From the Department of Medicine, National Jewish
Medical and Research Center, Denver, Colorado 80206 and the
¶ University of Colorado Health Sciences Center,
Denver, Colorado 80206
Received for publication, November 25, 2002, and in revised form, February 6, 2003
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ABSTRACT |
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Lipopolysaccharide (LPS) induces neutrophils to
synthesize and secrete pro-inflammatory cytokines and chemokines, which
are regulated at both the transcriptional and translational level. We
reported previously that neutrophils stimulated with LPS induce expression of genes typically expressed in response to stimulation with
antiviral type I interferons (IFN), such as myxovirus resistance-1 (MX1). However, we present evidence that this response of
neutrophils to lipopolysaccharide occurs in the absence of
interferon-dependent signaling.
Lipopolysaccharide-stimulated neutrophils do not phosphorylate the
interferon-associated transcription factors signal transducer and
activator of transcription-1 and -3, and medium from
lipopolysaccharide-stimulated cells was unable to induce
MX1 gene expression, suggesting a soluble factor is not
involved. Furthermore, LPS did not alter expression of IFNA
and IFNB genes. In contrast to neutrophils, LPS-stimulated human monocyte-derived macrophages induced the expression of
MX1, but IFNB was induced, and medium from
LPS-stimulated monocyte-derived macrophages supported MX1
induction. An inhibitor of p38 kinase blocked induction of
MX1 by lipopolysaccharide, but not IFN Neutrophils play a vital role in the innate immune
response by migrating to sites of infection, ingesting microorganisms, and producing antimicrobial agents. Activation of neutrophils can occur
by recognition of structural features of microbes, including the
bacterial cell wall component lipopolysaccharide
(LPS)1 (1). Exposure of
neutrophils and other immune cells to LPS primes the cell to respond to
secondary agonists with secretion of antimicrobial peptides and
formation of superoxide. In addition, it is now recognized that
neutrophils are potent synthetic cells and produce cytokines and
chemokines, such as TNF The toll-like receptor (TLR) family is implicated in the recognition of
bacteria and in the activation of cellular signaling pathways important
in host defense (4, 5). Genetic and biochemical data suggest that TLR4
is the major LPS receptor (4, 6-10), although TLR2 may also play a
role (11, 12). LPS stimulates a pathway leading to the activation of
the transcriptional regulator NF LPS activates tyrosine and serine/threonine kinases in cells (16-23).
Among these the MAP kinase family of serine/threonine kinases (p42/p44
MAP kinase, c-Jun N-terminal kinase, and p38) plays a major role in
cellular activation in a variety of cell types (24-26). However, in
neutrophils the p38 MAP kinase family is predominantly activated in
response to LPS and regulates the translation of cytokines, adhesion,
and migration (27-31). The role of p38 in regulation of gene
expression is less well understood, although transcription factors,
including activating transcription factor-2, SRF accessory protein-1,
and CCAAT-enhancer binding protein- The cellular response to viral infection includes the induction of
genes for the type I interferons, IFN Links between anti-bacterial and antiviral activity have been suggested
previously. In particular, the induction of IFN LPS from E. coli strain 0111:B4 was purchased from
List Biological Laboratories and contained minimal protein
contamination as determined by silver staining. Peptidoglycan (Fluka)
was prepared by extensive sonication followed by centrifugation.
Anti-STAT1 and -STAT3 were obtained from Transduction Laboratories, and
anti-phospho-STAT1 (Tyr-701), phospho-STAT3 (Tyr-705), and phospho-p38
were from Cell Signaling. Anti-p38 polyclonal antiserum was produced as described previously (27). IFN Cell Preparation and Stimulation--
Neutrophils were isolated
from citrated blood of healthy donors and purified on a Percoll
gradient, as described previously (44). Contamination with monocytic
cells is less than 5%. Cells (20-25 × 106 cells/ml)
were resuspended in neutrophil medium (RPMI containing 1%
heat-inactivated platelet-poor plasma and 10 mM HEPES, pH
7.6) and divided into 1.5-ml tubes. Heat-inactivated platelet-poor plasma is a necessary source of LPS-binding protein (45). RT-PCR analysis utilized 20 × 106 cells (in 1 ml). Similar
RT-PCR results were obtained at lower cell concentrations. Cells were
treated with the indicated concentration of LPS, IFN
Human monocyte derived macrophages were prepared by the method of Fadok
et al. (46). Briefly, peripheral monocytic cells from
healthy human volunteers were washed in Hanks' balanced salt solution
with calcium and magnesium, and 12 × 106 cells in
X-vivo medium (BioWhittaker) per well were seeded in 6-well plates. One
hour after plating, non-adherent cells were removed by washing, and
adherent cells were grown in X-vivo, 10% heat-inactivated human serum.
The medium was changed every 3 days thereafter, and experiments were
performed 7 or 8 days after plating. RAW264.7 cells, obtained from the
American Type Culture Collection, were cultured as described (47).
Affymetrix Oligonucleotide Array--
Preparation of total RNA
(5 µg) for analysis by oligonucleotide array (Affymetrix) was
performed as described (32). Data were analyzed using Affymetrix
GeneChip software. Time courses from three donors were analyzed.
Reverse Transcription (RT)-PCR--
cDNA was prepared by
reverse transcription using 2 µg of total RNA, derived from 20 × 106 neutrophils, or 0.5-2 µg of RNA from
monocyte-derived macrophages and treated as indicated. PCRs were
performed using specific primers for MX1, PKR,
TNFA, IFNA (48), IFNB, and
GAPDH. Murine-specific primers were used in experiments with
RAW264.7 cells.
