Departments of 1 Molecular and Experimental Medicine and 3 Immunology, The Scripps Research Institute, La Jolla 92037; and 2 La Jolla Institute for Allergy and Immunology, San Diego, California 92121
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ABSTRACT |
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Bacterial lipopolysaccharide
(LPS) is a powerful activator of the innate immune system. Exposure to
LPS induces an inflammatory reaction in the lung mediated primarily by
human blood monocytes and alveolar macrophages, which release an array
of inflammatory chemokines and cytokines including IL-8, TNF-,
IL-1
, and IL-6. The signaling mechanisms utilized by LPS to
stimulate the release of cytokines and chemokines are still
incompletely understood. Pretreatment with the protein tyrosine
kinase-specific inhibitors genistein and herbimycin A effectively
blocked LPS-induced NF-
B activation as well as IL-8 gene expression
in human peripheral blood monocytes. However, when genistein was added
2 min after the addition of LPS, no inhibition was observed. Utilizing
a coimmunoprecipitation assay, we further showed that LPS-stimulated
tyrosine phosphorylation of Toll-like receptor 4 (TLR4) may be involved
in downstream signaling events induced by LPS. These findings provide
evidence that LPS-induced NF-
B activation and IL-8 gene expression
use a signaling pathway requiring protein tyrosine kinase and that such
regulation may occur through tyrosine phosphorylation of TLR4.
inflammation; chemokine; lipopolysaccharide; signal transduction; monocytes
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INTRODUCTION |
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BACTERIAL
LIPOPOLYSACCHARIDE (LPS) is shed from bacteria during normal
bacterial growth and during bacteriolysis. Monocytes/macrophages bind
LPS and in response rapidly synthesize and secrete biologically active
products including cytokines that can mediate inflammation. LPS has
been shown to interact with a number of plasma elements including
complement proteins, coagulation factors, HDL, and LPS-binding protein
(LBP) (35). LBP is a 60-kDa serum glycoprotein synthesized in the liver, the concentration of which can increase 100 times during
acute-phase reactions, such as occurs during bacterial infections. LBP
binds to LPS on the surface of bacteria or LPS-coated particles and
facilitates attachment of these particles to monocytes/macrophages (43). This binding occurs via CD14 (42). CD14
is not a transmembrane protein and lacks the ability to transduce
cytoplasmic signal (38). Recently, the signal-transducing
receptor for LPS was identified as a member of the Toll-like receptor
family. Toll is a transmembrane receptor in Drosophila that
is involved in the induction of an antifungal response
(17). Activation of the Toll receptor results in the
stimulation of several signaling molecules that are homologous to
proteins involved in NF-B in mammalian cells (5).
Poltorak et al. (29) found that mutational inactivation of Tlr4 completely abolishes LPS signal transduction. These results document that Toll-like receptor 4 (TLR4) is the cellular
LPS receptor. There is increasing evidence that TLR4 mediates
LPS-induced signaling events, including activation of MAP kinases and
NF-
B (8-10).
LPS is a powerful activator of the innate immune system, stimulating
mononuclear phagocytes to synthesize an array of cytokines and
chemokines that recruit inflammatory cells to the involved tissue as
well as activating immune and inflammatory responses. Repeated exposure
to inhaled LPS also occurs as a consequence of its nearly ubiquitous
presence in environmental dust (23-25). Stimulation
of monocytes/macrophages with LPS induces several cellular functions,
including generation of a defined set of gene products, such as
interleukin (IL)-1, IL-1
, and IL-8. IL-8, a chemoattractant for
neutrophils and eosinophils, has recently been considered to play a key
role in the pathogenesis of lung inflammatory reactions (21,
26). The regulation of IL-8 gene expression in these cells is
governed by the activities of transcription factors. NF-
B is an
important transcription factor to immune cell function owing to its
ability to activate the transcription of many proinflammatory
immediate-early genes (4, 33). Numerous stimuli
can activate NF-
B, including the bacteria-driven chemoattractant formyl-Met-Leu-Phe, as well as other proinflammatory factors, including
IL-1, TNF-
, and LPS (6, 28).
Although the activation of NF-B has been extensively studied in
cultured cell lines of hematopoietic lineage, the signal transduction
pathways induced by LPS, in particular the early intracellular events,
that lead to monocyte/macrophage transcription activation, are still
not clearly understood. The presence of tyrosine protein kinase
activities, first described in several oncogene products of RNA tumor
viruses and growth factor receptors (14), has been shown
in neutrophils (12, 15), monocytes, and macrophages
(36, 40). It is likely that the protein tyrosine kinases
play an important role in transducing signals to the cell interior
(16, 32).
