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B in the Clostridium difficile Toxin A-Induced Up-Regulation and Secretion of IL-8 from Human Monocytes1
Departments of
*
Medicine and
Microbiology, Health Sciences Center, University of Virginia, Charlottesville, VA 22908
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B and AP-1, but not NF-IL-6. NF-B
inhibitors blocked the ability of toxin A to induce IL-8 secretion, and
supershift analysis indicated that the major isoform of NF-B
activated by the toxin is a p50-p65 heterodimer. This study is the
first to identify intracellular signaling pathways and transcription
factors involved in the C. difficile toxin-mediated
up-regulation of IL-8 synthesis and release by target cells. This
information should increase our understanding of the pathogenesis of
C. difficile colitis and the nature of IL-8 gene regulation
as well. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An intense inflammatory response with a marked neutrophil accumulation
is a key characteristic of the clinical pathophysiology of CDAD
(3, 19). Although inflammation is an important arm of the
immune system and represents an early line of defense against many
potential pathogens, unregulated inflammation can contribute to the
destruction of local host tissues, a phenomenon that may be relevant to
certain aspects of CDAD. The chemokine IL-8 plays an important role in
the pathogenesis of neutrophilic types of inflammation. Indeed, stools
and colonic mucosal biopsies from patients with C. difficile
colitis contain elevated levels of IL-8, implicating a role for this
chemokine in the pathophysiology of CDAD (20). IL-8 is a
CXC-type chemokine that binds to the cellular seven-transmembrane
domain G protein-coupled receptors known as CXCR1 and CXCR2
(21). IL-8 is a potent proinflammatory chemotactic factor
that predominantly exerts its effects on neutrophils (22, 23). Because IL-8 is such a powerful cytokine, its extracellular
release must be tightly regulated. In most cell types, transcriptional
control of IL-8 expression appears to be the most important regulatory
mechanism (24, 25, 26). The proximal 135 bp of the 5'-flanking
region of the IL-8 gene contains four elements known to be involved in
transcriptional regulation: AP-1, CCAAT/enhancer-binding protein
(C/EBP/NF-IL-6) and NF-B sites up-regulate transcription, and an
Octamer-1 (Oct-1) element that can repress transcription of the gene
(26, 27).
NF-B was originally identified as the transcription factor that
activates the promoter of the region of the Ig light chain locus in
B lymphocytes (28). Since its initial discovery,
investigators have demonstrated its involvement in the positive
regulation of a number of other genes including those for IL-8, IL-2,
IL-6, monocyte chemoattractant protein-1, G-CSF, TNF-
, the enzymes
inducible nitric oxide synthase and cyclooxygenase-2, the adhesion
molecules ICAM-1 and E-selectin, and its own repressor, IB
(28). NF-B exists as a family of homodimers and
heterodimers composed of different combinations of proteins
belonging to the NF-B/Rel family (29). In most cells,
NF-B is normally complexed with its inhibitor protein, IB. IB
constrains NF-B to the cytoplasm by masking its nuclear localization
signal (30, 31). Active NF-B is transported to the
nucleus after IB is phosphorylated by IB-kinase and degraded by
the proteasome complex (32).
C. difficile toxin A induces IL-8 secretion from a large
variety of cell types, including peripheral blood monocytes and the
monocyte cell line THP-1 (15), as well as the intestinal
epithelial cell lines HT-29 and T-84 (14, 33). Because the
mechanism through which toxin A induces IL-8 secretion is unknown, we
sought to characterize the nature of IL-8 up-regulation induced by
toxin A in human monocytes. In this study, we found that the induction
of IL-8 synthesis and secretion requires cellular uptake of toxin A and
cannot be stimulated by a recombinant toxin fragment containing only
the cell-binding domain. We also determined that toxin A induces IL-8
expression at the level of gene transcription and that this occurs
through a mechanism that requires intracellular calcium and likely
involves a calmodulin-sensitive step. Toxin A-induced IL-8 secretion
was partially attenuated by the tyrosine kinase inhibitor genistein,
but was unaffected by inhibitors of protein kinase C (PKC) and
phosphatidylinositol-3 kinase (PI-3 kinase). In addition, we determined
that toxin A activates nuclear translocation of NF-B and AP-1.