Enzyme-linked Immunosorbent Assay--
Secretion of IFN Conditioned Media Experiments--
Supernatants were isolated
from neutrophils or monocyte-derived macrophages stimulated for 4 h with 100 ng/ml LPS (LPS-CM) or non-stimulated cells (NS-CM). Naive
neutrophils (20 × 106) were suspended in 250 µl of
neutrophil medium and 750 µl of NS-CM or LPS-CM supplemented with 10 µg/ml polymyxin B. Monocyte-derived macrophages were stimulated for
4 h with NS-CM or LPS-CM supplemented with 10 µg/ml polymyxin B,
and 1.5 ml was added per well of naive monocyte-derived macrophages
cultured in a 6-well plate. Cells were incubated for 4 h at
37 °C; neutrophils were rotated continuously. RT-PCR was performed
as described above.
Whole Cell Extraction and Immunoblotting--
Neutrophils
(20 × 106) were stimulated for the indicated time,
washed once in ice-cold phosphate-buffered saline, pH 7.4, and resuspended in 400 µl of 20 mM imidazole, pH 7.4, 250 mM sucrose, 2.5 mM MgCl2, 5 mM EGTA, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 µM pepstatin, 1 mM phenylmethylsulfonyl
fluoride, 2 mM p-nitrophenyl phosphate, 10 mM p38 Kinase Reactions--
Whole cell extracts from stimulated
cells (250 µl or 400-500 µg from 20 × 106 cells)
were supplemented with 750 µl of 1% Triton X-100 in 50 mM Tris, pH 7.4, incubated on ice for 10 min, and
centrifuged for an additional 10 min. The supernatants from this spin
were incubated with anti-p38 for 1 h, and protein A-Sepharose was
added for an additional 30 min. The beads were washed, and kinase
activity was determined at 30 °C in 20 mM Tris, pH 7.4, 10 mM MgCl2, 50 µM
[ Expression of Interferon-stimulated Genes by LPS Is
IFN-independent--
We recently demonstrated changes in gene
expression in human neutrophils treated with LPS for 4 h using
oligonucleotide (32) and cDNA (38) microarray analysis.
Unexpectedly, increases of genes were observed that in other systems
are associated with activation by the type I interferons, IFN
Expression levels of two ISGs, MX1 and PKR, and
those of TNFA and GAPDH were confirmed by RT-PCR
(Fig. 1C). Induction of MX1 and PKR
paralleled the level of expression determined by oligonucleotide array,
with little expression after 2 h but clearly induced 4 h
after LPS. In contrast, TNFA expression was evident as early as 30 min after LPS exposure and was maintained at an elevated level
throughout the 4 h of LPS stimulation (Fig. 1C). The
expression of MX1 and TNFA was
dose-dependent and observed at LPS concentrations as low as
1 ng/ml (Fig. 1D). Levels of the ISG MX1 induced
by LPS were similar to those induced by IFN
Several early reports demonstrated the production of type I interferons
by LPS-stimulated cells (36, 37), a response that may depend on TLR4
(50). To our knowledge LPS is known to stimulate ISGs only through the
induction of type I interferons. Therefore, we explored the possibility
that LPS induced IFNA or IFNB in neutrophils. In
resting neutrophils, RT-PCR analysis indicated a low level of
IFNA and nearly undetectable levels of IFNB (Fig.
2A). After a 4-h exposure to
LPS, neutrophils failed to change the transcript level of either
cytokine (Fig. 2A). Furthermore, several IFNA species, IFNB and IFNW, were absent from
neutrophils and unchanged by LPS by gene expression analysis (data not
shown). Cells responded normally with the induction of MX1
and TNFA.
Although the expression of IFN Induction of MX1 Is Independent of STAT Activation and Release of a
Soluble Factor--
Although the data above indicate that type I
interferons are not produced in LPS-treated neutrophils, the finding
that LPS stimulation of neutrophils induces IFN-regulated genes
suggested that IFN-stimulated signaling pathways are activated. The
interferon-independent induction of ISG56 by dsRNA is regulated in part
by STAT1
Because chemokines can activate STAT proteins (52), we investigated if
LPS stimulated the release of a non-interferon factor in neutrophils
that mediates ISG induction. Our expression profile data and the data
of others (53) (data not shown) indicated that LPS regulates expression
of mRNA for oncostatin M. This gp130-activating ligand, associated
with activation of Jak/STAT pathways, is also released from already
synthesized pools (53). However, recombinant human oncostatin M failed
to induce MX1 (data not shown). To investigate further the
possibility that released factors were responsible for MX1
induction, cell-free supernatants from unstimulated and LPS-stimulated
neutrophils were added to naive neutrophils for 4 h, and
expression of MX1, TNFA, and GAPDH was
measured by RT-PCR. Conditioned medium from LPS-stimulated neutrophils
induced TNFA message but not that of MX1 (Fig.
2C). We have not characterized further the
TNFA-inducing factor released from LPS-stimulated neutrophils. Therefore, although conditioned medium from LPS-stimulated neutrophils is biologically active, it does not contain an ISG-inducing substance. These data indicate that LPS is able to induce
MX1 message, but in contrast to other cells types paracrine
factors are not sufficient for induction of MX1.
