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INFLAMMATION/IMMUNITY/MEDIATORS
Department of Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia 30322
Submitted 25 November 2002 ; accepted in final form 28 March 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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B in a MyD-88 dependent manner in response to flagellin.
Because TLRs, in general, are also thought to signal through members of the
MAPK family, we examined flagellin-induced MAPK activation (via examining its
phosphorylation status) and its subsequent role in expression of the chemokine
IL-8 in polarized intestinal epithelia. Flagellin, like other proinflammatory
stimuli (TNF-
, Salmonella typhimurium), activated p38 MAPK in
a TLR5-dependent manner, whereas aflagellate bacteria or EGF did not activate
this kinase. Although ERK1 and -2 were also observed to be activated in
response to flagellin, their activation was not restricted to proinflammatory
stimuli because they were also potently activated by aflagellate bacteria
(S. typhimurium or Escherichia coli) and EGF (neither of
which activate NF-B in these cells). Pharmacological inhibition of p38
MAPK (by SB-203580) potently (IC50 = 10 nM) reduced expression of
IL-8 protein (maximal inhibition, 75%) but had no effect on NF-B
activation, only slightly attenuated upregulation of IL-8 mRNA levels in
response to flagellin, and did not effect IL-8 mRNA stability. Together, these
results indicate that epithelial TLR5 mediates p38 activation and subsequently
regulates flagellin-induced IL-8 expression independently of NF-B,
probably by influencing IL-8 mRNA translation.
flagellin; nuclear factor-B; inflammation; toll-like receptor-5; mitogen-activated protein kinase; interleukin-8
We recently demonstrated (6, 9) that the primary means by which epithelial cells activate IL-8 expression in response to S. typhimurium is by the activation of epithelial toll-like receptor 5 (TLR5) via S. typhimurium flagellin monomers, which comprise flagella. Importantly, in T84 and Madin-Darby canine kidney model epithelia, expression of TLR5 is restricted to the basolateral membrane (6, 23), thus suggesting that TLR5-mediated proinflammatory gene expression may occur only on breach of the epithelium by bacteria or their products. TLR5 is one of 10 currently identified mammalian TLRs, which in general are thought to play a key role in innate immunity via the detection of conserved molecular patterns in microorganisms. In general, the 10 TLRs share much greater homology in their intracellular domains (toll-like IL-1 receptor domain) thought to be responsible for signaling than in their extracellular regions that are thought to function in ligand recognition (21). As such, there appears to be a considerable amount of conservation in the signal transduction pathways activated by different TLRs and, subsequently, in the changes in gene expression induced by the various members of this family. However, there are also some clear examples in differences in the signal transduction pathways used by different TLRs resulting in activation of distinct genes (27). Thus the signal transduction pathways need to be defined for each TLR and perhaps for different cell types.
Like other described TLRs, TLR5 utilizes the adaptor protein MyD88 and IL-1
receptor-associated kinase (IRAK) to activate a signal transduction cascade
that results in the activation of the transcription factor NF-B
necessary for flagellin-induced effects on gene expression
(6,
20). Although NF-B
family members play a diverse role in immune regulation and development and
thus can be activated by various classes of stimuli in some systems
(2), this transcription
factor's central role in intestinal epithelia is known to be primarily in
activation of proinflammatory gene expression
(4). TLR2, -4, and -9 have also
been demonstrated to activate members of the MAPK signaling cascade,
particularly ERK1 and -2 and p38MAPK
(21). In some experimental
systems, these MAPK proteins have been shown to lead to activation of
NF-B, whereas in others, p38 MAPK has been shown to potentiate
proinflammatory gene expression via posttranscriptional effects
(14). The goal of this study
was to examine the role of MAPK activation in flagellin-induced TLR5-mediated
proinflammatory gene expression focusing primarily on the chemokine IL-8. We
utilized both polarized model intestinal epithelia expressing endogenous TLR5
as well as MDCK and HeLa cells engineered to express TLR5, allowing both
physiologic and mechanistic assessments of this signaling pathway. We observed
that, whereas TLR5 activates both p38 and ERK1 and -2, p38 activation is more
restricted to proinflammatory signaling. Furthermore, we observed that such
p38 MAPK activation is not involved in TLR5-mediated NF-B activation
but rather regulates IL-8 expression by a posttranscriptional mechanism
analogous to that described for p38 regulation of TNF- expression
(14).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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was purchased from Invitrogen (Carlsbad, CA). Flagellin was
purified from S. typhimurium-conditioned media by anion/cation
exchange chromatography and purity verified as previously described
(9). Briefly, such flagellin
does not activate any TLR other than TLR5
(6) and has <50 pg/ml LPS
(18). Plasmid encoding
V5-tagged TLR5 was a gift of Jongdae Lee (Scripps Institute, La Jolla, CA),
and DN-TLR5 was made from this construct as previously described
(6). All other reagents were
purchased from Sigma (St. Louis, MO).
