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J. Biol. Chem., Vol. 277, Issue 25, 22414-22420, June 21, 2002
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From the Department of Microbiology and Immunology, Wake Forest
University School of Medicine,
Winston-Salem, North Carolina 27157
Received for publication, February 21, 2002, and in revised form, April 5, 2002
Flagellin from a number of Gram-negative
bacteria induces cytokine and nitric oxide production by inflammatory
cell types. In view of the evidence that flagellin responsiveness is
subject to modulation, we explored the possibilities that a prior
exposure to flagellin might result in a state of reduced flagellin
responsiveness or tolerance and that lipopolysaccharide (LPS) and
flagellin may induce a state of cross-tolerance to each other. Our
results demonstrate that a prior exposure to flagellin results in a
subsequent state of flagellin tolerance in human monocytes, THP1 cells,
Jurkat cells, and COS-1 cells. Tolerance occurs within 2 h after
addition of flagellin and does not require protein synthesis. Flagellin did not induce tolerance to LPS in monocytes and THP1 cells; however, LPS treatment of monocytes and THP1 cells resulted in a state of
flagellin cross-tolerance. Flagellin-induced self-tolerance is not the
result of a decrease in the steady-state level of toll-like receptor 5 (TLR5) or interleukin-1 receptor associated kinase (IRAK), but it is
associated with a block in the release of IRAK from the TLR5 complex in
flagellin-tolerant cells. Release is essential for IRAK activity
because the TLR5-associated IRAK lacks kinase activity. LPS-induced
cross-tolerance to flagellin is also associated with a block in IRAK
release from TLR5. These results provide evidence for a novel mechanism
of TLR signaling control.
Evidence from a large number of studies demonstrates the
importance of cytokine production in the protective response against Gram-negative pathogens. We (1, 2) and others (3) reported that
flagella from Gram-negative bacteria such as Salmonella
enteritidis and Pseudomonas aeruginosa induce cytokine
production (e.g.
TNF- Flagellin, like lipopolysaccharide (LPS) (13-16), signals via a
toll-like receptor (TLR)/IL-1 receptor-associated kinase
(IRAK)-dependent pathway (5, 8, 17). LPS signals via TLR4,
whereas flagellin utilizes TLR5 (8, 17). The activation of IRAK by
flagellin is a relatively rapid process; maximal activation of
IRAK activity in human and murine monocytes occurs within 5-10
min after the addition of flagellin to the cells (5).
In vitro studies with cultured monocytes and neutrophils as
well as analysis of these cell types from patients with Gram-negative sepsis have established that prior exposure to LPS induces a transient state of cellular tolerance to subsequent stimulation by LPS (see e.g. Refs. 18-20). LPS tolerance is present within hours
after an initial exposure to LPS (21-24). LPS tolerance may have
evolved as a mechanism to limit the mediator storm that is induced by LPS and which is responsible for the pathologic events associated with
LPS-induced septic shock. Because of the potential importance of LPS
tolerance in the host response to Gram-negative infection, the
underlying mechanism(s) that govern the induced state of LPS tolerance
continues to be a subject of intense investigation. Although the
mechanism for this effect has not been established definitively,
evidence has been obtained and arguments made in favor of a role for a
labile repressor (22), secretory leukocyte protease inhibitor (25), the
down-regulation of TLR4 expression (26) and decreased IRAK expression
(27). Investigators have also analyzed the potential for LPS,
lipoarabinomannan (from mycobacteria) and lipopeptides (from
mycoplasma) to induce a state of cross-tolerance to each other. The
available evidence indicates that LPS can induce tolerance to several
unrelated inducers including lipoarabinomannan (28) mycoplasma
lipopeptides (29) and IL-1 (20), all of which utilize
TLR-dependent signaling pathways.
In view of the evidence that flagellin responsiveness is subject to
modulation (2) and is TLR5/IRAK-dependent (5, 8, 17), we
explored the possibilities that a prior exposure to flagellin would
result in a state of reduced flagellin responsiveness and that LPS and
flagellin may induce a state of cross-tolerance to each other.
Cells and Reagents--
THP1 cells were obtained from the
American Type Tissue Culture Collection. Jurkat cells (a human T cell
line) constitutively expressing the SV40 large T antigen were kindly
provided by Dr. Amnon Altman, La Jolla Institute for Allergy and
Immunology, San Diego. These cell lines were maintained in RPMI 1640 containing 10% fetal bovine serum and 20 µg/ml gentamicin.