Different Mechanisms of ISG Induction in Neutrophils and
Macrophages--
In macrophages, IFN
The up-regulation of IFNB message in response to LPS
suggested that STAT signaling would be activated. To confirm the
activation of STAT proteins in LPS-stimulated monocyte-derived
macrophages, we measured the levels of phospho-STAT1 and -STAT3. LPS
failed to induce phosphorylation of either STAT1 or STAT3 after 30 min of exposure; however, prolonged exposure (4 h) of monocyte-derived macrophages to LPS stimulated the phosphorylation of STAT1 and STAT3
(Fig. 3B). Furthermore, a reproducible decrease in STAT proteins was observed in monocyte-derived macrophages stimulated with
LPS for 4 h.
To determine whether IFNB induction and delayed STAT
activation are associated with ISG induction in monocyte-derived
macrophages, we performed conditioned medium experiments. Media
harvested from resting and LPS-stimulated monocyte-derived macrophages
were treated with polymyxin B to inactivate LPS, added to naive
monocyte-derived macrophages for 4 h, and RT-PCR was used to
determine the activity of the conditioned medium. Consistent with the
transcriptional increase of IFNB levels and the activation
of STAT proteins in LPS-stimulated monocyte-derived macrophages,
conditioned medium from LPS-treated monocyte-derived macrophages
up-regulated MX1 mRNA to an equivalent level as control,
LPS-stimulated monocyte-derived macrophages (Fig. 3C).
Conditioned medium from LPS-treated monocyte-derived macrophages also
demonstrated a weak TNFA-inducing activity; the inactivation
of residual LPS by polymyxin B is also demonstrated by the weaker
induction of TNFA in conditioned medium than in control,
LPS-stimulated monocyte-derived macrophages. Therefore, whereas
monocyte-derived macrophages respond to LPS by induction of
IFNB, STAT phosphorylation, and transfer of
MX1-inducing activity into the supernatant, neutrophils fail
to display these traits.
MX1 Induction in Neutrophils Is Sensitive to p38
Inhibition--
Our recent report (32) on gene expression in
LPS-stimulated neutrophils suggests that the major transcriptional
target of the p38 inhibitor, SB203580, is the ISGs. Induction of ISGs
was primarily reduced in the presence of the inhibitor, whereas most genes were left unchanged. To explore the signal transduction pathways
utilized by LPS for induction of MX1, cells were treated with kinase inhibitors. Pretreatment with the p38 MAP kinase inhibitor SB203580 blocked induction of MX1, as shown by RT-PCR
analysis (Fig. 4A), in
agreement with oligonucleotide array data (32). The specificity of
SB203580 for p38 was supported by the findings that SB202474, an
inactive analog of SB203580, failed to inhibit MX1
induction, and another more specific p38 inhibitor, M39 (43), was also
active in inhibiting MX1 induction (Fig. 4B).
Furthermore, the dose-dependent inhibition of
MX1 induction by SB203580 was consistent with inhibition of
p38 (data not shown). In contrast to LPS, the expression of
MX1 by IFN
Differences in ISG induction described between neutrophils and
monocyte-derived macrophages prompted us to explore the role of p38 in
LPS-stimulated ISG induction in monocyte-derived macrophages. Expression of MX1 in response to LPS was not altered by
preincubation of monocyte-derived macrophages with SB203580 (Fig.
5A). In addition, transcription of TNFA was not inhibited by SB203580.
Macrophage-like RAW 264 cells also induced MX1 in response
to LPS and were similarly refractory to SB203580 (data not shown). The
lack of inhibition by SB203580 of MX1 induction in
monocyte-derived macrophages is not due to an inability to activate
p38, as LPS treatment leads to the time-dependent
phosphorylation of p38 (Fig. 5B), as seen in RAW 264 cells
(data not shown) (47). Therefore, these data support alternative
pathways for ISG induction by LPS in neutrophils and macrophages.
Protein Synthesis Is Required for MX1 Induction by LPS--
The
necessity for protein synthesis in the induction of MX1 by
LPS was explored using cycloheximide, a general protein synthesis inhibitor. Neutrophils pretreated with cycloheximide and stimulated with LPS did not induce MX1 message, whereas TNFA
levels were partially inhibited (data not shown). Thus, whereas LPS
does not induce the synthesis of a secreted ISG-inducing factor,
synthesis of an intracellular protein may be necessary for this response.
Induction of MX1 Is Specific for LPS and IFN and Is
TLR4-specific--
As shown above, both LPS and IFN
TLR4 is recognized as the LPS receptor. To determine whether other TLRs
can mediate the induction of ISGs, we stimulated neutrophils and
monocyte-derived macrophages with peptidoglycan (PGN), a TLR2-specific ligand, and determined gene expression using RT-PCR. In neutrophils, LPS and PGN stimulated the expression of TNFA, demonstrating
the biological activity of PGN on neutrophils (Fig.
6A). However, SB203580 had no
effect on the expression of TNFA, indicating the inhibition
of MX1 expression in LPS-stimulated neutrophils was specific. In contrast to the effect of LPS on neutrophils, PGN was
unable to induce expression of MX1 (Fig. 6A). As
shown previously, SB203580 inhibited MX1 induction by LPS,
but SB203580 did not modify the expression profile induced by PGN.
Similarly, lipoteichoic acid, another TLR2-activating ligand, failed to
induce MX1 (data not shown). Likewise, PGN-stimulated
monocyte-derived macrophages did not express MX1 (Fig.
6B), although TNFA was up-regulated by both LPS
and PGN. These data indicate that ISG induction is dependent on
TLR4-specific signaling events.