Model epithelia. Intestinal epithelial cell lines T84, HT-29cl19A,
or CACO-2BBE were cultured on collagen-coated permeable supports as previously
described (8). IL-8 secretion,
activation of NF-B-CAT reporter constructs, and IB-
levels were measured via ELISA as previously described
(5,
7).
MAPK assessments. Model epithelia were stimulated as described in figure legends, rinsed in cold HBSS, lysed (in PBS with 1% Triton X-100, 1 mM EDTA, 1 mM NaVO4,1mM NaF) cleared by centrifugation (10 min at 5,000 g at 4°C), and assayed for phospho-p38, phospho-ERK, or total P38 by SDS-PAGE immunoblotting. Where indicated, densitometry was performed via scanning with Scion Image densitometry software (Scion, Frederick, MD).
IRAK. IRAK activity was assayed via in vitro kinase activity on
the basis of the protocol by Croston et al.
(3). Briefly, stimulated or
control 5 cm2 model epithelia were lysed in 250 µl 1%
Triton-X-100, plus (in mM) 25 Tris · HCl, 150 NaCl, 1 DTT, 1 EDTA, 1
NaVO4, 1 NaF, 10 Na4P2O7, and
complete protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN).
Lysates were cleared via centrifugation (5,000 g; 10 min) and
subsequently via overnight incubation with 100 µl protein A-Sepharose beads
(Pierce Endogen, Rockford, IL). IRAK-1 was then immunoprecipitated via
overnight incubation with 10 µg anti-IRAK Ab (Santa-Cruz Bioscience, Santa
Cruz, CA) that had been preloaded onto 100 µl protein ASepharose beads
followed by four washes in the above-described lysis buffer followed by a wash
in (in mM) 50 HEPES, 10 NaCl, 1 DTT, 6 MgCl2, and 1
MnCl2. Immunoprecipitates were then incubated with 2 µg of
myelin basic protein and 0.5 µl ATP
32 (New England
Nuclear, Boston, MA). Phosphorlyated major basic protein was then
visualized/quantitated via SDS-PAGE autoradiography and Bio-Rad Gel Imaging
System (Bio-Rad Laboratories, Hercules, CA).
Exogenous expression of TLR5. MDCK cell and HeLa cells were
cultured in DMEM, supplemented with 10% FBS. For transient transfection, cells
were seeded in 6-well plates a day before transfection at 30% confluency.
Superfectin (Qiagen, Valencia, CA) was used to form DNA complexes, following
manufacturer's instructions. For each well, 0.5 µg of wild-type plasmid
combined with either 2 µg empty vector or 2 µg dominant-negative
(DN)-TLR5 plasmid was mixed with Superfectin reagent. Empty plasmid was used
as control. After 3 h incubation, normal medium was added for further
incubation overnight. Transfected cells were incubated overnight with and
experiments then performed within 18–24 h. For stable transfection,
pEF6/V5-TLR5 was linearized by ScaI. Linearized plasmid DNA (5 µg) was
added to MDCK cells in 100-mm culture dishes that were 
30% confluency
with superfectin as transfection reagent. At 24 h after transfection,
transfected cells were split at low density and cultured in DMEM supplemented
with 5–10 mg/ml blasticidin (Invitrogen) until the appearance of foci.
Foci were transferred to new a culture container and propagated. Expression of
TLR5 was confirmed by Western blotting with V5 antibody (Invitrogen).