COS-1 cells were obtained from Dr. Gregory Shelness, Wake Forest
University School of Medicine, and were grown in Dulbecco's modified
Eagle's medium with 10% fetal bovine serum and gentamicin. Human
peripheral blood monocytes were prepared from the blood of healthy
volunteers as described previously (2). Purified, endotoxin-free
recombinant flagellin was prepared as described previously (4). Rabbit antibodies against human TLR5 were prepared using two TLR5 peptides (residues 463-476 and 831-846) linked to keyhole limpet hemocyanin. Serum was obtained from the rabbit before immunization for use as a
control for antibody specificity. Affinity-purified goat anti-TLR5 was
purchased from Santa Cruz Biotechnology. Rabbit anti-IRAK antibody was
obtained from Upstate Biotechnology Co. and anti-FLAG antibody from Sigma.
Plasmids--
A wild-type IRAK cDNA (pcDNA3-IRAK) was
kindly provided by Glaxo Smith Kline. Because the human TLR5 gene does
not contain any introns, the coding region was cloned by PCR using
RPCI-11 human male BAC clone 18_A-_13 and appropriate primers. The
resultant PCR product was cloned into the p3XFLAG-CMV-14 expression
vector (Sigma). The pNF- Measurement of TNF- Western Blots and Analysis of IRAK Activity--
IRAK activity
was measured as described by Li et al. (27) with
modification. Cells (2.5-5 × 106/sample) were
incubated with or without stimulation for varying periods before lysis
in immunoprecipitation buffer containing 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.4% Nonidet P-40, 10% glycerol, 2 mM EDTA, 1 mM EGTA, 10 µg/ml soybean trypsin
inhibitor, 100 µg/ml leupeptin, 14 µM pepstatin A, 3 mM benzamidine, 1 × protease inhibitor mixture
(Calbiochem), 5 µM sodium orthovanadate, and 1 µM microcystin-LR. The resultant lysates were centrifuged
to remove particulate material and then incubated with anti-IRAK antibody and protein G-agarose (Invitrogen) for 2 h at 4 °C.
The immunoprecipitates were washed several times with
immunoprecipitation buffer and then with the IRAK buffer (20 mM Tris-HCl, pH 7.5, 20 mM Mg2Cl,
20 mM Transient Transfections and NF- Analysis of TLR5 Expression by Flow Cytometry--
Control and
flagellin-tolerant Jurkat cells were washed with 2% fetal bovine serum
in phosphate-buffered saline and then incubated with anti-TLR5 antibody
for 30 min at 4 °C. The cells were then washed, incubated for an
additional 30 min at 4 °C with a fluorescein isothiocyanate-conjugated rabbit antibody against goat IgG (Jackson ImmunoResearch Laboratories), washed, and fixed prior to analysis using
a Becton-Dickinson FACSCalibur flow cytometer.
Exposure of Cells to Flagellin Induces a Subsequent State of
Flagellin Tolerance--
We began by determining whether a primary
incubation of human monocytes or THP1 cells with flagellin would result
in a subsequent state of flagellin tolerance. In addition, we evaluated
the potential ability of flagellin and LPS to induce tolerance to each
other. Human monocytes and THP1 cells were incubated with or without 10 IRAK Kinase Activity and Protein Levels during Flagellin
Tolerance--
In view of these initial results with flagellin and the
observation that LPS tolerance may be linked, at least in part, to a
decrease in the level and activity of the IRAK (27), we analyzed the
effect of a primary exposure to flagellin or LPS on the subsequent activation of IRAK in THP1 cells. THP1 cells were incubated overnight with or without flagellin or LPS, washed, and then incubated in the
presence or absence of flagellin for 10 min or LPS for 30 min. As
expected, flagellin (Fig. 1A,
second lane, 0/F) (5) and LPS (third lane, 0/L)
(27) induced IRAK activation as measured by phosphorylation of myelin
basic protein. Quantitative analysis of the kinase results are shown in
Fig. 2 (black bars). In line with the TNF-
In confirmation of the results of Li et al. (27), a prior
exposure to LPS resulted in a marked decrease in the level of the IRAK
protein (Fig. 1B, seventh lane, and Fig. 2,
gray bars). Note that the lower prominent band in
Fig. 1B is the nonphosphorylated form of IRAK,
whereas the upper band is the phosphorylated form of the
protein. In contrast to the results with LPS, a prior exposure to
flagellin (fourth lane, F/0) did not result in a decrease in IRAK protein levels. However, the combination of overnight exposure to
flagellin and a 15-min incubation with LPS did result in a decrease in
the level of nonphosphorylated IRAK (sixth lane, F/L). However, there was clearly sufficient IRAK in the cells to obtain a
level of active IRAK which was similar to that obtained with cells
receiving only a single exposure to flagellin or LPS (Fig. 1A, second and third lanes). Because
the block in flagellin-induced IRAK activation occurs without any
evident change in IRAK protein expression, it seemed likely that the
level of IRAK protein is not a contributing factor in the induction of
flagellin tolerance. In addition, we concluded from these experiments
that LPS and flagellin tolerance may be mediated, at least in part, by
distinct mechanisms.
