UPEC. C3H/HeJ mice have a mutation in the mammalian LPSR TLR4 and consequently fail to respond to LPS (32, 33). Others have shown that C3H/HeJ mice are unable to clear bacteria from the urinary tract and fail to generate an appropriate inflammatory response after infection with UPEC (34, 35, 36, 37, 38). In addition, IL-6 is not present in the urine of C3H/HeJ mice after an infection with E. coli (39). Thus, it would seem that TLR4-mediated host responses to bacterial LPS are critical for inducing inflammation in the urinary tract. To further substantiate our in vitro findings, we analyzed the association between internalized bacteria and the activation of the bladder epithelium in these mice.
C3H/HeJ and C3H/HeN mice were infected with UTI 89, a type 1-piliated
strain of UPEC, and histological analysis of the bladder epithelium
using H&E and anti-E. coli immunofluorescence staining
was performed at 10 and 48 h after infection. In this study, the
recruitment of neutrophils into the epithelium was used as a measure of
epithelial activation. At 10 h after infection, both mouse strains
had similar numbers of bacteria in the bladder, but there were dramatic
differences in the appearance of the bladder epithelium (Figs. 6 and 7,
A–C, F, and G). In C3H/HeN mice,
large numbers of intracellular bacteria were found within the bladder
epithelium (Fig. 7, A and C). Also, at this time
point, neutrophils were found to be migrating into the epithelium and
specifically associating with infected cells. The influx of neutrophils
seemed to correlate with the destruction of the intracellular bacterial
foci (Fig. 7, A and B). Large foci of
intracellular bacteria were also observed in C3H/HeJ mice, but in
contrast to C3H/HeN mice, neutrophils were rarely present (Fig. 7F). By 48 h after infection, C3H/HeN mice had reduced
the number of bacteria in the bladder by >90%, and no intracellular
foci of bacteria in the epithelium were visible by histological
analysis (Figs. 6 and 7E). Moreover, large numbers of
neutrophils were present in both the epithelium and lamina propria of
these mice (Fig. 7, D and E). In C3H/HeJ mice, no
reduction in bacterial titers was observed at 48 h after infection
(Fig. 6). Large collections of bacteria remained within the bladder
epithelium (Figs. 6 and 7I) with a striking absence of
neutrophils in both the epithelium and lamina propria (Fig. 7, H and I). Together these data suggest a dynamic
sequence of events, whereby the bladder epithelium is activated by
TLR4/bacterial interactions at sites of internalized E.
coli.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). It has been suggested that direct
interactions between type 1 pili and specific host receptors on bladder
epithelial cells can activate host-signaling cascades leading to IL-6
transcription (11). In this report, we tested this
hypothesis by investigating the underlying mechanism of type 1
pilus-mediated augmentation of epithelial cytokine production. Recent studies have shown that latex beads coated with the type 1 pilus adhesin, FimH, specifically bind to and are internalized by bladder epithelial cells (9). By using these adhesin-coated beads, it was determined that type 1 pilus-host receptor interactions are not sufficient to activate epithelial cytokine production independent of other bacterial factors. These results suggest that type 1 pili facilitate cytokine production through an indirect mechanism or that other components of the pilus structure contribute to cytokine induction. The finding that AAEC185/put2002 (type1+, fimH-) does not illicit IL-6 production above levels seen after stimulation with AAEC185 (type 1-) demonstrates that other components of the type 1 pilus do not contribute to the activation of bladder epithelial cells in the absence of FimH.
Type 1 pilus-mediated invasion of bladder epithelial cells is associated with a survival advantage for UPEC in vivo (8). In this manuscript, we have shown that inhibition of bacterial invasion using cytochalasin D diminishes the boost in cytokine production observed in response to infection with type 1-piliated E. coli. Of interest, the activation of cytokine production by invasive bacteria is transient, even when the bacterial stimulus persists in the intracellular environment. These data demonstrate that type 1 pili enhance epithelial cytokine production by mediating bacterial invasion of bladder epithelial cells.