Real-time PCR. Total RNA was isolated from treated cells (TRIzol;
Invitrogen) and then reverse transcribed (1.0 µg) from random hexamer
primers by using Multiscribe Reverse Transcriptase (Applied Biosystems; Foster
City, CA). Real-time quantitative PCR analysis was performed in triplicate on
1 µl of this reverse transcribed cDNA by using the SYBRgreen Real-Time PCR
assay (Applied Biosystems, Foster City, CA). The PCR reactions and SYBRgreen
detections were carried out in an iCycler iQ Real-Time Detection System
(Bio-Rad) whose cycling conditions included preliminary incubations for 2 min
at 50°C and for 10 min at 95°C, followed by 40 cycles of a 15-s
denaturation at 95°C and a 1-min annealing/extension at 60°C. The 18S
ribosomal RNA gene was used as an endogenous control allowing for cDNA amounts
to be normalized. The primers for the IL-8 gene (forward:
5'-AAACCACCGGAAGGAACCAT-3', reverse:
5'-GCCAGCTTGGAAGTCATGT-3') were designed from the primer design
software PrimerExpress, whereas the primers for the 18S-rRNA gene were
obtained from the TaqMan Ribosomal RNA Control Reagents Kit (Applied
Biosystems, Foster City, CA). Real-time PCR analysis was performed on both
treated and untreated cells to establish relative levels of mRNA expression.
The level of IL-8 expression in individually treated and untreated samples was
first normalized by subtracting the mean value of the cycle threshold
(Ct) for the 18S-rRNA gene from that of the IL-8 gene
(
Ct). Relative levels of IL-8 expression were then
determined by 1) subtracting the individual Ct
values for untreated control samples from those of the treated cells
(Ct) and 2) expressing the final
quantitation value as 2-Ct. Standard
deviation of this relative mRNA level for a single treatment:
2(C±{SD[Ctreated]2+SD[Cuntreated]2}).
As variance of replicate analysis of each sample was far less than the
variance of values from separate experiments, data are displayed as the means
± SE of three separate experiments.
Il-8 mRNA stability. Model epithelia were treated as indicated and subsequently treated with actinomycin D (5 µg/ml) or vehicle (0.1% DMSO) to stop further transcription. At various times afterward, IL-8 mRNA was quantitated via hybridization-based assay purchased from R&D Systems (Minneapolis, MN) following the manufacturer's instructions.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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B is a major
regulator of proinflammatory gene expression induced by flagellin and many
other proinflammatory agonists, it has become apparent that MAPKs can also
play an important role in regulating proinflammatory gene expression. To begin
to examine the role of MAPKs in flagellin-induced proinflammatory gene
expression, we first examined which of the well-defined MAPK members were
activated by exposure to purified flagellin in polarized model intestinal
epithelia. Activation levels of p38 MAPK, ERK1 and -2 and JNK were assessed
via measuring their phosphorylation states via immunoblotting because this is
a well-defined indicator of their level of enzymatic activity. We observed
that either apical colonization by live S. typhimurium, which
expressed flagellin, or the basolateral addition of purified flagellin both
potently activated p38 MAPK, whereas colonization by a mutant strain that
lacked genes for flagellin (fliC/fljB) did not
(Fig. 1). The level of p38
activation induced by purified flagellin appeared similar to that induced by
the potent proinflammatory cytokine TNF-, whereas, in contrast, neither
the growth factor EGF nor Salmonella LPS, which like aflagellate
S. typhimurium does not induce IL-8 secretion
(7,
9), did not activate this
kinase. Analogous to what we have previously demonstrated for IL-8 secretion
(6), flagellin added
basolaterally but not apically, activated this kinase. The time course of
flagellin-induced p38 activation was consistently biphasic with slight, but
reproducibly detectable, activation at 5 min and a much more pronounced
activation by 45 min and persisting for at least 2 h. In contrast, although
the proinflammatory stimuli S. ty- phimurium, purified
flagellin, and TNF- all induced activation of ERK1 and -2
(Fig. 2), activation of this
kinase was not restricted to proinflammatory stimuli, because ERK1 and -2 were
activated at least as well by EGF, aflagellate S. typhimurium, or
E. coli although the aflagellate bacteria appeared to act somewhat
slower. Neither flagellin nor TNF- induced detectable activation of JNK
consistent with our observation detailed below that JNK inhibition does not
block IL-8 production, thus suggesting this kinase is not involved in
mediating responses to such proinflammatory agonists in these cells. Lastly,
we observed that the same pattern of MAPK activation was observed in multiple
intestinal epithelial cell lines (HT29 and CaCo-2), indicating this pattern is
a property of these stimuli's actions on intestinal epithelial cells in
general as opposed to one cell line in particular (data not shown). Thus,
whereas both ERK and p38 are activated in epithelia by flagellin, p38
activation was more closely associated with the proinflammatory responses
induced by flagellin.