Pharmacological inhibition of the activation of NF-B inhibited the
ability of toxin A to induce IL-8 secretion, suggesting an important
role for NF-B in this process. Supershift experiments indicated that
the isoform of NF-B activated by toxin A is the p50-p65 heterodimer.
These findings characterize an integral part of the mechanism of toxin
A-induced IL-8 secretion and serve as a model to further our knowledge
of the inflammatory response to enteric pathogens. Understanding the
mechanism through which toxin A induces a pathological inflammatory
response in CDAD may help lead to the development of a novel
nonantibiotic approach to therapy of this disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human PBMC were isolated from whole blood or buffy coat
fractions obtained from normal volunteers by density centrifugation
using Ficoll Histopaque 1077 (Sigma, St. Louis, MO). The PBMC were
enriched for monocytes by adherence to plastic tissue culture dishes
for 90 min at 37°C and used predominantly for the IL-8 assays
(according to the method of Linevsky et al.) (15). Based
on Giemsa staining, >80% of cells purified by this technique were
determined to be monocytes. For experiments requiring large numbers of
cells (e.g., gel-shift and supershift assays), monocytes were isolated
from PBMC using a CD-14 magnetic bead column-based, positive cell
selection technique (Miltenyi Biotec, Auburn, CA) and were determined
to be >90% monocytes. The two techniques for monocyte isolation
yielded identical results with respect to gel-shift and IL-8 assays.
Monocytes were allowed to rest in RPMI 1640 (Life Technologies, Grand
Island, NY) supplemented with 10% FBS, 100 U/ml penicillin, and 100
µg/ml streptomycin overnight before use. The discarded nonadherent or
column pass-through cells were predominantly lymphocytes and these
cellular fractions exhibited negligible or nondetectable IL-8 release
and activation of NF-B in response to toxin A (data not shown).
Toxins
Purified toxin A and TcdA.3 were gifts from D. Lyerly and T. Wilkins (Techlab, Blacksburg, VA). Several different lots of purified toxin A were used. Each was found to be free of LPS contamination and of equivalent activity. TcdA.3 is a recombinant GST-fusion polypeptide comprising the cell-binding domain of toxin A. GST alone did not have any effect on IL-8 production or EMSA results (data not shown). Because TcdA.3 is prepared from Escherichia coli, the preparations contained variable amounts of LPS as assayed by the E-TOXATE Limulus test (Sigma). For our studies, a polymyxin B agarose column (Sigma) was used to remove LPS from the TcdA.3 preparations prior to use. Different lots of TcdA.3 produced similar results.
IL-8 ELISA
Monocytes at 106 cells/ml were stimulated with PBS
alone or containing various concentrations of toxin A or TcdA.3 for
4 h at 37°C. In some experiments the cells were preincubated
with vehicle control or various inhibitors for 30 min prior to the
addition of toxin A. Compounds from Calbiochem (San Diego, CA) were
used at the following final concentrations: EDTA (2 mM),
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
tetra(acetoxymethyl) ester (BAPTA/AM) (30 µM), chloroquine (0.1 mM),
NH4Cl (20 mM), sulfasalazine (2 mM), curcumin (20 µM),
wortmannin (100 nM), calpastatin (200 nM), genistein (26 µM), and
bisindolylmaleimide (100 nM). The inhibitor W-7
(N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide
hydrochloride) was from Sigma and was used at a final concentration of
20 µM. For experiments using immobilized toxins, 10 µg of LPS,
toxin A, or TcdA.3 in 50 µl of binding buffer (1.5%
NaHCO3, pH 9.6) was added to 96-well Immulon IV plates
(Dynex, Chantilly, VA) overnight at 37°C, as previously described
(34). The wells were then washed twice with PBS to remove
unbound protein, and monocytes were incubated in the wells for 4
h. Cell supernatants were analyzed for IL-8 using the Quantikine ELISA
for human IL-8 (R&D Systems, Minneapolis, MN) or the IL-8 EASIA
(BioSource International, Camarillo, CA); results were equivalent using
either kit. The values displayed in Figs. 1
A, 2, 4, 5, 6,
and 7 represent the mean and SDs from representative experiments as
described in the figure legends.