Bacterial invasion has also been implicated in the stimulation of intestinal epithelial cell cytokine production (2, 18). However, the means through which bacterial invasion leads to this phenomenon are unclear. Purified LPS is a potent inducer of cytokine production in macrophages; however, LPS is generally a poor stimulator of epithelial cytokine production. Consequently, it has been suggested that this molecule is not involved in the induction of cytokine production by these cells in response to bacterial infection (17, 18, 26). 5637 bladder epithelial cells respond to LPS in a dose dependent manner between 0.5 ng/ml and 50 µg/ml with a threshold of activation between 5 ng/ml and 50 ng/ml. However, 5637 cells required 50 µg/ml of LPS to achieve the same IL-6 response as human PBLs stimulated with 5 ng of LPS (unpublished data).
The functional role of LPS in the activation of epithelial IL-6 was further investigated using the LPS inhibitors polymyxin B and detoxified LPS. Polymyxin B is an antibiotic that binds to the lipid A moiety of LPS and prevents its interaction with LPS-binding protein (17, 40). Detoxified LPS is generated by mild alkaline hydrolysis of LPS removing the fatty acid side chains from lipid A, which are responsible for the toxic activity of LPS (41). Previous reports have indicated that LPS molecules with various deacylated forms of lipid A are capable of antagonizing biological responses to intact LPS by interacting with host cells (42, 43). Therefore, we reasoned that this molecule might also be able to antagonize the recognition of LPS by bladder epithelial cells. Polymyxin B and detoxified LPS, at concentrations of 1 µg/ml and 100 µg/ml, respectively, inhibited both purified LPS-mediated responses and responses to type 1-piliated and nonpiliated E. coli, demonstrating that LPS is the critical bacterial molecule recognized by 5637 bladder epithelial cells. These data indicate that bacterial invasion coupled with LPS recognition enhance epithelial responsiveness to E. coli and suggest that the role of bacterial associated LPS in epithelial cytokine production should be considered for other invasive pathogens. Interestingly, P pilus-mediated induction of cytokines from a kidney epithelial cell line has been shown to occur in an LPS-independent manner (17). P pili do not mediate bacterial invasion of kidney or bladder epithelial cells (9). Thus, it seems that multiple mechanisms exist for the initiation of inflammation in the urinary tract.
In this report, bacterial invasion of bladder epithelial cells was
demonstrated to enhance the responsiveness of bladder epithelial cells
to E. coli via an LPS-dependent mechanism. It is possible
that this occurs through the up-regulation of an LPSR after type 1
pilus-mediated invasion or potentially via synergy between LPS and
invasion-mediated signaling cascades. However, these possibilities seem
unlikely due to the observation that the coaddition of LPS and
FimCH-coated beads fails to enhance the LPS responsiveness of 5637
cells (unpublished data). Furthermore, there is no delay in the
augmentation of IL-6 after stimulation with type 1-piliated bacteria
(Fig. 1B), suggesting that up-regulation of an LPSR is not
required for this response. It is also possible that bacterial invasion
leads to the clustering of an LPSR at sites of bacterial
internalization or that a pool of LPSRs are located in an intracellular
compartment. In support of such models, the maximal IL-6 response of
5637 cells to AAEC185 (type1-) is only 25% that
of the maximal cytokine response to AAEC185/pSH2 (type
1+), although the same amount of LPS is present
in both circumstances. This observation argues that type 1
pilus-mediated invasion alters the interaction between bacterial
associated LPS and the relevant LPSR or the accessibility of the LPSR.
Evidence for receptor clustering has already been demonstrated for the
pathogen-pattern recognition receptor TLR2, which transiently clusters
around phagosomes during the internalization of yeast particles by
macrophages (44). The possibility that invasion enhances
the response to an as yet unidentified bacterial molecule in an
LPS-dependent manner has not been excluded.
TLR4 has recently been identified as the primary mammalian LPSR
(14, 32, 45, 46, 47, 48, 49). The observation that LPS is the stimulus
for IL-6 production by 5637 bladder epithelial cells suggests that
TLR4, and potentially mCD14, may be involved in the activation of
bladder epithelial cells in response to E. coli.