Flagellin Tolerance Is Not Restricted to Monocytes--
To
determine whether flagellin tolerance is restricted to cells of the
monocytic lineage, we determined whether this condition could be
induced in Jurkat cells, a human T cell line. Using an NF- Time Course for the Onset of Flagellin Tolerance--
To determine
the minimal time required for the onset of flagellin tolerance, we
stimulated Jurkat cells with flagellin for 4, 12, 18, and 24 h
prior to a second 15-min exposure to flagellin. After the second
incubation, the level of IRAK activity was assessed. Control cells were
incubated with flagellin for a single 15-min period before the
measurement of IRAK activity. As shown in Fig. 3A, flagellin tolerance was
maximal within 4 h after an initial exposure to flagellin and was
sustained over the ensuing 24 h. In view of the relatively rapid
onset of flagellin tolerance, we next determined whether protein
synthesis was required. Jurkat cells were incubated in the presence or
absence of 10 µg/ml cycloheximide for 15 min before a subsequent 2- or 4-h incubation in the presence of flagellin. The cells were then
washed and incubated with flagellin for 15 min before the measurement
of IRAK activity. Control cells were incubated with or without
flagellin for a single 15-min period. As presented in Fig.
3B, cycloheximide treatment did not block the onset of
flagellin tolerance. Furthermore, these results also demonstrate that
flagellin tolerance occurs as early as 2 h after an initial
exposure to the protein.
Lack of an Effect of High Level IRAK Expression on Flagellin
Tolerance--
Although the results presented in Fig. 1 are consistent
with the notion that flagellin tolerance is not caused by a decline in
IRAK expression, we tested this hypothesis by transiently transfecting Jurkat cells with the NF- Flagellin Tolerance and TLR5 Expression--
In addition to a
decrease in IRAK expression, LPS tolerance has also been associated
with the down-regulation of TLR4 expression (26). Because flagellin
tolerance might simply be the result of the inhibition of TLR5
expression, we analyzed the steady-state level of TLR5 in control and
flagellin-tolerant cells using flow cytometry. Jurkat cells were
incubated overnight in the presence or absence of 10 TLR5/IRAK Association during Flagellin
Tolerance--
In view of the observations that flagellin tolerance
does not involve changes in the level of TLR5 or IRAK, we evaluated the possibility that flagellin tolerance may develop because of a reduced
ability of IRAK to associate with the TLR5 receptor complex. Previous
work has demonstrated that the association of IRAK with the IL-1
receptor complex is dependent on stimulation of cells with IL-1
(34-36). Similarly, the association of IRAK with TLR2 appears to be
dependent on cellular stimulation (37). We began by assessing TLR5-IRAK
association in TLR5-positive, flagellin-responsive COS-1 cells that
were transiently transfected with TLR5-FLAG and IRAK expression
plasmids. The cells were transfected, rested for 24 h, and then
incubated overnight in the presence or absence of flagellin.
Subsequently, the cells were washed and then incubated with flagellin
for 0-30 min. Cell lysates were prepared, and the TLR5-FLAG was
immunoprecipitated with anti-FLAG antibody. After electrophoresis and
transfer of proteins to PVDF membranes, the membranes were probed for
the presence of IRAK using an anti-IRAK antibody. The blots were then
stripped and reprobed for TLR5 using the anti-FLAG antibody. In all
cases, the level of TLR5 was the same in each lane (data not shown). In
marked contrast to the earlier studies with the IL-1 receptor or TLR2,
IRAK was associated with TLR5 in unstimulated control cells (Fig.
5A). Stimulation of the
cells with flagellin for 0.5-30 min did not dramatically change the
level of associated IRAK. Similar results were obtained with
flagellin-tolerant COS-1 cells. In all cases, the TLR5-associated IRAK
migrated in the gel in a manner consistent with the phosphorylated form
of the protein (IRAK-P). The migration of this band was shifted after
treatment with the serine/threonine-protein phosphatase 2A,
i.e. the protein phosphatase 2A-treated IRAK migrated more rapidly that IRAK-P as a broad band, the majority of which migrated with the nonphosphorylated form of the protein (data not shown).