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Unfractionated RNA was purified from monocytes and was analyzed
by slot blotting and Northern hybridization essentially as described
previously (35). A specific 30-base ssDNA oligonucleotide
(Operon, Alameda, CA) corresponding to a region in the 5'-untranslated
region of IL-8 mRNA was end-labeled with [
-32P]ATP
(ICN, Costa Mesa, CA). Using the conditions outlined here, this probe
recognizes a single band corresponding to IL-8 mRNA in fractionated
cellular RNA (Ref. 35 , and data not shown). Briefly, total mRNA was
extracted from monocytes using RNA STAT-60 (Tel-Test, Friendswood, TX),
and 15 µg of RNA was immobilized on a Nytran+ (Schleicher
& Schuell, Keene, NH) membrane using a slot-blot apparatus (Life
Technologies). The blot was hybridized overnight at 60°C, washed in
1x SSC, 0.1% SDS, and autoradiographed at -80°C for 24 h. The
results were quantified by densitometric analysis (NIH-Image computer
program) (36). Fig. 1
B displays results
representative of three separate experiments.
Nuclear extracts
Nuclear extracts were prepared essentially as previously
described by Smith et al. (37). Monocytes at
107 cells/ml were stimulated with 10 µg/ml LPS or various
concentrations of toxin A for 30 min at 37°C. A time course indicated
that maximal NF-B activity was observed at 30 min after the addition
of toxin A (data not shown). In some experiments toxin A was
preincubated with a neutralizing toxin A affinity-purified polyclonal
goat antiserum (a gift from T. Wilkins, VPI, Blacksburg, VA) for 30 min
prior to addition of the toxin to monocytes (34). In other
experiments, monocytes were preincubated with the NF-B inhibitors
sulfasalazine (2 mM) or curcumin (20 µM) for 30 min prior to toxin A
treatment. Nuclear extracts were prepared as previously described and
either used immediately for EMSA or stored at -80°C for future use
(37).
EMSA and supershift assays
EMSA were performed essentially as described by Smith et al.
(37). Consensus NF-B or AP-1 oligonucleotide probes
were from Promega (Madison, WI) and the NF-IL-6 probe
5'-TCGATCAGTTGCAAATCGT-3' was custom synthesized (IDT, Coralville, IA).
All probes were end-labeled with [-32P]ATP (ICN).
Reaction mixtures consisted of 2 µg of nuclear extract, binding
buffer (20 mM HEPES, 1 mM DTT, 0.1 mM EDTA, 50 mM KCl, 5% glycerol,
200 µg/ml BSA), 1 µg poly(dI-dC) (Sigma), and 0.05 µg of
sonicated salmon sperm (Stratagene, La Jolla, CA). Anti-Rel Abs for
supershifts were purchased from Santa Cruz (Santa Cruz, CA): -p50
(sc-114), -p65 (sc-109), -cRel (sc-70), and -RelB (sc-226); 2
µl were added per sample. Ten nanograms of cold NF-B or AP-1
consensus probe were added to the control samples as specific and
nonspecific competitors. The reactions were incubated on ice for 30
min, 1 ng of 32P-labeled probe was added, incubated for
10 min at room temperature, and immediately subjected to nondenaturing
4% PAGE. The gels were dried and autoradiographed at -80°C for
18 h. The images were analyzed semiquantitatively by densitometry
using the NIH-Image computer program. Figs. 8, 9, 10, and 11 display
representative experiments as described in the figure legends.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Linevsky et al. previously demonstrated by RT-PCR that toxin A
up-regulates IL-8 gene expression (15). We sought to
confirm and extend this finding and to determine whether or not
transcriptional up-regulation of the IL-8 gene is the predominant
mechanism through which toxin A induces IL-8 secretion in monocytes.