The ability of SB203580 to block ISG induction in LPS-stimulated
neutrophils indicates that p38 plays a role and suggests that the
inability of PGN to induce ISG expression might be explained, at least
in part, by inadequate p38 activation. Therefore, we determined p38
activity in response to PGN. PGN enhanced p38 activity in a
time-dependent manner, with p38 activity observed after 10 min and maximal activity at 40 min; p38 activity declined by 60 min
post-stimulation (Fig. 6C). However, the extent of
PGN-stimulated p38 activity was less than that observed with LPS (Fig.
6C), and the time course of PGN-stimulated p38 activation
was delayed compared with LPS.
We previously identified ISGs as p38-dependent gene
targets in LPS-stimulated neutrophils (32). The present study was
undertaken to clarify the role of p38 in LPS-stimulated ISG induction
and the mechanism of this response. The delayed expression of ISGs in
LPS-stimulated neutrophils suggested that LPS was activating a
secondary, indirect response, as had been observed in macrophage(-like) cells (36, 37). However, several lines of evidence point toward a
mechanism in neutrophils distinct from those known to be involved in
the expression of ISGs in macrophages. First, LPS did not induce expression of IFNA or IFNB mRNA, as
determined by RT-PCR and gene expression analysis. Furthermore,
secretion of IFN LPS is well known to enhance the expression of type I IFNs in
macrophages, particularly of IFN Although non-interferon ligands can tyrosine-phosphorylate and activate
STAT proteins (52), we have found no evidence for this in
LPS-stimulated neutrophils. Another post-translational modification of
STAT1, arginine methylation, was reported to be important for STAT1
transactivation (55). However, the observation that tyrosine
phosphorylation of STATs is still required for transactivation and
translocation to the nucleus suggests that STAT activation by these
additional mechanisms is not responsible for induction of ISGs.
Together with the lack of detectable type I interferon release or the
secretion of a paracrine factor, the failure of LPS to enhance STAT
phosphorylation in neutrophils points toward a unique mechanism of ISG
induction. This finding is reminiscent of the observation that certain
viruses and dsRNA induce ISGs in the absence of IFN secretion and
independently of STAT activation (40, 41). Induction of ISGs by virus
is dependent on members of the IRF family of transcriptional regulators
including IRF3, IRF5, and IRF7 (34, 35, 56). IRF family members are
phosphorylated in response to virus on C-terminal Ser residues (48,
56-59), a modification necessary for transactivation (60), and
identified by a retardation of electrophoretic mobility of IRF3 (60).
These investigators were unable to observe activation of IRF3 by LPS in
numerous cell types. However, LPS does alter the electrophoretic mobility of IRF3 in
neutrophils,2 suggesting the
involvement of IRF3 in LPS signal transduction in neutrophils, but this
gel shift is not altered by SB203580.2 A recent publication
has indicated the importance of IRF3 and NF LPS Induction of ISGs Is p38-dependent--
The p38
pathway has a role in cytokine production, adhesion, and migration of
LPS-stimulated neutrophils (27, 28, 30, 31, 62). The finding that
transcription factors are substrates for p38 (or
p38-dependent kinases) implicates p38 in the regulation of
gene expression (26). Furthermore, STAT1 phosphorylation on Ser-727, a
modification necessary for the transactivation potential of STAT1, is
dependent on p38 in some cell types (63). However, p38 inhibition with
SB203580 had no effect on IFN
A definite role of p38 is obscured by the possibility of other targets
for SB203580, including Raf, JNK2
The p38 kinase pathway may also regulate the activity of other
components necessary for the transcriptional complex, such as a non-IRF
transcription factor and/or co-activators. Kinase pathways are also
known to modify chromatin and thus are implicated in chromatin
remodeling. p38 phosphorylates histone H3 on Ser-28 (67), and the
p38-activated kinase MSK-1 phosphorylates H3 on Ser-10 and HMG-14 (68).
Recently, nucleosome remodeling was shown to occur as a consequence of
TLR2 activation (69). A role for p38 in histone acetylation, another
chromatin modification, has not been described, although ATF-2, a p38
substrate, displays histone acetyltransferase activity (70). At
present, we cannot distinguish the mechanism(s) by which SB203580
inhibits ISG expression.
ISG Induction by LPS Requires Protein Synthesis--
The lack of a
secreted ISG-inducing factor from LPS-stimulated neutrophils and the
sensitivity of this response to cycloheximide suggest that the
synthesis of an intracellular protein is required. One candidate is
IRF7, a protein induced by IFN The Induction of ISG by LPS in Neutrophils Occurs through the
Activation of p38 and TLR4 but Not TLR2--
Stimulation of
neutrophils with a TLR2-activating ligand, PGN, had no effect on
transcript levels of MX1; however, PGN was active, as
demonstrated by the induction of TNFA. A similar observation was made recently in murine macrophages (50). In addition, the induction of MX1 in human monocyte-derived macrophages is
also mediated by TLR4 but not TLR2. Therefore, induction of ISGs is confined to activation of a subset of TLR family members. However, the
mechanisms by which MX1 induction occurs is different
between the two cell types. Induction of MX1 is sensitive to
SB203580 only in neutrophils. Although PGN activates p38 in
neutrophils, it is to a lesser degree than does LPS. This can be
interpreted to suggest that the level or duration of p38 activity by
PGN and TLR2 is insufficient to support ISG induction; however, whereas low LPS concentrations induced MX1 expression (Fig.
1C), this dose of LPS activated p38 poorly (28). Thus, our
findings do not exclude the possibility that a non-p38, TLR4-specific
pathway is required and that TLR2, while activating p38 sufficiently, does not activate this putative second signal. Although p38 plays a
necessary role in LPS-induced ISG expression in neutrophils, p38
activation may not be sufficient for ISG expression. As described above
and suggested previously (61) (data not shown), IRF3 may fulfill this role.