In view of the earlier studies on IRAK association with the IL-1
receptor and TLR2, we were concerned that the association of IRAK with
the TLR5 receptor complex in unstimulated cells might be the result of
the overproduction of IRAK in transfected cells. Therefore, we assessed
the association of endogenous IRAK with TLR5 in cells transiently
transfected with only TLR5-FLAG. As shown in Fig. 5B,
endogenous IRAK was associated with TLR5 in unstimulated control and
tolerant cells, and stimulation did not appear to change the level of
TLR5-associated IRAK dramatically. The constitutive association of IRAK
with TLR5 was also observed in untransfected THP1 cells (see Fig.
7).
The results presented in Fig. 5 raised the possibility that flagellin
signaling via TLR5 might be different from the process observed with
the IL-1 receptor or TLR2. Perhaps IRAK activation and signaling in
response to flagellin is not dependent on IRAK release from TLR5. To
evaluate this possibility, we took advantage of the observation that
TLR5/IRAK complexes are very poorly immunoprecipitated with the
anti-IRAK antibody.2
Apparently, the epitope recognized by the anti-IRAK antibody is not
accessible when the protein is associated with the TLR5 complex.
Therefore, we prepared cell lysates from unstimulated and
flagellin-stimulated THP1 cells and incubated the lysates with
anti-IRAK antibody (Fig. 6,
left and middle lanes) or anti-TLR5 antiserum
(right lane). The resultant immunoprecipitates were then
assayed for kinase activity using myelin basic protein as the
substrate. As expected, anti-IRAK immunoprecipitates from flagellin-stimulated cells exhibited a higher level of IRAK activity than samples from unstimulated cells (left and middle
lanes). However, there was little if any IRAK activity in the
immune complexes obtained with anti-TLR5 antibodies even though IRAK-P
is present (Fig. 5; see also Fig. 7).
This finding clearly indicates that IRAK-P is not active when
associated with TLR5. Thus flagellin stimulation via TLR5 must be
associated with the release of an active form of IRAK.
In view of the observation that flagellin stimulation did not produce a
detectable change in the level of TLR5-associated IRAK (Fig. 5), we
considered the possibility that the flagellin-induced release of IRAK-P
may involve an exchange reaction that replaces IRAK-P with the
nonphosphorylated form of IRAK. Because the TLR5-associated IRAK is
always found in the phosphorylated form, the newly bound IRAK would
have to be phosphorylated rapidly. This type of exchange reaction would
provide a reasonable explanation for the ability of LPS to render cells
cross-tolerant to flagellin (Table I; Figs. 1 and 2). By decreasing the
level of free IRAK (Fig. 1B and Ref. 27), LPS would be
expected to impair the release of IRAK-P severely. If this were the
case, then we would expect to find IRAK-P still associated with TLR5 in
LPS-tolerant cells, even though the level of free IRAK was reduced
dramatically. Therefore, we made THP1 cells tolerant to flagellin or
LPS and analyzed the levels of TLR5-associated IRAK (using anti-TLR5
antibodies in the immunoprecipitation reaction) and free IRAK (using
anti-IRAK antibody) after a second 30-min incubation with flagellin.
Control cells were incubated with flagellin for only the 30-min period. As shown in Fig. 7, IRAK-P was associated with TLR5 in control cells
(first lane). Almost all of the IRAK in the anti-IRAK
immunoprecipitate was nonphosphorylated (second lane).
Similar results were obtained with the lysates from flagellin-tolerant
cells (third and fourth lanes). However, a very
different pattern was observed with lysates from LPS-tolerant cells
(fifth and sixth lanes). As expected, the level
of free IRAK (as assessed by its immunoprecipitation with anti-IRAK
antibody) was reduced dramatically (sixth lane). However,
the level of TLR5-associated IRAK was not markedly different from
control or flagellin-tolerant cells (fifth lane). In
conjunction with the results presented in Figs. 5 and 6, these results
provide strong support for the hypothesis that flagellin self-tolerance is caused by a block in the release of IRAK-P from the TLR5 complex, thus preventing IRAK-P from expressing its kinase and signaling activities.
We and other investigators (5, 8, 17) have established that
Gram-negative flagellin signals via a TLR5/IRAK-dependent pathway in a variety of cell types including human and murine monocytes
as well as promonocytic, T cell, and epithelial cell lines. The
activation of this signaling pathway is relatively rapid as evidenced
by the maximal activation of IRAK within 5 min after exposure of cells
to flagellin (5). The results presented in this paper demonstrate that
exposure of human monocytes, THP1 cells, Jurkat, and COS-1 cells to
Gram-negative flagellin results in a subsequent state of flagellin
nonresponsiveness or tolerance. Like flagellin, LPS can also induce a
state of flagellin hyporesponsiveness (Fig. 1 and Table I). However,
flagellin does not possess the ability to induce tolerance to LPS. As
noted earlier, the development of tolerance to a potent
immunostimulatory molecule such as flagellin may have evolved as a
mechanism to prevent the sustained activation of proinflammatory cells
and thus the cytokine-induced events that are associated with the
septic response. There is a strong correlation among decreases in
TNF- Unlike the situation with the IL-1 receptor (34-36) or TLR2 (37),
IRAK-P is constitutively bound to TLR5 (Figs. 5 and 6). Indeed, the
level of IRAK associated with TLR5 does not appear to change after
stimulation with flagellin (Fig. 5). As shown in Fig. 6,
TLR5-associated IRAK is not enzymatically active. Thus essentially all
of the measurable IRAK enzyme activity in cells is associated with
released IRAK-P. Because IRAK enzyme activity is inducible (Figs.