Pretreatment of monocytes with the transcriptional inhibitor
actinomycin D completely abolished the ability of toxin A to induce
IL-8 secretion (Fig. 1A),
indicating that IL-8 secretion is dependent upon transcription. To
further characterize the nature of the IL-8 response, we performed
Northern analysis on RNA isolated from monocytes treated with various
concentrations of toxin A. Minimal IL-8 message was detected in resting
monocytes. Toxin A treatment of monocytes, however, stimulated a
marked, dose-dependent increase in detectable IL-8 mRNA (Fig. 1B). By semiquantitative densitometric analysis we
determined that 0.1 nM toxin A resulted in an 
13-fold increase in
IL-8 message, and 1.0 nM and 10 nM toxin A induced 19- and
30-fold increases, respectively. Overall, these results indicate
that toxin A up-regulates IL-8 secretion primarily through
transcriptional up-regulation of the IL-8 gene in monocytes.
Toxin A-induced IL-8 secretion requires mobilization of intracellular Ca2+ and activation of calmodulin
Excluding effects on Rho GTPases, little is known about the
intracellular signaling pathways induced or altered by toxin A. Toxin A
has been reported to induce release of Ca2+ from
intracellular stores in neutrophils (38, 39) and rat
pancreatic acini (40). Because Ca2+ ionophores
have been shown to elicit IL-8 secretion from monocytes
(41), and an intracellular Ca2+ flux is
associated with induction of IL-8 secretion by certain signals, such as
IL-1 (42), we next sought to determine whether the effect
of toxin A on IL-8 secretion required mobilization of intracellular
Ca2+. We used EDTA to deplete extracellular
Ca2+ and used the cell-permeable agent BAPTA/AM to chelate
releasable intracellular stores of Ca2+. Monocytes
incubated in EDTA alone showed a decrease in the level of toxin
A-induced IL-8 secretion by approximately 40% compared with control
monocytes (Fig. 2A). When
monocytes were incubated in EDTA and BAPTA/AM, the ability of toxin A
to activate IL-8 secretion was completely inhibited (Fig. 2A). Because a major effect of intracellular
Ca2+ flux is the activation of calmodulin, we next examined
whether the inhibition of calmodulin could diminish toxin A-induced
secretion of IL-8. W-7 is a potent and specific inhibitor of calmodulin
activity and we observed that treatment with this agent abrogated
toxin-mediated IL-8 secretion by 83% (Fig. 2B). These
results indicate that both intracellular Ca2+ and
calmodulin are required for toxin A-induced IL-8 secretion in
monocytes.
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Toxin A binds to target cells through its carboxyl-terminal
repeating domain (Fig. 3), although the
identity of the human cell surface receptor is not yet known
(43). A recombinant GST-fusion polypeptide comprising this
cell-binding domain of toxin A has been reported to induce
intracellular Ca2+ flux in rabbit brush border epithelial
cells (43). Using this information in conjunction with our
observation that the toxin A-induced up-regulation of IL-8 secretion
contained a Ca2+-sensitive step, we next sought to
determine whether treatment of monocytes with the cell-binding domain
of toxin A alone (referred to as TcdA.3) could induce IL-8 secretion.
Monocytes were treated with either native holotoxin A or recombinant
TcdA.3 for 4 h. In agreement with a previous report
(15), we found that toxin A-induced IL-8 secretion is
dose-dependent with an optimal dose of 10-9 M (Fig. 4). Somewhat unexpectedly, however, we
found that the TcdA.3 fragment did not induce appreciable levels of
IL-8 production from monocytes (Fig. 4). Even after 24 h, TcdA.3
did not induce significant levels of IL-8 release (data not shown).