Indirect evidence supports a role for neutrophils in antiviral defense.
Viral infection promotes neutrophilic inflammation (75, 76), and
neutrophils express anti-viral defensins (77). Neutrophils exposed to
LPS also synthesize and secrete large quantities of the CC-chemokines
MIP-1
In summary, gene expression analysis of LPS-stimulated neutrophils has
led to the detection of an unexpected set of antiviral genes (32, 38).
In contrast to other cell types (36, 37, 83), such as macrophages, the
LPS induction of ISGs in neutrophils does not involve secretion of
mediators, including IFN, in neutrophils,
and induction of MX1 was dependent on protein synthesis.
LPS, but not IFN
, substantially activated p38. In contrast, the
induction of MX1 by LPS in monocyte-derived macrophages was
insensitive to p38 inhibition, although p38 is phosphorylated in
LPS-stimulated but not IFN
-stimulated monocyte-derived macrophages. The expression of MX1 in neutrophils and monocyte-derived
macrophages is mediated by TLR4 but not TLR2. The data presented here
indicate that lipopolysaccharide activates novel interferon-independent signaling pathways in neutrophils and that induction of antiviral genes
is a consequence of exposure of neutrophils to bacterial products.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and IL-1
, which are coded for by the
TNFA and IL-1
genes (2, 3). Production of
these immunomodulatory proteins in response to LPS is controlled at
both the transcriptional and translational level.
B in many cell types (4, 7, 8, 13,
14), including neutrophils (15). Whereas NF
B plays a pivotal role in
regulating the expression of cytokine and chemokine genes, other
transcriptional regulators may also be involved in signaling from TLRs.
In contrast to many other cell types, transcription factor activation
is poorly understood in neutrophils.
(26), are known substrates of
p38. The role of p38 in activating these transcription factors in
neutrophils has not been explored. However, inhibitors of p38 have
little effect on the expression of cytokine genes in neutrophils (28,
32).
and IFN
, which act in an
autocrine or paracrine manner to stimulate the induction of
interferon-stimulated genes (ISGs) (33). Induction of IFN
/
protein by virus requires the activation of the IRF family of transcriptional regulators by phosphorylation, and subsequent gene
expression of IFNA and IFNB (34, 35), through
poorly understood pathways. IFN
/
binds to the cell surface IFN
receptor and activates the Jak1 and Tyk2 tyrosine kinases leading to
the phosphorylation of the STAT1 and STAT2 transcription factors and subsequent dimerization (33). The major regulatory factor involved in
IFN
/
action, ISGF3, is a complex of STAT1, STAT2, and
IRF9-p48-ISGF3
. ISGF3 binds to a conserved sequence in the 3'
region of IFN-responsive genes known as the interferon-stimulated
response element. ISGs, such as MX1, are regulated primarily
by transcriptional activation of this element, and the antiviral
activity of IFN
/
resides in their ability to induce ISGs.
by LPS has been well
described (36, 37). In this way, antiviral gene expression is
accomplished in response to bacterial infection. We recently described
(32, 38) the regulation of ISGs in LPS-treated neutrophils. Here we
investigated the mechanisms by which neutrophils induce ISGs in
response to LPS. LPS-stimulated neutrophils do not increase
IFNA or IFNB gene expression, secrete a soluble
mediator to induce ISGs, or phosphorylate STAT proteins; in contrast,
monocyte-derived macrophages increase IFNB expression,
secrete a soluble ISG-inducing factor(s), and phosphorylate STAT
proteins. Furthermore, the induction of ISGs by LPS in neutrophils, but
not monocyte-derived macrophages, is sensitive to inhibitors of p38,
and these effects are mediated by TLR4 but not TLR2. Whereas both
IFN-dependent and -independent induction of ISGs by dsRNA
and virus has been described (39-42), to our knowledge this is the
first report of IFN-independent ISG induction by LPS. These data
suggest a potential new role of neutrophils in innate immunity by
inducing an antiviral genotype and suggest that distinct pathways are
activated by LPS in neutrophils and monocyte-derived macrophages.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
A/D was purchased from PBL Biomedical Laboratories. Cycloheximide, lipoteichoic acid (from
Staphylococcus aureus), and polymyxin B were obtained from
Sigma. SB203580, SB202474, and PD98059 were from Calbiochem.
(S)-5-[2-(1-Phenylethylamino)pyrimidin-4-yl]-1-methyl-4-(3-trifluoromethylphenyl)-2- (4-piperidinyl)imidazole
(M39) was from Merck (43).
/D, and PGN and
rotated continuously at 37 °C for up to 4 h. In some
experiments, cell supernatants were retained for conditioned media
experiments or enzyme-linked immunosorbent assay (see below).
and
IFN
was quantified by enzyme-linked immunosorbent assay kits as
directed by the manufacturer (R & D Systems).
-glycerol phosphate, and 200 µM sodium
orthovanadate. After incubation on ice for 5 min, cell suspensions were
sonicated using a Braun-Sonic 200 sonicator twice for 10 s,
extracted with 400 mM NaCl for 20 min at 4 °C, and
clarified by centrifugation at 18,000 × g for 20 min
at 4 °C. Monocyte-derived macrophages were treated as indicated,
washed in phosphate-buffered saline, and lysed in SDS sample buffer.
Protein samples were separated by SDS-PAGE, transferred to
nitrocellulose, and probed with the indicated antibody.