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Flagellin-induced p38 MAPK activation mediated by TLR5/IRAK. Two different molecules have been reported to function as receptors for flagellin, namely TLR5 and the ganglioside ASGM1 (6, 10, 17). Thus we then sought to determine what aspect of flagellin-induced p38 activation could be accounted for by flagellin activation of TLR5. We generated MDCK cells that stably express TLR5 and compared their p38 activation to control MDCK transformed with antibiotic resistance gene only (Fig. 3A). MDCK express some endogenous TLR5 and thus, as expected, exhibited detectable p38 activation in response to flagellin. However, the magnitude of the response was much greater in MDCK expressing TLR5 indicating flagellin-induced p38 MAPK activation was, at least in part, directly attributable to TLR5. We then sought to use our previously described DN-TLR5 construct to investigate whether functional TLR5 signaling is necessary for flagellin-induced p38 MAPK activation. First, we attempted to generate MDCK cells that stably expressed DN-TLR5. However, cells that survived initial drug selection (indicating they expressed DN-TLR5) consistently died a couple of days later suggesting that DN-TLR5 may interfere with a necessary growth/survival signaling pathway in these cells. Thus we utilized transient transfection to verify the role of TLR5 in mediating flagellin-induced p38 activation. Specifically, we utilized HeLa cells that allow high transfection rates and do not seem to have a functional endogenous TLR5 signaling pathway. HeLa cells were transfected with either control vector [red fluorescence protein (RFP) to monitor transfection efficiency], TLR5 alone, or TLR5 and DN-TLR5. Counting of fluorescent cells indicated a transfection efficiency of 55–60%. RFP transfectants exhibited enhanced p38 phosphorylation in response to TNF but not flagellin, whereas either agonist activated p38 in cells expressing TLR5 (Fig. 3B). Such TLR5 conferment of p38 activation in response to flagellin was blocked by DN-TLR5, whereas TNF-induced p38 activation (negative control) was unaffected, confirming the role of TLR5 in flagellin-induced MAPK activation.
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Flagellin activation of ASGM1 can be simulated by ligation of ASGM1 by use of a commercially available anti-ASGM1 antibody (22). Because ASGM1 can be on either the apical or basolateral membrane, we investigated whether we might ligate this molecule and activate p38 with either flagellin or anti-ASGM1. Although very high concentrations of apical, or of course basolateral, activated p38 (albeit modest activation for apical flagellin), neither apical nor basolateral anti-ASGM1 (even when used at high concentrations) activated p38 (Fig. 4) although immunoblotting with the rabbit antibody, and subsequent detection by an anti-rabbit secondary antibody, revealed that such anti-ASGM1 had bound both the apical or basolateral surface with sufficient avidity to resist extensive washing. These results indicate that flagellin's activation of p38 MAPK in such model intestinal epithelia is indeed mediated by activation TLR5 rather than ASGM1.
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We then sought to investigate the relationship between p38 MAPK activation and other signals generated by interaction of TLR5 and flagellin. First, we examined the relationship between P38 MAPK and IRAK-1, a kinase associated with TLR signaling in general (11) and that has been shown to be activated by TLR5 in monocytic cells (20). We observed that, indeed, IRAK-1 is also activated in response to flagellin in polarized epithelial cells as measured by an in vitro kinase assay (Fig. 5). IRAK-1 activation is generally thought to be a more proximal signal than MAPK activation. Consistent with this notion, the time course of IRAK activation appeared fairly similar to that of p38 activation with modest activation after 5 min and more robust activation by 1 h. Furthermore, activation of IRAK-1 was not affected by the p38 inhibitor SB-203580 (average inhibition was <5% in 2 experiments) suggesting that activation of IRAK-1 is not dependent on p38 MAPK activation.
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p38 regulates IL-8 expression via a posttranscriptional mechanism.
We then considered the consequences of MAPK activation on the epithelial
expression of the proinflammatory chemokine IL-8. We observed that the
well-characterized p38 inhibitor SB-203580 exhibited a concentration-dependent
inhibition of flagellin-induced IL-8 secretion
(Fig. 6). A similar level of
inhibition was observed for IL-8 secretion induced by TNF-, which, like
flagellin, induces activation of p38 MAPK.