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In order to exert their cytotoxic effects, toxins A and B must be
internalized by the target cell (44, 45). Lysosomotropic
agents such as chloroquine and NH4Cl can inhibit
toxin-mediated cytotoxicity, suggesting that endosomal acidification is
involved in internalization (45). In order to determine
whether internalization of toxin A is also required for the
toxin-mediated induction of IL-8 secretion, we pretreated monocytes
with these agents prior to the addition of toxin A and observed that
both chloroquine and NH4Cl effectively inhibited toxin
A-induced secretion of IL-8 (Fig. 5A). We assessed the
importance of toxin internalization, using an additional approach.
Tissue culture plates were coated with toxin A or TcdA.3 to supply the
stimulus for IL-8 release. Monocytes that attach and spread on this
solid phase support containing toxin A would be able to bind, but not
internalize toxin A (46). As a positive control, we
observed that monocytes allowed to adhere to LPS-coated wells secreted
significant amounts of IL-8 (Fig. 5B). Monocytes adherent to
toxin A-coated wells, however, failed to secrete detectable IL-8. Taken
together these results strongly suggest that cellular uptake and
internalization through an endosome-like pathway is required for toxin
A to effectively induce IL-8 secretion from monocytes.
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The reversible phosphorylation of serine, threonine, and tyrosine
residues acts as a molecular switch to translate signals from the
extracellular environment into specific cell functions. Protein kinase
C (PKC) is a serine/threonine kinase that has been shown to play a role
in the induction of IL-8 secretion by Helicobacter pylori,
PMA, and IL-1ß (47, 48, 49). In addition, intracellular
Ca2+ flux activates "classical" forms of PKC. However,
we found that treatment of monocytes with the PKC inhibitor
bisindolylmaleimide did not diminish the effect of toxin A and, in
fact, actually caused a slight increase in basal and toxin-induced
levels of IL-8 release (Fig. 6).
Induction of IL-8 secretion by bisindolylmaleimide has been observed in
human synovial fibroblasts (50). Similarly, although
phosphatidylinositol-3 kinase (PI-3 kinase) is known to be an important
effector of a variety of intracellular signaling pathways in monocytes,
the PI-3 kinase inhibitor wortmannin did not block the up-regulation of
IL-8 by toxin A and seemed to cause an increase in the basal level of
IL-8 secretion (Fig. 6). IL-8 production in response to certain agents,
such as IL-1ß, has been reported to involve protein tyrosine kinase
(PTK) activity (49). When used in our system, we observed
that the tyrosine kinase inhibitor genistein did decrease toxin
A-induced release of IL-8 by about 75% (Fig. 6), indicating a
potential role for tyrosine kinase-dependent signaling pathways in the
up-regulation of IL-8 release stimulated by toxin A.
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B abrogate the toxin
A-mediated activation of IL-8 secretion
NF-B is a potent transcriptional activator of various
inflammatory mediators. For many types of stimuli, including certain
microbial pathogens, NF-B appears to be required for inducible
transcription of the IL-8 gene (51, 52). Therefore, we
next examined whether there was evidence for a role of NF-B in toxin
A-induced IL-8 secretion. Sulfasalazine and curcumin are well-described
inhibitors of NF-B activity in a variety of cell types. Each agent
is believed to act by a different mechanism: sulfasalazine inhibits
phosphorylation of IB (53), whereas curcumin
interacts directly with the p50 subunit of NF-B, thereby blocking
degradation of IB (54). Pretreating monocytes with
these agents, we observed that sulfasalazine completely abolished the
ability of toxin A to induce IL-8 release and curcumin markedly
diminished (by 75%) the effect of toxin A as well (Fig. 7). These results suggest that activation
of NF-B is required for the toxin-induced up-regulation of IL-8
expression.