-32P]ATP (4 µCi/assay), 2 µg of GST-ATF-2, 1 mM dithiothreitol, and 10 mM
p-nitrophenyl phosphate. Reactions were stopped after 20 min
by addition of 2× SDS sample buffer. Phosphorylation of ATF-2 was
visualized after SDS-PAGE on a STORM 860 PhosphorImager (Amersham Biosciences), and quantification was performed using ImageQuant (Amersham Biosciences).
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
or
IFN
(49). Further analysis of the time course of expression levels
of these ISGs by oligonucleotide array indicated that LPS-induced ISG
induction occurred after a prolonged lag (Fig.
1A). Little ISG induction was
observed until 4 h of LPS stimulation; examples of the expression of three such genes are shown in Fig. 1A. In contrast,
expression of TNFA and IL-1
, NF
B-regulated
genes for proinflammatory cytokines known to be expressed in
LPS-stimulated neutrophils, increased rapidly and was sustained for
4 h (Fig. 1B).
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Fig. 1.
Up-regulation of interferon-stimulated genes
MX1, ISG15, and PKR in LPS-stimulated
neutrophils. A, transcript levels of MX1,
ISG15, and PKR. Total RNA was isolated from human
neutrophils stimulated with 100 ng/ml LPS for the indicated times.
Biotinylated cRNA was synthesized by the protocol provided by
Affymetrix and hybridized to a Hu6800FL GeneChip. The average
difference is a measure of RNA levels. The graph is a
representative example of analysis from three donors. B,
transcript levels of cytokines TNFA and IL-1
as assessed by Affymetrix GeneChip analysis, as described above.
C, RT-PCR analysis of MX1, PKR,
TNFA, and GAPDH expression in neutrophils
stimulated with 100 ng/ml LPS for the indicated times. D,
dose-response relationship of gene expression in LPS-stimulated
neutrophils by RT-PCR. Neutrophils were treated for 4 h with the
indicated concentration of LPS or IFN
(100 units/ml).
(Fig. 1D), a
known inducer of MX1, yet IFN
caused little enhancement
of TNFA levels (Fig. 1D). Furthermore, the
induction of MX1 by LPS was blocked by pretreatment with
polymyxin B, an inhibitor of LPS signaling (data not shown).
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Fig. 2.
LPS-stimulated neutrophils do not induce
interferons, activate STAT1 and STAT3, or secrete an ISG-inducing
factor. A, neutrophils were stimulated for 4 h
with LPS (100 ng/ml), and gene expression was assessed by RT-PCR using
primers to the indicated genes. B, neutrophils were treated
for the indicated times with LPS (100 ng/ml) and IFN (100 units/ml)
or left untreated (NS). Whole cell extracts were prepared,
and STAT1, STAT3, and phospho-STAT (pSTAT) levels were
determined by Western blotting. The asterisk indicates a
probable degradation product of STAT1 in IFN
-stimulated cells.
C, conditioned media (cond med.) from neutrophils
left unstimulated (
) or treated with 100 ng/ml LPS
(+) for 4 h were treated with 10 µg/ml polymyxin B to
adsorb any free LPS and added to fresh neutrophil preparations for
4 h. RT-PCR was performed to determine expression of
MX1, TNFA, and GAPDH. Fresh
neutrophils were also left unstimulated (NS) or stimulated
with LPS (100 ng/ml) for 4 h.
/
protein is tightly regulated by
IFN gene expression, the possibility exists that neutrophils release
preformed IFN protein upon stimulation by LPS. However, IFN
and
IFN
were not detected in the supernatants of resting and
LPS-stimulated neutrophils (data not shown). Together, these data
indicate that induction of ISGs in LPS-stimulated neutrophils is
independent of the transcription and production of type I interferons.
(51). IFN
/
activate the STAT family of
transcriptional regulators by Jak1/Tyk2-mediated phosphorylation of
STATs on tyrosine (36, 37). Whole cell extracts were probed for STAT1,
STAT3, and tyrosine-phosphorylated STAT1 and STAT3. STAT1
(p90) and
STAT3
(p92) were the predominant species detected in neutrophils.
Exposure of neutrophils to LPS for 30 min and 4 h did not increase
the level of phospho-STAT1 isoforms or phospho-STAT3 isoforms (Fig.
2B). Exposure of neutrophils to LPS for 30 min was
consistently observed to decrease the basal phosphorylation level of
both STAT proteins. In contrast, IFN
stimulation of cells led to a
robust, time-dependent phosphorylation of both STAT
proteins (Fig. 2B). Stimulation of neutrophils with IFN
,
but not LPS, for 4 h resulted in the loss of STAT1 but not STAT3.
production by LPS mediates ISG
induction (36, 37). In order to clarify the difference between
neutrophils and macrophages, we produced human monocyte-derived
macrophages and stimulated them with LPS. Monocyte-derived macrophages
treated with LPS increased MX1 transcript levels (Fig.
3A). Like neutrophils, resting
monocyte-derived macrophages expressed little IFNA, and LPS
did not modulate IFNA levels (Fig. 3A), as
determined by RT-PCR. However, LPS-stimulated monocyte-derived
macrophages up-regulated IFNB (Fig. 3A),
consistent with previous studies (36) with murine macrophages.
View larger version (22K):
[in a new window]
Fig. 3.