Our hypothesis is that the protein tyrosine kinases play a key role in
the LPS-stimulated signaling events that lead to NF-B activation and
proinflammatory cytokine gene expression in human peripheral blood
monocytes. In this report we show that LPS-stimulated NF-
B
activation and subsequent IL-8 gene expression are accompanied by
tyrosine phosphorylation of TLR4 in peripheral blood monocytes and that
inhibition of protein tyrosine kinases blocks LPS-stimulated NF-
B
activation and IL-8 gene expression. Utilizing a coimmunoprecipitation assay, we further show that LPS-stimulated tyrosine phosphorylation of
TLR4 may be involved in downstream signaling events induced by LPS.
These results indicate that protein tyrosine kinase activity is an
important signal transducer for LPS-induced NF-
B activation and
proinflammatory cytokine gene expression and that such regulation may
occur through the tyrosine phosphorylation of TLR4 in activated human
peripheral blood monocytes.
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EXPERIMENTAL PROCEDURES |
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Reagents.
LPS isolated from Salmonella minnesota Re595 was a gift from
R. Ulevitch (Scripps Research Institute). Recombinant murine TNF-
was kindly provided by V. Kravchencko (Scripps Research Institute).
Actinomycin D, cycloheximide, and phorbol 12-myristate 13-acetate (PMA)
were purchased from Sigma (St. Louis, MO). Recombinant human IL-1
,
pyrrolidine dithiocarbamate (PDTC), herbimycin A, and genistein were
obtained from Calbiochem (San Diego, CA). An antibody against a
COOH-terminal peptide (residues 289-317) of I
B-
was a gift
from Dr. W. C. Greene (University of California, San Francisco,
CA). A rabbit polyclonal antibody against TLR4 was from Torrey Pines
Biolabs (San Diego, CA), and an MAb against phosphotyrosine was
purchased from Upstate Biotechnology (Lake Placid, NY).
Preparation of monocytes from peripheral blood. Heparinized human peripheral blood from healthy donors was fractionated on Percoll (Pharmacia) density gradients. Mononuclear cells and neutrophils were initially separated by centrifugation through a 55%/74% discontinuous Percoll gradient. Monocytes were further prepared from the mononuclear cell population by adherence to tissue culture flasks (27). The purity of monocytes was >80-90% as determined by staining with the anti-CD14 monoclonal antibody (Coulter Immunology, Miami, FL), and cell viability was >95% as measured by trypan blue exclusion. Monocytes were resuspended in RPMI 1640 medium (Irvine Scientific, Santa Ana, CA) with 10% (vol/vol) heat-inactivated fetal bovine serum, penicillin (100 units/ml), streptomycin (100 µg/ml), and L-glutamine (2 mM; Irvine Scientific). All reagents were tested by Limulus amebocyte lysate assay (BioWhittaker) and contained <0.005 ng/ml of endotoxin.
Detection of immunoreactive IL-8. Monocytes were stimulated with LPS at a concentration of 10 ng/ml for various times up to 8 h. The conditioned media were collected, and secreted IL-8 was measured by enzyme-linked immunosorbent assay (ELISA) using a commercial kit (Genzyme) according to the manufacturer's recommended protocol. The quantities of secreted IL-8 in the test samples were determined by a standard curve generated with purified IL-8.
Preparation of nuclear extracts.
Nuclear extracts were prepared by a modified method of Dignam et al.
(10). Monocytes were separately plated at a density of
1 × 106 cells in six-well plates. After stimulation,
cells were washed three times with ice-cold PBS, harvested, and
resuspended in 0.4 ml of buffer A [10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, and 0.5 mM
phenylmethylsulfonyl fluoride (PMSF)]. After 10 min, 23 µl of 10%
Nonidet P-40 were added and mixed for 2 s. Nuclei were separated
from cytosol by centrifugation at 13,000 g for 10 s and
then resuspended in 50 µl of buffer B (20 mM HEPES, pH
7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, and 0.1 mM PMSF). After 30 min
at 4°C, lysates were separated by centrifugation (13,000 g, 30 s), and the supernatants containing nuclear
proteins were transferred to new vials. The protein concentration of
extracts was measured with a protein dye reagent (Bio-Rad) with bovine
serum albumin as the standard, and the samples were diluted to an equal
concentration in buffer B for use directly or storage at
80°C.