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ERK1 and -2 inhibitor U-0126 also blocked flagellin-induced IL-8 secretion
suggesting that, although not specific to proinflammatory stimuli, the ERK
signaling pathway is nonetheless required for such induced proinflammatory
gene expression. Consistent with the failure of flagellin to induce JNK
phosphorylation, JNK inhibition did not reduce flagellin-induced IL-8
expression indicating this kinase's activity is not crucial in this signaling
pathway. We then considered possible mechanisms whereby blockade of MAPK might
so attenuate IL-8 expression focusing on p38 MAPK, the MAPK most specific to
proinflammatory stimuli. In light of a number of reports that MAPKs regulate
the NF-B pathway and the major role of this transcription factor in
regulating IL-8 transcription, we measured whether p38 MAPK inhibition
affected activation of the NF-B pathway in model epithelia. As such,
epithelia are very difficult to transfect, the activation of the NF-B
pathway is best assessed in such epithelia by examining the signaling events
that immediately precede and regulate the activation of this proinflammatory
transcription factor, namely the phosphorylation and degradation of
IB-. We observed that the concentration of SB-203580 (10 µM)
that maximally inhibited IL-8 secretion had no effect on these signaling
events that mediate NF-B activation
(Fig. 7). Furthermore, by using
HeLa cells transiently transfected with TLR5 and a synthetic NF-B
reporter gene construct whose activity is regulated solely by this
transcription factor, we observed that neither the NF-B activation
induced by TNF- nor by flagellin in TLR5 transfected cells was
significantly inhibited by SB-203580. These results indicate that inhibition
of p38 MAPK blocks IL-8 expression independent of effects on NF-B
activation.
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Activation of p38 MAPK has been shown to increase both the translation rate
and mRNA stability of genes such as TNF-, which contain a 5'
AU-rich element (ARE) (1,
12,
14). Because the IL-8 gene
also contains a 5'ARE, we investigated whether p38 inhibition suppressed
IL-8 expression via affecting levels of IL-8 mRNA level via quantitative
real-time PCR. IL-8 mRNA levels are very low in unstimulated epithelia but
increased 150-fold 90 min after the addition of purified basolateral
flagellin or TNF-. By 3 h after such stimulation, IL-8 mRNA levels had
moderately declined in the flagellin-treated cells but remained fully elevated
in the TNF--treated cells. For neither stimulus at neither time point
did we observe a decline in IL-8 mRNA levels significant enough to explain the
reduction in IL-8 secretion in epithelia under p38 inhibition
(Fig. 8). Because p38
inhibition had only a modest effect on the induction of IL-8mRNA, we then
examined the effect of p38 inhibition on Il-8 mRNA stability. Model epithelia
were treated with flagellin in the presence or absence of SB-203580 for 90
min, treated with actinomycin D to shut off further transcription and IL-8
mRNA quantitated by a well-characterized commercial hybridization-based
quantitative assay. We did not observe a difference in the relative decay
rates of IL-8 transcript in such cells
(Fig. 9) indicating that p38
inhibition was not significantly affecting IL-8 mRNA half-life. Lastly, we
utilized trypan blue exclusion to assess whether p38 inhibition was simply
toxic to these cells. SB-203580 did not have a substantial effect on trypan
blue exclusion by T84 cells (cells showing positive trypan blue staining was 6
vs. 8% for control cells vs. those treated with 10 mM SB-203580 for 5 h,
respectively). Together, these results indicate p38 inhibition likely
suppresses TLR5-mediated IL-8 expression via reducing IL-8 translation
analogous to observations made about genes such as TNF that also have a
5'ARE (14).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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B (DN-IRAK blocked flagellin-induced
NF-B activation in TLR5-transfected HeLa cells by 80%). The ability of flagellin and various other agonists to activate p38 MAPK correlated with their ability to induce expression of IL-8. In contrast, we observed that these proinflammatory agonists as well as a cytokine agonist and aflagellate bacteria, which do not induce IL-8, all activated ERK. This observation is consistent with the notion that p38 MAPK is a "stress-induced kinase" (one if its previously accepted names), whereas ERK activation is a more general signal that may function in both growth factor and stress-induced signaling. By such a paradigm, ligation of basolateral intestinal epithelial TLR5 would indeed seem an appropriate stress signal, because it would indicate that the gut epithelium has been breached by enteric microbes. However, a growth factor-type signal might be an appropriate "all is well" signal in response to apical colonization by commensal microbes, thus providing a rationale for why aflagellate and commensal bacteria might elicit only ERK activation. However, whereas ERK signaling is used broadly by many signaling pathways, it is likely still necessary for IL-8 expression because ERK inhibitors potently blocked IL-8 expression induced by flagellin or TNF.