LPS-stimulated macrophages induce
interferons, activate STAT1 and STAT3, and secrete an ISG-inducing
factor. A, monocyte-derived macrophages were stimulated
for 4 h with LPS (100 ng/ml), and gene expression was assessed by
RT-PCR using primers to the indicated genes. B,
monocyte-derived macrophages were treated for the indicated times with
LPS (100 ng/ml) and IFN (100 units/ml) or left untreated
(NS). Whole cell extracts were prepared, and STAT1, STAT3,
and phospho-STAT (pSTAT) levels were determined by Western
blotting. C, conditioned media (cond med.) from
monocyte-derived macrophages left unstimulated (
) or
treated with 100 ng/ml LPS (+) for 4 h were treated
with 10 µg/ml polymyxin B and added to fresh monocyte-derived
macrophages for 4 h. RT-PCR was performed to determine expression
of MX1, TNFA, and GAPDH. Fresh
monocyte-derived macrophages were also left unstimulated
(NS) or stimulated with LPS (100 ng/ml) for 4 h.
was not affected by SB203580 (Fig.
4A). Consistent with the lack of effect of SB203580, IFN
stimulated little p38 phosphorylation and did so with delayed kinetics
(Fig. 4C). However, LPS leads to the rapid and sustained phosphorylation of p38 in neutrophils (Fig. 4B) (28). We
have shown previously (27) that phosphorylation of p38 correlates with
the activation of p38 kinase in LPS-stimulated neutrophils (data not
shown). Levels of p38 protein are not changed by the treatments. An
inhibitor of the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/MAP kinase pathway, PD98059, failed to
alter levels of MX1 in LPS-stimulated cells (data not
shown). Therefore, the activation of p38 is an important signal for ISG induction by LPS but is dispensable for signaling by IFN
.
View larger version (33K):
[in a new window]
Fig. 4.
Induction of MX1 by LPS is
sensitive to SB203580 in neutrophils. A, neutrophils
were left unstimulated or preincubated with SB203580 (SB; 10 µM) for 15 min and treated with LPS (100 ng/ml) for
4 h. Cells were also treated with IFN (100 units/ml) for 4 h. RT-PCR was performed to determine the expression of MX1
and GAPDH. B, neutrophils were pretreated with
p38 inhibitors SB203580 (10 µM) and M39 (1 µM) (inhibitors 1 and 3) or inactive analog SB202474 (10 µM) (inhibitor 2) prior to stimulation with LPS (100 ng/ml), as indicated. RT-PCR was performed as described previously.
C, whole cell extracts of neutrophils treated with LPS (100 ng/ml) or IFN
(100 units/ml) for 30 min and 4 h or from
non-stimulated neutrophils (NS) were probed with antibody to
phospho-p38 (pp38), and the same blot was reprobed with
antibody to p38.
View larger version (37K):
[in a new window]
Fig. 5.
Induction of MX1 by LPS is
insensitive to SB203580 in macrophages. A,
monocyte-derived macrophages were left unstimulated or pretreated with
SB203580 (SB; 10 µM) or vehicle and stimulated
with LPS (100 ng/ml) for 4 h. RT-PCR was performed to determine
the expression of MX1, TNFA, and
GAPDH. B, lysates from monocyte-derived
macrophages treated with LPS (100 ng/ml) or IFN (100 units/ml) for
30 min and 4 h or from non-stimulated cells (NS) were
probed with antibody to phospho-p38 (pp38), and the same
blot was reprobed with antibody to p38.
stimulate the
expression of MX1 message. The ability of other relevant
ligands to induce MX1 in neutrophils was determined. The
pro-inflammatory cytokines TNF
and IL-1
, fMet-Leu-Phe, and OSM
failed to induce MX1 expression (data not shown).
View larger version (23K):
[in a new window]
Fig. 6.
Ligands for TLR4, but not TLR2, stimulate
MX1 induction. A, neutrophils were
pretreated with SB203580 (SB; 10 µM) or
vehicle and stimulated with LPS (100 ng/ml) or PGN (10 µg/µl) for
4 h. MX1, TNFA, and GAPDH
expressions were determined by RT-PCR. B, monocyte-derived
macrophages were stimulated with LPS (100 ng/ml) or PGN (10 mg/µl)
for 4 h. MX1, TNFA, and GAPDH
expressions were determined by RT-PCR. C, neutrophils were
stimulated with either PGN (10 µg/ml) for the indicated times or LPS
(100 ng/ml) for 20 min. The kinase activity of immunoprecipitated p38
was assessed using ATF-2 as a substrate. A representative
autoradiograph is depicted in the upper panel.
Kinase activity was quantified by PhosphorImager analysis, and the
means ± S.E. of three experiments is shown in the
lower panel; the mean fold change is indicated
below the graph.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
or IFN
was not observed in the supernatants of
LPS-treated neutrophils. Second, phosphorylation of STAT proteins, a
well characterized marker of STAT activation, was not observed in
neutrophils stimulated with LPS. Finally, the supernatant from
LPS-stimulated neutrophils was unable to support induction of
MX1, indicating that a paracrine mechanism was not
responsible. In contrast, LPS stimulation in other cell types is
associated with interferon-dependent induction of ISGs and
signaling events (36, 37).
(36, 37). Data presented here
support the enhanced expression of the IFNB gene by LPS, enhanced STAT phosphorylation that is delayed with respect to STAT
phosphorylation in IFN
-treated monocyte-derived macrophages, and the
ability of the supernatant from LPS-stimulated monocyte-derived macrophages to support MX1 induction in naive
monocyte-derived macrophages. That LPS stimulates induction of ISGs in
neutrophils in the absence of type I interferons or
interferon-dependent signaling has not, to our knowledge,
been observed previously. Therefore, a clear distinction exists between
induction of ISGs in neutrophils and monocyte-derived macrophages after
exposure to LPS. We conclude that in neutrophils LPS activates a novel
and intrinsic ISG-inducing signaling pathway. Interferon-independent
induction of ISGs can occur in response to dsRNA, although the
dependence on IFN
/
is cell type-dependent (40, 51,
54).