EMSA.
We performed EMSA by incubating 2.5 µg of the nuclear extract in 12 µl of binding buffer [5 mM HEPES, pH 7.8, 5 mM MgCl2, 50 mM KCl, 0.5 mM dithiothreitol, 0.4 mg/ml poly(dI-dC) (Pharmacia), 0.1 mg/ml sonicated double-stranded salmon sperm DNA, and 10% glycerol]
for 10 min at room temperature. Then ~20-50 fmol of 32P-labeled oligonucleotide probe (30,000-50,000
counts per min) were added, and the reaction mixture was incubated for
10 min at room temperature. The samples were analyzed on 6% acrylamide gels, which were made in 50 mM Tris-borate buffer containing either 1 mM EDTA or 50 mM Tris/380 mM glycine/2 mM EDTA, and were
pre-electrophoresed for 2 h at 12 V/cm. Electrophoresis was
carried out at the same voltage for 2-2.5 h. Gel contents were
transferred to Whatman DE-81 paper, dried, and exposed for 3-5 h
at 80°C with an intensifying screen. Using this method, one
sometimes sees a nonspecific DNA-protein complex of unknown origin in
the autoradiograph.
Immunoprecipitation and immunoblotting. Approximately 10 µg of cytoplasmic extracts, collected after the Nonidet P-40 lysis and centrifugation steps (see Preparation of nuclear extracts above), were incubated with an appropriate amount of antibody for 3 h and then precipitated following absorption onto protein A-Sepharose. Precipitates were washed three times, separated by SDS-PAGE, and transferred to Hybond-ECL nitrocellulose (Amersham). Filter strips were incubated with primary antibody for 30 min at room temperature, followed by addition of peroxidase-conjugated IgG at 1:10,000 for 30 min, and analysis with enhanced chemiluminescence reagents (DuPont-NEN).
Luciferase activity assay.
The plasmid pIL-8(B)LUC (WT-IL-8-LUC) contains a
B site from the
promoter region of the IL-8 gene, and a separate plasmid pIL-8(Mu)LUC
(MU-IL-8-LUC) has a nonfunctional mutant
B site. Both constructs
were kindly provided by Dr. N. Mackman (Scripps Research Institute)
(18). The plasmid pCMV
(Clontech) was used as a control
for monitoring the transfection efficiency by the expression of
-galactosidase. THP1 cells were transiently transfected with
diethylaminoethyl-dextran (19) and were cultivated for 48 h before a 4-h stimulation with media or LPS (10 ng/ml).
Luciferase activity was determined by use of the luciferase assay kit
(Promega) and the Monolight 2010 luminometer (Analytical Luminescence,
San Diego, CA).
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RESULTS |
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LPS-stimulated production of IL-8 involves transcription
activation.
To assess the relationship between LPS stimulation and synthesis of
proinflammatory cytokines, we examined the effects of LPS on IL-8
synthesis in monocytes. IL-8, a prototypic proinflammatory chemokine,
induces the expression of a variety of genes whose products are
involved in acute and chronic inflammatory conditions. Unstimulated
human peripheral blood monocytes produced little IL-8. Addition of LPS
(10 ng/ml) resulted in a time-dependent production of IL-8 as measured
by ELISA. Pretreatment with either actinomycin D or cycloheximide
completely inhibited LPS-induced IL-8 protein synthesis (not shown).
These results indicate that LPS stimulates de novo IL-8 protein
synthesis in monocytes, which is consistent with what has been
previously reported. We previously demonstrated that LPS stimulates
NF-B activity in monocytes (28), suggesting that
activation of NF-
B may be involved in LPS-stimulated IL-8 gene
expression. To test this hypothesis, we examined the effect of PDTC on
both IL-8 gene expression and NF-
B activation. PDTC is an
antioxidant that blocks the dissociation of I
B from the cytoplasmic
NF-
B, thus preventing the activation and nuclear translocation of
NF-
B (2). As shown in Fig.
1A, PDTC treatment of
monocytes significantly inhibited LPS-induced expression of IL-8. The
same treatment almost completely blocked the activation of NF-
B by
LPS (Fig. 1B), suggesting that NF-
B activation is required for LPS-stimulated IL-8 gene expression.