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B and AP-1 but not
NF-IL-6
Based on our results using the NF-B inhibitors, it seemed
likely that toxin A would be able to activate and induce nuclear
translocation of NF-B. Activation and nuclear translocation of
NF-B can be detected with EMSA. Using a NF-B-specific probe to
perform EMSA we found that resting monocytes contain a low level of
nuclear NF-B (Fig. 8, lane
1). Within 30 min of stimulation with 1 nM toxin A, we observed a
roughly sixfold increase in the amount of nuclear NF-B
(lane 2). LPS induces a similar increase in the
amount of nuclear NF-B (lane 3). We confirmed the
specificity of the complex that was induced by toxin A through
competition analysis; a 10-fold excess of unlabeled NF-B
oligonucleotide resulted in a reduction of the predominant complex
induced by toxin A (lane 4), whereas a 10-fold excess
of an unrelated oligonucleotide (AP-1) failed to compete
(lane 5). The toxin A-induced NF-B activation
occurred in a concentration-dependent manner and could be blocked by
preincubation with a specific antitoxin A polyclonal Ab (data not
shown).
The IL-8 promoter contains three elements known to be involved in
transcriptional up-regulation. The NF-B element is believed to be
the main regulator of inducible expression of the IL-8 gene; however,
for certain signals, expression of the IL-8 gene has been reported to
depend upon activation of a second transcription factor, either AP-1 or
NF-IL-6, in addition to NF-B (51, 52). For example,
human cytomegalovirus-induced transcriptional up-regulation of the IL-8
gene is dependent upon both AP-1 and NF-B (51). We
therefore investigated the effect of toxin A on AP-1 and NF-IL-6. Using
an AP-1-specific probe, we found that the basal level of AP-1 in
monocytes was fairly high (Fig. 9,
lane 1), but we observed a modest, approximately twofold,
increase in the amount of nuclear AP-1 in toxin-treated nuclear
extracts (lane 2). We confirmed the specificity of
the complex detected by EMSA through competition analysis; a 10-fold
excess of unlabeled AP-1 oligonucleotide resulted in a reduction of the
complex induced by toxin A (lane 3), whereas a
10-fold excess of an unrelated oligonucleotide (NF-B) failed to
compete (lane 4). In contrast to the results observed
for NF-B and AP-1, we detected no significant increase in nuclear
localization of the transcription factor NF-IL-6 in response to toxin A
(data not shown).
Based on the results observed in the IL-8 experiments summarized in
Fig. 7, we predicted that pharmacological inhibitors of NF-B should
diminish the effect of toxin A on activation of NF-B as well.
Indeed, we found that sulfasalazine (Fig. 10, lane 4) and curcumin
(lane 6) were each able to markedly inhibit toxin
A-induced nuclear localization of NF-B. Overall, then, our results
indicate a central role for NF-B, likely in conjunction with AP-1,
in the up-regulation of IL-8 gene expression induced by toxin
A.
Toxin A activates the p50/p65 NF-B heterodimer
NF-B is actually a complex of homo- or heterodimers comprised
of members of the Rel family of proteins. The p50/p50 NF-B homodimer
lacks a transcriptional activation domain and, in some instances, acts
as a transcriptional repressor (55, 56). The p65 Rel
subunit possesses a transcriptional-activating domain, hence the
p50/p65 form of NF-B usually functions as a powerful inducible
transcriptional activator (55). In order to identify the
subunits present in the toxin A-activated isoforms of NF-B, we
performed supershift assays using Abs specific for each of the known
Rel proteins (Fig. 11). Abs to p50
(lane 5) and p65 (lane 6) induced a
partial shift in mobility, and a partial loss of the predominant
toxin-inducible NF-B complexes. When the p50 and p65 Abs were added
to extracts simultaneously, the toxin-induced NF-B complex was
almost completely shifted (lane 7). Abs to c-Rel
(lane 8) and Rel-B (lane 9) did not
induce a detectable shift or loss of the toxin-induced NF-B complex.