1-3), we conclude that flagellin signaling via TLR5 must involve the
release of IRAK-P from TLR5. How might we reconcile the sustained
association of TLR5 and IRAK-P with the need for IRAK-P to dissociate
from the TLR5 complex to have kinase and signaling activity? We have
considered two explanations for these results. First, the level of
IRAK-P released in response to flagellin may be extremely low relative
to the total amount of IRAK-P·TLR5 complexes in cells. Thus
our assay system may not have been sensitive enough to detect a very
small difference in IRAK-P release between unstimulated and
flagellin-stimulated cells. In view of the low concentrations of
flagellin which activate the IRAK·TLR5 signaling pathway (4), it is
indeed possible that only a small number of receptor complexes may be
engaged. Alternatively, the relatively constant level of IRAK-P-TLR5
association may be the result of a flagellin-induced exchange reaction
between IRAK-P and IRAK that allows for the release of IRAK-P and thus maintains a constant level of TLR5-associated IRAK. A lack of IRAK
exchange would provide an explanation for the cross-tolerance induced
by LPS (Table I and Figs. 1 and 2). In LPS cross-tolerance to
flagellin, the exchange of TLR5-associated IRAK-P may not occur because
of a marked reduction in the availability of free IRAK (Fig. 6; see
also Refs. 5 and 27). However, it is also possible that LPS, like
flagellin, may block IRAK-P release by a mechanism that is not linked
directly to the level of free IRAK. In light of our findings, it will
be of considerable interest to determine whether IRAK exchange is also
impaired in TLR4 receptor complex in LPS-tolerant cells. Such studies
would allow us to determine whether a block in IRAK release as a
mechanism for tolerance is TLR5-specific or is applicable to other TLRs.
How might the release of IRAK-P from TLR5 be blocked during flagellin
tolerance? The induction of flagellin tolerance is relatively rapid in
onset (Fig. 3) and does not appear to require protein synthesis as
evidenced by the inability of cycloheximide to prevent the onset of
tolerance (Fig. 3B). Preliminary results with an IRAK mutant
do not support a role for IRAK autophosphorylation in the induction of
tolerance. Furthermore, we have not detected any difference in the
electrophoretic mobility of TLR5 from control and tolerant
cells,2 a finding that may rule out TLR5 modification as
the basis for the block in IRAK release from TLR5. Based on the results
of our studies and the requirements for signaling by other TLRs, we
have considered several mechanisms that might explain the block in IRAK
release from TLR5. The initial exposure to flagellin may induce the
down-regulation of a TLR5 coreceptor that is essential for flagellin
signaling. Such a molecule may be equivalent to TLR4-associated CD14 or
MD-2 (38-40). Alternatively, an initial exposure to flagellin may not
only result in the release of IRAK, but also the irreversible release
of a factor that participates in IRAK exchange, perhaps an IRAK-binding
protein that participates in IRAK signaling. Alternatively, flagellin
tolerance may involve the binding of a protein that inhibits IRAK
release. Such a protein would have to be present in cells and undergo
some form of post-translational modification to bind to the TLR5
complex. Studies are current under way to evaluate each of these possibilities.
On the basis of the results obtained with the IL-1 receptor and TLRs,
it appears that different TLRs may share a number of common signaling
features such as IRAK and MyD88, but they may also exhibit substantial
differences in cofactor requirements which result in relatively unique
signaling mechanisms. Furthermore, it is quite likely that TLR
cross-talk (this study as well as Refs. 20, 29, and 41) may have a
profound impact on the ability of a cell to respond over a protracted
period to diverse microbial products.
We thank Pameeka Smith for outstanding
technical assistance, Drew Cawthon for help with the flow cytometry,
and Dr. Griffith Parks for reviewing this manuscript.
*
This work was supported by National Institutes of Health
Grants AI-38670 and AI-51319 (to S. B. M.).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.