Interestingly, we report here that 5637 bladder epithelial cells
express mRNA for TLR4. C3H/HeJ mice express a nonfunctional form of
TLR4 and consequently fail to respond to LPS, making this an ideal
system to investigate the in vivo role of type 1 pilus-mediated
bacterial invasion and LPS in the activation of the bladder epithelium.
Previous studies have demonstrated that after infection with UPEC,
C3H/HeJ mice fail to produce macrophage-inflammatory protein-2, leading
to a defect in neutrophil recruitment to the urinary tract, and
subsequent failure to clear bacteria (34, 35, 36, 37).
Furthermore, we have shown here that C3H/HeJ mice are unable to clear
type 1-piliated UPEC from the bladder during the first 2 days after
infection. These results, as with our in vitro data, state that host
responses to type 1-piliated bacteria in the bladder occur through an
LPS-dependent pathway. UPEC is able to invade bladder epithelial cells
leading to the formation of large intracellular foci of bacteria (Fig. 7) (8) (M. A. Mulvey, J. D. Schilling, and
S. J. Hultgren, manuscript in preparation). Based on the data
presented here, it would be predicted that invasion of these epithelial
cells in vivo would strongly activate the bladder epithelium in an LPS-
dependent manner. For our analysis, neutrophil recruitment into the
epithelium was used as a measure of epithelial activation. Consistent
with this hypothesis were the results with the C3H/HeJ mice, which
revealed a defect in the influx of neutrophils into the epithelium
despite high levels of bacterial invasion of bladder epithelial cells.
In contrast, C3H/HeN mice had large numbers of neutrophils associating
with foci of intracellular bacteria in the bladder epithelium. These
results demonstrate that bacterial LPS and TLR4 are involved in the
activation of bladder epithelial cells in response to type 1-piliated
UPEC during an in vivo UTI.
Over the past decade it has become increasingly apparent that
epithelial cells can participate in innate responses to pathogens
(2, 18, 50). The results presented here indicate that type
1 pili augment bladder epithelial cytokine responses to E.
coli by mediating bacterial invasion, not through a direct
mechanism as proposed previously (11, 51). Furthermore,
this study reveals that LPS is required for bacterial invasion to
augment epithelial responses to E. coli, which may be a more
general theme in bacterial epithelial interactions. The role of LPS in
this response and the results of experiments using C3H/HeJ mice (TLR4
mutant) implicate TLRs and, in particular, TLR4 as the probable
epithelial receptor involved in responses to E. coli.
Consistent with this prediction, 5637 cells express TLR4 mRNA. In
addition, recent studies have demonstrated that epithelial cells can
express TLRs at the protein level and that mutations in the
extracellular domain of TLR4 affect the LPS responsiveness of these
cells (52, 53). TLRs lead to the activation of NF-B and
the subsequent production of inflammatory cytokines and chemokines,
such as IL-6 and IL-8 (20, 52, 54). However, numerous
other inflammatory molecules are regulated by NF-B and future work
investigating the role of TLRs in the induction of epithelial mediators
will be vital to our understanding of epithelial cells as effectors of
the innate immune system.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Scott Hultgren, Department of Molecular Microbiology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110.
3 Abbreviations used in this paper: UTI, urinary tract infection; UPEC, uropathogenic E. coli; mCD14, membrane-bound CD14; TLR, Toll-like receptor; TMP-SMZ, trimethoprim-sulfamethoxazole; H&E, hematoxylin and eosin.