This result suggests that toxin A induces nuclear localization of the
p50/p65 heterodimer isoform of NF-B.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Intracellular calcium flux is an important second messenger that plays
a role in a diverse array of cellular processes, including nuclear
signaling. Certain extracellular signals can induce rapid mobilization
of intracellular stores and/or promote influx of extracellular
Ca2+. Ca2+ pumps revert the level of cytosolic
Ca2+ to its normal state and the resulting Ca2+
oscillations produced by this on/off mechanism can impart specificity
of the signal as it is translated by the cell (59).
Signaling through Ca2+ mobilization has been implicated in
the activation of NF-B as well as the up-regulation of IL-8 by
stimuli such as NO and thapsigargin (42, 59, 60). Our
findings implicate key roles for intracellular Ca2+ flux
and calmodulin in the activation of IL-8 release induced by toxin A.
Other investigators have reported mobilization of intracellular
Ca2+ by toxin A (38, 39, 40, 43). Our study,
however, is the first to link an intracellular Ca2+ signal
to any of the known cellular effects of toxin A. It is unlikely that
Ca2+ and calmodulin are required for binding or
internalization of toxin A because a previous study demonstrated that
they are not necessary for toxin A-mediated cytotoxicity in rat
intestinal crypt cells (61). In addition toxin-induced
cytotoxicity is known to be dependent on cellular internalization of
the toxin (44, 45). In our study, complete inhibition of
the effects of toxin A required both EDTA and BAPTA/AM, indicating that
mobilization of intracellular Ca2+ and influx of
extracellular Ca2+ are each required for effective
activation of IL-8 gene transcription. In addition, the specific
calmodulin inhibitor W-7 abrogated IL-8 secretion, suggesting that the
effect of toxin A requires the activation of calmodulin. Together,
these results strongly implicate the involvement of an intracellular
Ca2+/calmodulin-dependent signaling pathway in toxin
A-induced secretion of IL-8. Potential roles for Ca2+ and
calmodulin may involve the regulation of endosomal maturation and
activation of kinases involved in the pathway NF-B signaling
pathway.
Our study also demonstrates that the carboxyl-terminal repeating portion of toxin A alone (TcdA.3) was not able to induce IL-8 secretion from monocytes. In some ways, this result was unexpected as TcdA.3 has been reported to be sufficient to induce intracellular Ca2+ flux in neutrophils and rabbit brush border epithelial cells (42, 43). One possible explanation is that the toxin A-dependent signaling pathways are different for monocytes as compared with other cell types. Alternatively, the binding portion may actually direct the signaling necessary to induce the IL-8 response, but the recombinant fusion protein, TcdA.3, does not retain the proper signaling conformation. Another possibility is that a signal in addition to Ca2+ mobilization is required to induce IL-8 expression, a signal that is transduced by the holotoxin, but not the TcdA.3 fragment. If this is the case, then some other portion of the toxin molecule may be required to up-regulate expression of IL-8. We also found that internalization and endosomal acidification are necessary prerequisites to toxin-mediated IL-8 secretion. Barroso et al. used deletional analysis of the toxin B gene to determine that a 50-amino acid hydrophobic portion of the middle domain of the toxin is involved in toxin internalization (62). It is therefore possible that TcdA.3 lacks IL-8-inducing activity because it is not properly internalized without the central domain.
Transcriptional activation of cytokine genes such as IL-8 occurs when a
stimulus interacts with the cell in such a way that its message is
relayed to the transcription factors directly responsible for
transcriptional activation of the gene. This signal transduction
pathway generally involves the sequential activation of a cascade of
intracellular protein kinases. In an attempt to characterize the
signaling pathway through which toxin A induces IL-8 secretion in
monocytes we employed inhibitors for three important families of
kinases: PKC, PI-3 kinase, and protein tyrosine kinase (PTK).
