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Department of Immunology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio
Correspondence: Yoshihiro Ohmori, D.D.S., Ph.D., Department of Immunology, NB30, Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195. Email: ohmorih{at}ccf.org
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and KC (a C-X-C chemokine) mRNA in LPS-treated cell cultures were
unaffected. A similar deficiency in LPS-induced gene expression was
observed in livers and spleens from Stat1-/- mice. The
reduced LPS-stimulated gene expression seen in Stat1-/-
macrophages was not the result of reduced activation of nuclear factor
B. LPS stimulated the delayed activation of both IFN-stimulated
response element and IFN-
-activated sequence binding activity in
macrophages from wild-type mice. Activation of these STAT1-containing
transcription factors was mediated by the intermediate induction of
type I IFNs, since the LPS-induced IP-10, IRF-1, and iNOS mRNA
expression was markedly reduced in macrophages from
IFN-/ßR-/- mice and blocked by cotreatment with
antibodies against type I IFN. These results indicate that indirect
activation of STAT1 by LPS-induced type I IFN participates in promoting
optimal expression of LPS-inducible genes, and they suggest that STAT1
may play a critical role in innate immunity against gram-negative
bacterial infection.
Key Words: transcriptional regulation • type I IFNs
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The finding that the Toll-like receptor-4 is the primary signaling
receptor for LPS in mice has aided progress in elucidating the
intracellular pathways that mediate response to LPS
[5
6
7
]. After interaction with LPS, Toll-like receptor-4
appears to initiate an ordered recruitment of adapter molecules and
kinases including MyD88, interleukin (IL)-1 receptor-associated kinase,
tumor necrosis factor (TNF) receptor-associated factor 6, nuclear
factor-B (NF-B)-inducing kinase, and IB kinases
[8
]. These adapter molecule-kinase interactions lead to
phosphorylation, ubiquitination, and degradation of IB with
associated release and nuclear translocation of NF-B
[9
, 10
]. Members of the Rel family such as
RelA (p65), NFkB1 (P50), and c-Rel have been shown to be activated by
LPS in macrophages [11
]. These Rel family members
functionally and physically interact with members of other
transcription factor families and cooperatively regulate the
transcriptional activation of many LPS-inducible genes
[12
, 13
]. LPS is also known to induce de
novo synthesis of transcription factors and to enhance transacting
functions of factors such as PU.1 and signal transducer and activator
of transcription 1 (STAT1) through selective phosphorylation
[4
, 14
, 15
]. LPS may also act
indirectly through the intermediate expression of cytokines, which can
alter macrophage gene expression cooperatively or antagonistically
through autocrine or paracrine loops [16
,
17
]. This diversity of mechanisms creates substantial
complexity in the pattern of LPS-induced gene expression during
bacterial infection.
The JAK/STAT signaling pathway has been shown to be an essential signaling component for cytokine-mediated gene expression in immune responses [18 ]. Indeed, an obligate role for STAT1 in interferon (IFN)-dependent biological responses has been demonstrated using mice in which the STAT1 gene has been deleted, although no obvious defects in sensitivity or response to other cytokines known to activate STAT1 have been noted [19 , 20 ]. These mice have been shown to be highly susceptible to viral and some forms of bacterial infections. It is interesting that LPS and IFN are known to induce expression of a common set of genes in sensitive cell types such as macrophages [2 , 4 ]. Although STAT1 appears to play an essential role in various forms of innate immunity, its participation in LPS-inducible gene expression is incompletely understood. In addition to the serine-directed phosphorylation of STAT1 mentioned above [15 ], three roles are possible. First, some or all LPS-inducible changes in gene expression may be independent of STAT1 activation. Second, LPS may directly activate one or more STAT molecules; this has been suggested in several studies although others have reported that LPS signaling does not directly involve the JAK/STAT paradigm [21 22 23 24 25 ]. Finally, since LPS is well known to activate the expression of multiple cytokine genes including type I IFNs [17 , 26 , 27 ], these genes may be indirectly responsible for some portion of LPS-induced response. The present study was undertaken to distinguish between these scenarios and to explore the mechanisms involved. The results demonstrate that STAT1 is essential for maximum expression of LPS-stimulated IFN-inducible 10-kDa protein (IP-10), IFN regulatory factor-1 (IRF-1), and inducible nitric oxide synthase (iNOS) gene expression through the indirect activation of STAT1 by intermediate production of type I IFNs.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(M-23),
STAT2 (C-18), interferon stimulated gene factor-3 (ISGF3)-p48),
NFB1 (NLS), and RelA (A) were obtained from Santa Cruz Biotechnology
(Hercules, CA). Rabbit antiserum to mouse type I IFN (IFN- and
IFN-ß) was obtained from Lee BioMolecular Research Laboratory (San
Diego, CA).
Mice and Cell Culture
Homozygous STAT1 mutant (Stat1-/-) mice in which
the Stat1 gene has been deleted by homologous recombination
and wild-type 129/B6 mice were kindly provided by R. D. Schreiber
(Washington University School of Medicine, St. Louis, MO)
[19
]. Homozygous type I IFN receptor mutant mice
(IFN-/ßR-/-) and wild-type 129Sv/Ev mice were
obtained from B & K Universal (Hull, U.K.) [28
].
Specific pathogen-free C57Bl/6 mice 9 to 12 weeks of age were purchased
from the Jackson Laboratory (Bar Harbor, ME). Thioglycollate
broth-elicited peritoneal macrophages were prepared as described
previously [29
] and cultured in RPMI 1640 medium
containing L-glutamine, penicillin, streptomycin, and 5%
fetal bovine serum.