Received for publication March 22, 2000. Accepted for publication October 13, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B through toll-like receptor 4 (TLR-4) in cultured human dermal endothelial cells: differential expression of TLR-4 and TLR-2 in endothelial cells. J. Biol. Chem. 275:11058.B through interleukin-1 signaling mediators in cultured human dermal endothelial cells and mononuclear phagocytes. J. Biol. Chem. 274:7611.This article has been cited by other articles:
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  J. Song, B. L. Bishop, G. Li, R. Grady, A. Stapleton, and S. N. Abraham TLR4-mediated expulsion of bacteria from infected bladder epithelial cells PNAS, September 1, 2009; 106(35): 14966 - 14971. [Abstract] [Full Text] [PDF]
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B. K. Billips, S. G. Forrestal, M. T. Rycyk, J. R. Johnson, D. J. Klumpp, and A. J. Schaeffer Modulation of Host Innate Immune Response in the Bladder by Uropathogenic Escherichia coli Infect. Immun., November 1, 2007; 75(11): 5353 - 5360. [Abstract] [Full Text] [PDF]
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E. C. Hagan and H. L. T. Mobley Uropathogenic Escherichia coli Outer Membrane Antigens Expressed during Urinary Tract Infection Infect. Immun., August 1, 2007; 75(8): 3941 - 3949. [Abstract] [Full Text] [PDF]
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L. E. Mansson, P. Kjall, S. Pellett, G. Nagy, R. A. Welch, F. Backhed, T. Frisan, and A. Richter-Dahlfors Role of the Lipopolysaccharide-CD14 Complex for the Activity of Hemolysin from Uropathogenic Escherichia coli Infect. Immun., February 1, 2007; 75(2): 997 - 1004. [Abstract] [Full Text] [PDF]
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G. Gatti, V. Rivero, R. D. Motrich, and M. Maccioni Prostate epithelial cells can act as early sensors of infection by up-regulating TLR4 expression and proinflammatory mediators upon LPS stimulation J. Leukoc. Biol., May 1, 2006; 79(5): 989 - 998. [Abstract] [Full Text] [PDF]
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 Y. Jiang, P. Oliver, K. E. Davies, and N. Platt Identification and Characterization of Murine SCARA5, a Novel Class A Scavenger Receptor That Is Expressed by Populations of Epithelial Cells J. Biol. Chem., April 28, 2006; 281(17): 11834 - 11845. [Abstract] [Full Text] [PDF]
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 K Harada, K Isse, and Y Nakanuma Interferon {gamma} accelerates NF-{kappa}B activation of biliary epithelial cells induced by Toll-like receptor and ligand interaction J. Clin. Pathol., February 1, 2006; 59(2): 184 - 190. [Abstract] [Full Text] [PDF]
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D. A. Hunstad, S. S. Justice, C. S. Hung, S. R. Lauer, and S. J. Hultgren Suppression of Bladder Epithelial Cytokine Responses by Uropathogenic Escherichia coli Infect. Immun., July 1, 2005; 73(7): 3999 - 4006. [Abstract] [Full Text] [PDF]
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A. L. Kau, S. M. Martin, W. Lyon, E. Hayes, M. G. Caparon, and S. J. Hultgren Enterococcus faecalis Tropism for the Kidneys in the Urinary Tract of C57BL/6J Mice Infect. Immun., April 1, 2005; 73(4): 2461 - 2468. [Abstract] [Full Text] [PDF]
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A. Sato and A. Iwasaki From The Cover: Induction of antiviral immunity requires Toll-like receptor signaling in both stromal and dendritic cell compartments PNAS, November 16, 2004; 101(46): 16274 - 16279. [Abstract] [Full Text] [PDF]
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M. Ueta, T. Nochi, M.-H. Jang, E. J. Park, O. Igarashi, A. Hino, S. Kawasaki, T. Shikina, T. Hiroi, S. Kinoshita, et al. Intracellularly Expressed TLR2s and TLR4s Contribution to an Immunosilent Environment at the Ocular Mucosal Epithelium J. Immunol., September 1, 2004; 173(5): 3337 - 3347. [Abstract] [Full Text] [PDF]