Published, JBC Papers in Press, April 12, 2002, DOI 10.1074/jbc.M201762200
2
S. B. Mizel and J. A. Snipes, unpublished observations.
The abbreviations used are:
TNF-
Gram-negative Flagellin-induced Self-tolerance Is Associated with
a Block in Interleukin-1 Receptor-associated Kinase Release from
Toll-like Receptor 5*
and
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 and IL-1) by human
monocytes. Genetic evidence pointed to a role for flagellin, the major
structural protein of the flagellum (1). Subsequently, we demonstrated
that purified recombinant flagellin is an extraordinarily potent
inducer of cytokine and nitric oxide production by monocytes (4, 5).
Half-maximal stimulation of monocytes and THP1 cells was achieved with
picomolar concentrations of flagellin. The proinflammatory action of
flagellin has been confirmed by other investigators using Caco-2 cells
and model epithelial systems (6-12).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B-Luc reporter vector was purchased from
Stratagene and the Renilla luciferase control vector,
pRL-TK, from Promega.
Production--
The human TNF-
enzyme-linked immunosorbent assay kit was obtained from PharMingen (San
Diego) and used according to the manufacturer's instructions. Cells
were incubated in the presence or absence of flagellin for 4 h;
and then culture supernatants were collected, centrifuged to remove
cells, and assayed for TNF- content.
-glycerophosphate, 1 mM sodium
orthovanadate, 1 µM microcystin-LR, and 5 µM ATP. Myelin basic protein (2 µg/reaction) and 10 µCi of [32P]ATP were added, and the reactions were
allowed to proceed for 30 min at 37 °C. The samples were then
electrophoresed in 15% SDS-polyacrylamide gels and exposed to x-ray
film. The level of IRAK protein was assessed using Western blots.
Samples of the anti-IRAK immunoprecipitates were electrophoresed in a
7.5% SDS gel and transferred electrophoretically to an Immunoblot PVDF membrane (Bio-Rad). The membrane was incubated sequentially with the
anti-IRAK antibody, an anti-rabbit horseradish peroxidase-conjugated antibody (Jackson Immunochemicals), and a chemiluminescent substrate (Pierce Chemical Co.). Quantitative analysis of Western blots was done
with the Alpha Innotec Imaging System. To assess the association of
IRAK with TLR5, cell lysates were incubated with anti-FLAG antibody,
and the resultant immunoprecipitates were electrophoresed and the
proteins transferred to a PVDF membrane. The membrane was then probed
with the anti-IRAK antibody. The blots were stripped with Restore
Western blot stripping buffer (Pierce) and reprobed with anti-FLAG
antibodies to verify the efficiency of immunoprecipitation in each
sample. In those instances in which endogenous TLR5/IRAK association
was being evaluated, anti-TLR5 antibodies were used to generate the immunoprecipitates.
B-dependent
Reporter Assays--
Jurkat or COS-1 cells (5 × 106
cells/sample) were transiently transfected using the Effectene
transfection reagent according to the manufacturer's instructions
(Qiagen). NF-B-dependent reporter assays involved
transient transfection of the pNF-B-Luc reporter vector (Stratagene)
and the Renilla luciferase control vector, pRL-TK (Promega).
In some experiments, the cells were also transfected with a wild-type
IRAK expression vector and incubated overnight in the presence or
absence of flagellin. Otherwise, the cells were rested overnight before
a 4-h incubation in the presence or absence of flagellin. The cells
were then harvested, and lysates were prepared using the lysis buffer
provided with the Promega Dual Luciferase Reporter Assay System.
Renilla luciferase activity was used to correct for
differences in transfection efficiency among the individual samples.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
9 M flagellin or 100 ng/ml LPS for ~18 h,
washed, and then incubated for an additional 4 h in the presence
or absence of flagellin or LPS. Culture supernatants were collected and
analyzed for TNF- content. As shown in Table
I, unstimulated human monocytes and THP1
cells (0/0) did not produce detectable levels of TNF-. As expected,
both types of cell generated high levels of TNF- in response to a
single exposure to flagellin or LPS (0/F and 0/L, respectively). As
expected, a primary exposure of cells to LPS resulted in a marked
reduction in subsequent responsiveness to LPS (L/L). In addition, a
primary exposure to LPS also dramatically reduced the response to
flagellin (L/F). This result differs from our earlier observation that
LPS-tolerant human monocytes retain some responsiveness to intact
flagella (2). We believe that the difference in magnitude of the
post-LPS response to purified flagellin versus intact
flagella may be caused by the strength of signal provided by intact
flagella as opposed to purified flagellin because intact flagella may
possess the ability to cross-link a far greater number of receptors
than flagellin. Like LPS, flagellin also induced a state of subsequent
self-tolerance (F/F) in human monocytes and THP1 cells. However,
flagellin did not markedly reduce the response to LPS (F/L). These
findings raised the interesting possibility that flagellin and LPS may
modulate TLR signaling potential by different mechanisms.