B in the induction of
ISGs by LPS (61). However, our studies differ in several respects.
Doyle et al. (61) describe a set of early
"interferon-stimulated genes" whose induction is dependent on
NF
B, including IFNB. The early response is additionally
modulated by IRF3; however, subsequent late ISG induction (including
that of MX1) is dependent on IFN
(61) as seen in
virus-infected cells (58). Due to complications in genetically
manipulating neutrophils, we are unable at this time to modulate this
pathway. Further experiments are necessary to resolve the role of IRF3, and other IRF family members, in the transcriptional response of
neutrophils to LPS. Although we have not determined the importance of
NF
B on the response in human neutrophils, IFN
production does not
appear to be involved. However, it is possible that the protein
synthesis-sensitive factor is a newly synthesized
NF
B-dependent gene (see below).
-stimulated MX1 induction in
neutrophils, further indicating that p38 activation and STAT regulation
are independent signaling events in neutrophils. IFN
is a weak
inducer of p38 phosphorylation in neutrophils, which again indicates
that the LPS effect on ISG induction differs from that stimulated by
IFN
. p38 has been implicated in the induction of ISG54 in
response to LPS in astrocytes, and IRF3 was shown to translocate to the
nucleus after several hours of LPS exposure (64); however, these
investigators did not link IRF3 activation to p38 activity or comment
on IFN
/
production.
, TGFBR-I and -II, and Lck (43,
65). However, the IC50 values of SB203580 for Lck (20 µM) and TGFBR-I (40 µM) are greater than
that used in these studies (10 µM), and a JNK inhibitor,
SP600125, did not alter MX1 gene expression (data not
shown). Whereas Raf is inhibited in vitro at a lower
concentration of SB203580 (IC50 2 µM), no inhibition is evident in vivo (66), and an inhibitor of the Raf substrate mitogen-activated protein kinase/extracellular
signal-regulated kinase kinase, PD98059, has no effect on
MX1 induction by LPS. Finally, SB202474, an inactive analog
of SB203580, was ineffective at blocking MX1 induction,
whereas a more specific inhibitor of p38, M39 (43), also was effective
at inhibiting MX1 induction. Although it is necessary at
present to use inhibitors to modulate kinases in neutrophils, the data
indicate that SB203580 is acting by the inhibition of p38.
/
and virus (71, 72) and necessary
for a full genetic response to infection (71-73). However, we were
unable to detect IRF7 under any conditions in neutrophils.
Alternatively, the maintenance of a constitutively expressed, but
labile, protein may be the target of cycloheximide. The
interferon-independent induction of ISGs by dsRNA is generally independent of de novo protein synthesis (54, 74). The known inhibitory effect of SB203580 on cytokine synthesis supports the possibility that SB203580 and cycloheximide act to inhibit production of the same factor.
and MIP-1
that may compete with viral co-receptors.
Macrophages exposed to LPS display antiviral activity to human
immunodeficiency virus by secretion of MIP-1
and MIP-1
(78, 79)
and down-regulation of CCR5 (80). The induction of antiviral ISGs by
LPS described here suggests that exposure of neutrophils to bacterial
infection primes neutrophils for an antiviral role. However, this study
shows that macrophages and neutrophils have different mechanisms of ISG
induction by LPS, suggesting that antiviral mechanisms may also differ.
The finding that LPS induces antiviral ISGs suggests that
anti-bacterial pathways and antiviral pathways are linked in
neutrophils. Alternatively, ISG products could function in the more
established antibacterial activity of neutrophils. Recently, the F
protein of respiratory syncytial virus was shown to recognize TLR4
(81); whether this affected induction of ISGs was not determined. This
may not represent a universal mechanism of antiviral response because
many viruses require internalization and replication before ISGs are
induced (41, 82).
/
, or STAT activation. Furthermore, the
signal transduction pathways necessary for induction of ISGs in
neutrophils involves p38, is activated by a TLR4 ligand, and is not
activated by a TLR2 ligand. Together, our results suggest a new
mechanism for neutrophils in antiviral defense and that novel signaling
pathways are activated in response to LPS, which differ substantially
from pathways activated by LPS in macrophages. These studies may aid
our understanding of the mechanisms of TLR activation and of innate immunity.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Sally Billstrom and Carol Sable for careful reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant HL61407 (to G. S. W.).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.
§ To whom correspondence should be addressed: National Jewish Medical and Research Center, 1400 Jackson St., Denver, CO 80206. Tel.: 303-398-1640; Fax: 303-398-1381; E-mail: malcolmk@njc.org.
Published, JBC Papers in Press, February 20, 2003, DOI 10.1074/jbc.M212033200
2 K. C. Malcolm, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: LPS, lipopolysaccharide; dsRNA, double-stranded RNA; IFN, interferon; IRF, IFN-regulatory factor; ISG, IFN-stimulated gene; M39, (S)-5-[2-(1-phenylethylamino)pyrimidin-4-yl]-1-methyl-4-(3-trifluoromethylphenyl)-2-(4-piperidinyl)imidazole; MX1, myxovirus-resistance gene-1; PGN, peptidoglycan; PKR, dsRNA-dependent protein kinase; STAT, signal transducer and activator of transcription; TLR, toll-like receptor; GAPDH, glyceraldehyde-3- phosphate dehydrogenase; MAP, mitogen-activated protein; RT, reverse transcription.
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