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Protein tyrosine kinase activity is required for LPS-induced
NF-B activation and chemokine gene expression.
Because protein tyrosine kinases play an important role in
intracellular signal processes linked to diverse receptor types, we
examined the role of protein tyrosine kinases in LPS-activated NF-
B
and IL-8 gene expression. Genistein and herbimycin A have both been
shown to specifically inhibit protein tyrosine kinases in multiple cell
types with distinct and different modes of action (1, 37).
After pretreatment with genistein, herbimycin A, or medium control,
monocytes were stimulated with LPS. NF-
B activation was assessed by
EMSA and IL-8 protein production was measured by ELISA. LPS-induced
NF-
B activation (Fig. 4) and IL-8
production (not shown) were completely inhibited in monocytes
pretreated with genistein (Fig. 4, lane 5) or herbimycin A
(Fig. 4, lane 7), and genistein inhibited LPS-stimulated
NF-
B activation in a dose-dependent manner (Fig.
5A). These results suggest
that protein tyrosine kinase activity is required for LPS-induced
NF-
B-mediated cytokine gene transcription. However, the step at
which the tyrosine kinase functions and the molecules are tyrosine
phosphorylated is not known. We attempted to dissect the transcription
activation pathway temporally by intervening pharmacologically at
different time points after LPS challenge. Monocytes were stimulated
with LPS, and then genistein (100 nM) was added at different times (Fig. 5B). It was observed that genistein blocked NF-
B
activation when added within 60 s of the LPS addition, but after
this time the addition of genistein failed to inhibit the NF-
B.
These results indicate that a tyrosine kinase-phosphorylated
intermediate is important for the LPS-induced NF-
B activation and
that the tyrosine phosphorylation of this molecule(s) occurs at very
early stage to mediate this response.
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LPS stimulates tyrosine phosphorylation of TLR4.
To determine whether LPS stimulates tyrosine phosphorylation of its
receptor, TLR4, we treated cell lysates from control and LPS-stimulated
monocytes with antibody against TLR4, and the immunoprecipitates were
resolved by SDS-PAGE and then detected using either antiphosphotyrosine antibody or anti-TLR4 antibody (Fig.
6A). As shown in Fig.
6A, LPS stimulated tyrosine phosphorylation of TLR4 in a
time-dependent manner. To further confirm the effect of tyrosine
phosphorylation, we pretreated monocytes with genistein (100 nM) for 10 min and significantly inhibited LPS-induced TLR4 tyrosine
phosphorylation (Fig. 6B, lane 4). When monocytes
were stimulated with LPS for 4 min and then genistein (100 nM) was
added, the tyrosine phosphorylation of TLR4 was not significantly
inhibited by genistein (Fig. 6B, lane 5).
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DISCUSSION |
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The major outer membrane component of gram-negative bacteria, LPS,
is one of the most important activators of the innate immune system,
which involves the host defense against infection (20, 34). Monocytes/macrophages are among the cells that are most sensitive to LPS. Although a great deal has been learned during the
past few years about the synthesis and release of proinflammatory cytokines by monocytes/macrophages, relatively little is known about
the intracellular events that lead to cytokine gene transcription. To
study the signaling mechanisms for LPS-stimulated cytokine gene
expression, we have chosen to use NF-B-driven IL-8 synthesis in
monocyte as our primary model system. Using purified peripheral blood
monocytes, we showed that PDTC, an antioxidant inhibitor of NF-
B,
not only reduced LPS-induced NF-
B activation (Fig. 1A)
but also abolished the IL-8 protein secretion (Fig. 1B).
These observations suggest that LPS-stimulated IL-8 gene expression could be a consequence of NF-
B activation. To further confirm this
hypothesis, we used IL-8 gene promoter-reporter constructs to assess
the effect of LPS-induced NF-
B activation on the transcription of
IL-8 gene in THP-1 monocyte-like cells. LPS stimulated luciferase activity when the promoter region contained a functional
B site but
not when the
B site was mutated and nonfunctional (Fig. 3). Together
with the previous data, these results demonstrate that LPS-stimulated
IL-8 gene expression is a consequence, at least in part, of NF-
B activation.
Stimulation of human blood leukocytes with LPS is known to result in an
increase of tyrosine phosphorylation (13, 39), which may
contribute to the activation of proinflammatory cytokine gene
expression (11). We therefore examined the role of protein tyrosine kinase in LPS-induced NF-B activation IL-8 gene expression. Genistein and herbimycin A have both been shown to specifically inhibit
protein tyrosine kinase with distinct and different modes of action.