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J. A. Feulner, M. Lu, JohnM. Shelton, M. Zhang, J. A. Richardson, and R. S. Munford Identification of Acyloxyacyl Hydrolase, a Lipopolysaccharide- Detoxifying Enzyme, in the Murine Urinary Tract Infect. Immun., June 1, 2004; 72(6): 3171 - 3178. [Abstract] [Full Text] [PDF]
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M. J. Duncan, G. Li, J.-S. Shin, J. L. Carson, and S. N. Abraham Bacterial Penetration of Bladder Epithelium through Lipid Rafts J. Biol. Chem., April 30, 2004; 279(18): 18944 - 18951. [Abstract] [Full Text] [PDF]
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S. S. Justice, C. Hung, J. A. Theriot, D. A. Fletcher, G. G. Anderson, M. J. Footer, and S. J. Hultgren From the Cover: Differentiation and developmental pathways of uropathogenic Escherichia coli in urinary tract pathogenesis PNAS, February 3, 2004; 101(5): 1333 - 1338. [Abstract] [Full Text] [PDF]
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L. Falzano, M. G. Quaranta, S. Travaglione, P. Filippini, A. Fabbri, M. Viora, G. Donelli, and C. Fiorentini Cytotoxic Necrotizing Factor 1 Enhances Reactive Oxygen Species-Dependent Transcription and Secretion of Proinflammatory Cytokines in Human Uroepithelial Cells Infect. Immun., July 1, 2003; 71(7): 4178 - 4181. [Abstract] [Full Text] [PDF]
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 R. Pawlinski, B. Pedersen, B. Kehrle, W. C. Aird, R. D. Frank, M. Guha, and N. Mackman Regulation of tissue factor and inflammatory mediators by Egr-1 in a mouse endotoxemia model Blood, May 15, 2003; 101(10): 3940 - 3947. [Abstract] [Full Text] [PDF]
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J. D. Schilling, S. M. Martin, C. S. Hung, R. G. Lorenz, and S. J. Hultgren Toll-like receptor 4 on stromal and hematopoietic cells mediates innate resistance to uropathogenic Escherichiacoli PNAS, April 1, 2003; 100(7): 4203 - 4208. [Abstract] [Full Text] [PDF]
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J. D. Schilling, S. M. Martin, D. A. Hunstad, K. P. Patel, M. A. Mulvey, S. S. Justice, R. G. Lorenz, and S. J. Hultgren CD14- and Toll-Like Receptor-Dependent Activation of Bladder Epithelial Cells by Lipopolysaccharide and Type 1 Piliated Escherichia coli Infect. Immun., March 1, 2003; 71(3): 1470 - 1480. [Abstract] [Full Text] [PDF]
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M. Christodoulides, B. L. Makepeace, K. A. Partridge, D. Kaur, M. I. Fowler, R. O. Weller, and J. E. Heckels Interaction of Neisseria meningitidis with Human Meningeal Cells Induces the Secretion of a Distinct Group of Chemotactic, Proinflammatory, and Growth-Factor Cytokines Infect. Immun., August 1, 2002; 70(8): 4035 - 4044. [Abstract] [Full Text] [PDF]
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A. Fox-Marsh and L. C. Harrison Emerging evidence that molecules expressed by mammalian tissue grafts are recognized by the innate immune system J. Leukoc. Biol., March 1, 2002; 71(3): 401 - 409. [Abstract] [Full Text] [PDF]
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I. U. Mysorekar, M. A. Mulvey, S. J. Hultgren, and J. I. Gordon Molecular Regulation of Urothelial Renewal and Host Defenses during Infection with Uropathogenic Escherichia coli J. Biol. Chem., February 22, 2002; 277(9): 7412 - 7419. [Abstract] [Full Text] [PDF]
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M. A. Mulvey, J. D. Schilling, and S. J. Hultgren Establishment of a Persistent Escherichia coli Reservoir during the Acute Phase of a Bladder Infection Infect. Immun., July 1, 2001; 69(7): 4572 - 4579. [Abstract] [Full Text] [PDF]
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) or absence (
) of the invasion inhibitor cytochalasin
D (CD). At 6 h after infection, IL-6 production was assessed by
ELISA. B, IL-6 production was monitored in 5637 cells
left unstimulated (
) at 2, 6, and 24 h postinvasion.
Experiments were repeated at least three times in triplicate. *,
p 
0.05 according to a Student’s
t test (determined using GraphPad Prism).
). D,
Similar assays were performed in the presence (