Transcription factor NF-B is known to play a major role in
regulating the expression of many proinflammatory genes in general and IL-8
expression in model intestinal epithelia in particular. Whereas some studies
have reported a role of MAPKs in activating NF-B, particularly studies
that overexpress these kinases, our results do not indicate such a role for
the MAPK activity induced by flagellin or TNF-. Specifically, we
observed that inhibition of p38 activity suppresses production of IL-8 but yet
did not affect NF-B activation nor the signaling events immediately
proximal to its activation. Furthermore, p38 inhibition appeared to have only
modest effects on IL-8 mRNA levels. The modest effect of p38 inhibition of
IL-8 mRNA induction could result through an effect of p38 on IL-8 mRNA
stability as has been reported for TNF-
(1) and another AU-rich element
containing transcripts or could result from effects of the p38 pathway on
elements in the IL-8 promoter other than those responsive to NF-B. In
support of the latter possibility, reporter assays done with the native IL-8
promoter (instead of synthetic NF-B construct) were moderately
suppressed by p38 inhibition (by 50%). In contrast to this modest
suppression of IL-8 mRNA induction by p38 inhibition, we and others
(7) have previously
demonstrated that blockade of NF-B activation concomitantly inhibits
both IL-8 secretion and IL-8 mRNA accumulation. Thus our results indicate
that, whereas NF-B regulates IL-8 transcription, the p38 MAPK pathway
acts downstream, and perhaps parallel, of this process. These observations are
consistent with the recent report that activation of p38 is able to increase
the efficiency of translation of genes that contain 3' untranslated
AU-rich elements (14).
Whereas we and others (5, 7) have previously observed a very strong correlation between levels of IL-8 secretion and IL-8 mRNA, it seems expedient for cells, particularly intestinal epithelial cells to exert an additional level of regulation over such a gene. While IL-8 plays an important role in recruiting neutrophils that clear mucosal infections, these neutrophils are also largely responsible for inducing the clinical manifestations of self-limiting, locally contained, infections such as human Salmonellosis. Thus intestinal epithelial cells would seek to recruit polymorphonuclear neutrophils as soon as possible once a pathogen is detected but also shut down continued neutrophil recruitment once the perturbing agonist is cleared. Having p38 MAPK regulate translation would seem to provide a means to boost translation rates and, conversely, shut off production more quickly than would be possible for a gene whose expression was controlled solely at the transcriptional level. Such rapid control would seem to prevent systemic infection and the uncontrolled inflammation that characterizes some chronic diseases.
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DISCLOSURES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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K. J. Wright, P. C. Seed, and S. J. Hultgren Uropathogenic Escherichia coli Flagella Aid in Efficient Urinary Tract Colonization Infect. Immun., November 1, 2005; 73(11): 7657 - 7668. [Abstract] [Full Text] [PDF]
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 M. Rimoldi, M. Chieppa, P. Larghi, M. Vulcano, P. Allavena, and M. Rescigno Monocyte-derived dendritic cells activated by bacteria or by bacteria-stimulated epithelial cells are functionally different Blood, October 15, 2005; 106(8): 2818 - 2826. [Abstract] [Full Text] [PDF]
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A. M. Torok, A. H. Bouton, and J. B. Goldberg Helicobacter pylori Induces Interleukin-8 Secretion by Toll-Like Receptor 2- and Toll-Like Receptor 5-Dependent and -Independent Pathways Infect. Immun., March 1, 2005; 73(3): 1523 - 1531. [Abstract] [Full Text] [PDF]
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 R. T. M. Boudreau, D. W. Hoskin, and T.-J. Lin Phosphatase inhibition potentiates IL-6 production by mast cells in response to Fc{varepsilon}RI-mediated activation: involvement of p38 MAPK J. Leukoc. Biol., November 1, 2004; 76(5): 1075 - 1081. [Abstract] [Full Text] [PDF]
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