Genistein is a competitive inhibitor binding to the ATP-binding site of
the protein tyrosine kinase (1); herbimycin A irreversibly
inactivates protein tyrosine kinase by binding to the reactive SH
domain of the kinase (37). Preincubation of
monocytes with either genistein or herbimycin A completely abrogated
LPS-induced NF-
B activation as well as IL-8 protein synthesis (both
inhibitors alone had no effect on cell activation). These results
demonstrate that protein tyrosine kinase is important for LPS-induced
NF-
B activation. However, if genistein is added 60 s or
more after the LPS challenge, the inhibition is not seen. Inhibition of
tyrosine kinase after this time was too late to stop the relay of the
signaling. These results suggest that within this time, LPS stimulates
a tyrosine kinase involved in NF-
B activation and that early
inhibition of the tyrosine kinase abolishes this response. The
inability of genistein to block LPS-induced NF-
B activation when
added more than 60 s after LPS implies that additional signaling
pathways may be involved in LPS induced NF-
B activation. There is
increasing evidence that TLR4 mediates LPS-induced signaling events,
including activation of MAP kinases and NF-
B (7, 22,
45). On the basis of these results, we hypothesized that
tyrosine phosphorylation of TLR4 may be involved in LPS-induced NF-
B
activation in monocytes. To address this issue, we immunoprecipitated LPS-stimulated monocytes with antibody against the TLR4 and then detected them using anti-phosphotyrosine antibody. Our results demonstrate that LPS stimulates tyrosine phosphorylation of the TLR4
and that this effect of LPS inhibited by genistein (Fig. 6).
The mechanisms by which LPS induces tyrosine phosphorylation of TLR4
are not clear. Recent evidence from different groups has indicated that
several molecules can autophosphorylate on tyrosine residues (8,
31, 44). Therefore, LPS-induced tyrosine phosphorylation of TLR4
may occur by an LPS-stimulated autophosphorylation mechanism,
independently of other protein tyrosine kinases. Alternatively, increased TLR4 tyrosine phosphorylation could be a consequence of
LPS-activated protein tyrosine kinases. The ability of genistein to
inhibit both LPS-induced tyrosine phosphorylation and activation of
NF-B suggests that an activated protein tyrosine kinase is necessary
for these LPS responses. Genistein inactivates protein tyrosine kinases
by binding to the ATP-binding site of the tyrosine kinase. It is known
that I
B phosphorylation involves two serine kinases, IKK-
and
-
(9, 30, 41). These kinases have been shown to
phosphorylate serine residues S32 and S36 of I
B following stimulation with the molecules known to activate NF-
B. These results
suggest that genistein does not directly inhibit NF-
B. Thus it seems
most likely that genistein inhibits an upstream protein tyrosine kinase
that is necessary for the LPS-induced tyrosine phosphorylation of TLR4
and thereby activation of NF-
B.
The results presented above make two important points. First,
LPS-stimulated IL-8 gene expression is a consequence of NF-B activation in human peripheral blood monocytes, and this function of
LPS requires protein tyrosine kinase. Second, LPS-stimulated tyrosine
phosphorylation of TLR4 may contribute to the downstream signaling
events induced by LPS. These results suggest that stimulation of LPS
causes a rapid production of signals that induce the activation of
NF-
B and that such regulation may occur through the tyrosine phosphorylation of TLR4.
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ACKNOWLEDGEMENTS |
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The authors thank Drs. N. Mackman and R. Medzhitov for providing DNA constructs.
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FOOTNOTES |
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This work was supported by United States Public Health Service Grants AI-43524 and HL-69425 to Z. K. Pan and National Institutes of Health (NIH) Training Grant T32 AI-07469 to L.-Y. Chen. This work was also supported in part by the Sam and Ross Stein Charitable Trust and NIH Grant M01RR00833 provided to the General Clinical Research Center of the Scripps Research Institute. This is publication 14269-MEM from The Scripps Research Institute.
Address for reprint requests and other correspondence: Z. K. Pan, Dept. of Molecular and Experimental Medicine, Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037 (E-mail: zkpan{at}scripps.edu).
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.
First published December 20, 2002;10.1152/ajplung.00116.2002
Received 17 April 2002; accepted in final form 11 December 2002.
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