Induction of tolerance by flagellin and LPS in human monocytes and THP1
cells
9 M
flagellin (F) or 100 ng/ml LPS (L). The cells were then washed and
incubated in the absence or presence of flagellin or LPS for 4 h.
The culture medium was then harvested, centrifuged to remove cells, and
assayed for TNF- content. The values represent the mean ± S.D.
of triplicate samples.
results presented in Table I, a prior exposure to
flagellin almost completely inhibited the activation of IRAK (F/F) but
had no effect on the response to LPS (F/L). As expected, a prior
exposure to LPS blocked the subsequent response to flagellin (L/F).
View larger version (30K):
[in a new window]
Fig. 1.
Effect of flagellin-induced tolerance on IRAK
activation and level in THP1 cells and Jurkat cells.
A and B, 5 × 106
THP1 cells were incubated overnight in the absence
(0) or presence of 10 9 M flagellin
(F) or 100 ng/ml LPS (L), washed, and then
incubated for 10 min with flagellin or for 30 min with LPS. The cells
were then analyzed for the level of IRAK activity using myelin basic
protein (MBP) as a substrate (A) and IRAK protein
level using a Western blot (B). C, 5 × 106 Jurkat cells were incubated overnight in the absence or
presence of 109 M flagellin, washed, and then
incubated for 10 min with or without flagellin. The cells were then
analyzed for the level of IRAK activity.
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Fig. 2.
Quantitative analysis of IRAK activity
and protein level during tolerance in THP1 cells. The data in this
figure were derived from the quantitation of the results in Fig.
1 using the Alpha Innotec Imaging System. F,
flagellin; N, no addition.
B-Luc
reporter construct, we established that Jurkat cells are highly
responsive to flagellin, with 50% of the maximal response being
achieved with ~7 × 1012 M flagellin
(data not shown). As with monocytic cells (5), flagellin induced a
marked increase in IRAK enzyme activity in Jurkat cells (Fig.
1C, 0/F). However, overnight incubation of these cells
with flagellin completely blocked the subsequent ability of to induce
IRAK activation (fourth lane, F/F). In addition to Jurkat
cells, we have found that flagellin tolerance can be induced in COS-1
and HEK293 cells (data not shown). Thus the ability of flagellin to
induce a subsequent state of nonresponsiveness to itself is not
restricted to cells of the monocytic lineage but rather appears to be a
general property of TLR5-positive, flagellin-responsive cells.
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Fig. 3.
Time course for the induction of flagellin
tolerance in Jurkat cells. A, Jurkat cells were
incubated with flagellin for 4, 12, 18, or 24 h before a second
15-min incubation with flagellin. Control cells were incubated with or
without flagellin for 15 min. All of the cultures were harvested after
the second flagellin incubation and analyzed for IRAK activity.
B, Jurkat cells were incubated in the presence or absence of
10 µg/ml cycloheximide for 15 min before the addition of flagellin
for an additional 2 or 4 h. Control cells were incubated with or
without flagellin for 15 min. All of the cultures were harvested after
the second flagellin incubation and analyzed for IRAK activity.
B-Luc reporter construct and an IRAK cDNA under the transcriptional control of a constitutively active cytomegalovirus promoter. Because the Jurkat cells express the SV40
large T antigen and the IRAK expression vector contains an SV40 origin
of replication, we expected that the transfected cells would
constitutively express very high levels of IRAK. If flagellin tolerance
is not linked to the level of IRAK, then flagellin should induce
self-tolerance even though the cells express a relatively high level of
IRAK protein. A representative experiment is presented in Table
II. Because flagellin has a
relatively rapid effect on the expression of luciferase, there was a
significant level of residual luciferase activity in cells incubated
overnight in the presence of flagellin (F/0). However, a second
incubation with flagellin did not result in an increase in luciferase
activity (F/F). This was the case whether or not the cells were
transfected with the IRAK-FLAG expression plasmid. The elevated level
of NF-B-dependent luciferase expression in unstimulated
cells transfected with an IRAK expression vector has been observed by
other investigators (30-33). Using Western blots, we determined that
flagellin had no effect on the level of IRAK protein expression in the
transfected cells (data not shown). Taken together, the results
presented in Figs. 1 and 2 and Table II establish that flagellin
tolerance does not involve an effect on IRAK expression.