Bisindolylmaleimide is a selective inhibitor active against PKC-,
-ßI, -ßII, -, -
, and -
isoforms
(63). We found that the treatment of monocytes with this
agent did not inhibit toxin A-induced IL-8 secretion. In agreement with
this finding, Hippensteil et al. reported that toxin B does not
activate PKC and may actually inhibit PMA-mediated PKC activity
(64). We also found that the PI-3 kinase inhibitor
wortmannin was unable to block toxin A-mediated IL-8 secretion. In
support of this finding, it has been reported that toxin B does not
up-regulate PI-3 kinase in monocytes and actually inhibits LPS-induced
PI-3 kinase activation (65). The PTK inhibitor genistein,
however, did abrogate toxin A-induced IL-8 secretion in this study.
Precedent exists for this finding, as the PTK inhibitor erbistatin was
found to inhibit toxin A-induced secretion in rabbit ileal loops
(R. L. Guerrant, University of Virginia, personal communication)
and PTKs have been implicated in IL-1ß- and H.
pylori-induced IL-8 secretion (49, 66). This report,
however, is the first to implicate a PTK-sensitive pathway for a
C. difficile toxin-mediated cellular effect.
Activation of NF-B by enteric bacterial pathogens such as H.
pylori and enteropathogenic E. coli has been reported
by others, but our report is the first to demonstrate the activation of
this important proinflammatory transcription factor by toxins from
C. difficile (67, 68). We found that toxin A
induces rapid nuclear translocation of the p50/p65 isoform of NF-B
in monocytes. In addition, our findings that the NF-B inhibitors
sulfasalazine and curcumin abrogate toxin-mediated IL-8 secretion
suggests that NF-B plays a functional role in this process and is
not a simple concurrent but unrelated phenomenon. The time course of
the toxin-mediated activation of NF-B in relation to IL-8 secretion
also supports this hypothesis. Our data are consistent with the
observations that, for most stimuli, the activation of NF-B is
essential for inducible expression of the IL-8 gene and, in some
instances, has been noted to occur following Ca2+ flux and
preceding IL-8 production (26, 54, 59).
Even though NF-B appears to be the most important transcription
factor for inducible expression of the IL-8 gene, most stimuli must
also activate AP-1 or NF-IL-6 to elicit IL-8 expression. Our study
demonstrates that AP-1 is also activated by toxin A. Since certain
kinases such as MEKK-1 are common to the pathways leading to activation
of NF-B and AP-1, some stimuli including TNF- can induce both
transcription factors (69). Furthermore, several stimuli
such as human cytomegalovirus have been shown to induce IL-8 expression
through the activation of both NF-B and AP-1 (51).
In summary, C. difficile toxin A up-regulates IL-8
expression at the transcriptional level and intracellular
Ca2+/calmodulin, a PTK, and the transcription factor
NF-B play important roles in this effect. Cell surface binding of
toxin A is not sufficient to induce IL-8 secretion; internalization
appears to be required. The activation of NF-B by toxin A may play
other roles in addition to the expression of IL-8 because NF-B is
also involved in the transcriptional activation of other cytokines
known to be induced by toxin A, including TNF-, IL-1, and IL-6,
(13, 16). Current therapy for CDAD requires treatment with
the antibiotic vancomycin or metronidazole although relapse occurs in
20% of cases (70). Because antibiotic therapy inhibits
reconstitution of normal colonic flora it may contribute to the high
rate of CDAD recurrences. Our finding that NF-B plays a role in the
toxin A-mediated neutrophilic inflammatory response could potentially
provide a target for the development of novel nonantibiotic treatment
for CDAD. In addition, further investigation of the biochemical
pathways leading to the toxin A-mediated up-regulation of IL-8 may
uncover novel aspects of the nature of IL-8 gene expression itself.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David Bobak, Box 485, Health Sciences Center, University of Virginia, Charlottesville, VA 22908. E-mail address:
3 Abbreviations used in this paper: CDAD, C. difficile-associated disease; PKC, protein kinase C; PTK, protein tyrosine kinase; PI-3 kinase, phosphatidylinositol-3 kinase; BAPTA/AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester; TcdA.3, cell-binding domain of toxin A.
Received for publication July 8, 1999. Accepted for publication August 25, 1999.
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