Effect of constitutive IRAK expression on the induction of flagellin
tolerance in Jurkat cells
B-Luc
and pRL-TK. One set of cells was also transfected with IRAK. The cells
were incubated overnight in the absence (0) or presence of
109 M flagellin (F). The cells were then washed
and incubated in the presence or absence of flagellin for 4 h. The
cells were harvested and assayed for inducible luciferase as well as
constitutive Renilla luciferase activity. The values have
been normalized using the Renilla luciferase activity in
each sample. The percent tolerance was calculated using the following
formula: Percent tolerance = 100% B([F/F 
0/0] [F/0 0/0]/[0/F 0/0] × 100).
9
M flagellin and then analyzed for surface expression of
TLR5. The samples were incubated sequentially with an affinity-purified goat antibody raised against a peptide within the extracellular domain
of TLR5 and a fluorescein isothiocyanate-labeled rabbit anti-goat IgG
and then analyzed using a FACSCalibur flow cytometer (Fig.
4). As is evident from the flow cytometry
data, overnight incubation with flagellin did not result in a decrease
in the steady-state level of surface TLR5. Identical results were
obtained using fixed, permeabilized control and tolerant Jurkat cells
and a rabbit antibody directed against a peptide in the cytoplasmic domain of TLR5 (data not shown).
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Fig. 4.
Flagellin-tolerant Jurkat T cells do not
express a reduced level of surface TLR5. Jurkat T cells were
incubated overnight in the presence (solid line) or absence
(dotted line) of 10 9 M flagellin.
The cells were prepared for flow cytometry as described under
"Experimental Procedures." Control cells are represented by the
lighter, dotted line and tolerant cells by
the darker line.
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Fig. 5.
Constitutive association of IRAK with the
TLR5 complex in COS-1 cells. A, COS-1 cells were
transfected with IRAK and TLR5-FLAG expression plasmids, rested
overnight, and then incubated for 24 h in the presence or absence
of 10 9 M flagellin. The cells were then
washed and incubated in the presence or absence of flagellin for
0.5-30 min. Cell lysates were prepared and incubated with anti-FLAG
antiserum and protein G to immunoprecipitate TLR5 and any associated
proteins. After electrophoresis, the proteins were transferred to PVDF
membranes and probed for IRAK as outlined under "Experimental
Procedures." B, COS-1 cells were transfected with
only TLR5-FLAG, rested overnight, and then incubated for 24 h in
the presence or absence of 109 M flagellin.
The cells were then washed and incubated in the presence or absence of
flagellin for 3 min. Cell lysates were prepared and incubated with
anti-FLAG antiserum and protein G to immunoprecipitate TLR5 and any
associated proteins. After electrophoresis, the proteins were
transferred to PVDF membranes and probed for IRAK as outlined under
"Experimental Procedures." All of the blots were stripped and
reprobed for TLR5. In all cases, the level of TLR5 was the same.
View larger version (11K):
[in a new window]
Fig. 6.
TLR5-associated IRAK lacks kinase
activity. THP1 cells were incubated in the presence (F)
or absence (0) of flagellin for 15 min before the
preparation of cell lysates. The lysates were incubated with either
anti-TLR5 or anti-IRAK antibody and protein G. The immunoprecipitates
were then analyzed for IRAK activity using myelin basic protein
(MBP) as a substrate.
View larger version (12K):
[in a new window]
Fig. 7.
Phosphorylated IRAK is associated with the
TLR5 complex in flagellin- and LPS-tolerant THP1 cells. THP1 cells
were incubated overnight in the absence (0) or presence of
flagellin (F) or LPS (L) before preparation of
cell lysates. Aliquots of the cell lysates were immunoprecipitated with
either anti-TLR or anti-IRAK antibodies and the immunoprecipitates were
analyzed for the presence of IRAK by Western blot.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
production (Table I), IRAK activation (Figs. 1 and 2), and
NF-B-dependent gene expression (Table II) in
flagellin-tolerant cells. This action of flagellin is not the result of
a decrease in the steady-state level of surface TLR5 (Fig. 4) or
intracellular IRAK (Fig. 1).
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Dept. of Microbiology
and Immunology, Wake Forest University School of Medicine, Medical
Center Blvd., Winston-Salem, NC 27157. Tel.: 336-716-2216; Fax:
336-7169928; E-mail: smizel@wfubmc.edu.
ABBREVIATIONS
, tumor
necrosis factor ;
CMV, cytomegalovirus;
IL-1, interleukin-1;
IRAK, IL-1 receptor-associated kinase;
IRAK-P, phosphorylated IRAK;
LPS, lipopolysaccharide;
Luc, luciferase;
NF-B, nuclear factor B;
PVDF, polyvinylidene difluoride;
TK, thymidine kinase;
TLR, toll-like
receptor.
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