Preparation of RNA and Northern Hybridization Analysis
Total RNA was extracted by the guanidine isothiocyanate-cesium
chloride method as describe previously [30
]. Northern
hybridization analysis and cDNA probes for mouse IFN- IP-10, IRF-1,
iNOS, TNF-, KC (a C-X-C chemokine), and rat
glyceraldehyde-3-phosphate dehydrogenase were described previously
[13
, 29
, 31
, 32
].
cDNA fragment for mouse IFN-ß was prepared by reverse
transcriptase-polymerase chain reaction using a set of primers
corresponding to the mouse IFN-ß cDNA sequence obtained from the
GenBank data base [33
]. Northern blots were also
quantified using phosphorescence detection. The relative magnitude of
expression was determined for each gene and normalized to values for
glyceraldehyde-3-phosphate dehydrogenase expression in the same
experiment.
Preparation of Nuclear Extracts
Nuclear extracts were prepared using a modification of the
method of Dignam et al. [34
] as described previously
[13
]. After stimulation, the cells were washed with
ice-cold phosphate-buffered saline three times, harvested, and
resuspended in 300 µL of hypotonic buffer A (10 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid [HEPES; pH 7.9], 10 mM KCl, 0.1 mM ethylenediaminetetraacetic
acid [EDTA], 0.1 mM
ethyleneglycol-bis(ß-aminoethylether)-N,N'-tetraacetic
acid, 1 mM dithiothreitol [DTT], 1 mM phenylmethylsulfonyl fluoride,
and 10 µg/mL of leupeptin, antipain, aprotinin, and pepstatin) for 10
min on ice. The cells were then lysed in 0.6% Nonidet P-40 by
vortexing for 10 s. Nuclei were separated from cytosol by
centrifugation at 12,000 x g for 30 s, washed
with 300 µL of buffer A, and resuspended in buffer C (20 mM HEPES
[pH 7.9], 25% glycerol, 0.4 M NaCl, 1mM EDTA, 1mM
ethyleneglycol-bis(ß-aminoethylether)-N,N'-tetraacetic
acid, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/mL of
leupeptin, antipain, aprotinin, and pepstatin) and briefly sonicated on
ice. Nuclear extracts were obtained by centrifugation at 12,000 x
g for 10 min. Protein concentration was measured by the
method of Bradford [35
] by using the protein dye reagent
(Bio-Rad).
Electrophoretic-Mobility Shift Assay (EMSA)
The following oligonucleotides were used in EMSA (sense strand):
IP-10 IFN-stimulated response element (ISRE) [36
]:
5'-gatctCTCACGCTTTGGAAAGTGAAACCTACCTCACTCa-3', IRF-1
IFN--activated sequence (GAS) [37
];
5'-tcgaGCCTGATTTCCCCGAAATGAGGC-3', IP-10-B2
[11
];
5'-gatcGAGGGGAGAGGGAAATTCCAAGTTCATG-3'. Underlined
sequences represent the consensus sequence for the ISRE, GAS, and B,
respectively. For binding reactions, nuclear extracts (5 µg of
protein) were incubated in 12.5 µL total volume containing 20 mM
HEPES (pH 7.9), 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 5% glycerol, 200
µg/mL of bovine serum albumin, and 1.25 µg of poly(dI-dC) for 15
min at room temperature. 32P-labeled oligonucleotide (0.5
ng, 5 x 105 cpm) was then added to the reaction
mixture and incubated for 15 min at room temperature. The reaction
products were analyzed by electrophoresis in a 5% polyacrylamide gel
with 0.25 x TBE buffer (22.3 mM Tris, 22.2 mM borate, 0.5 mM
EDTA). In some experiments, rabbit antibody to NF-B1 (p50); RelA
(p65); and STAT1, STAT2, and ISGF3- (p48) were added prior to
electrophoresis. The dried gels were analyzed by autoradiography and by
phosphorescence detection.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and KC mRNA was
equivalent in both wild-type and Stat1-/- macrophages
(Fig. 1C)
.
|
B
[38
]. Since the IP-10, IRF-1, and iNOS genes each
contain one or more NF-B binding sites in their promoter regions
[13
, 36
, 37
,
39
40
41
], the impaired LPS-stimulated expression of these
genes in STAT1-deficient peritoneal macrophages might be caused by a
defect in LPS-mediated activation of NF-B. To determine whether
NF-B activation was impaired in STAT1-deficient macrophages, nuclear
extracts were prepared using LPS-stimulated peritoneal macrophages from
wild-type or STAT1-deficient mice and analyzed for specific DNA-binding
activities by EMSA. LPS rapidly induced NF-B binding activities
within 30 min, which persisted for at least 4 h (Fig. 2A
). A similar pattern of complex formation was observed in nuclear
extracts from LPS-stimulated STAT1-deficient macrophages. Antibody
supershift assay showed that the composition of B-binding complexes
in LPS-stimulated STAT1-deficient macrophages was almost identical to
that seen in wild-type macrophages (Fig. 2B)
; while the anti-NFkB1
(p50) antibody supershifted a portion of the upper complex and the
entire lower complex, anti-RelA antibody (p65) supershifted only the
upper complex. Thus, the lower complex contained NFkB1 (p50) homodimer
and upper complex contained NFkB1 (P50)/RelA (p65) heterodimer.
|
B and STAT1 synergize to
induce transcription of genes which contain both STAT1 and NF-B
DNA-binding sites in their promoters [13
,
39
, 42
], the requirement for STAT1 in
LPS-induced gene expression could reflect the ability of LPS to
activate DNA-binding activities that recognize such sites. As shown in
Fig. 3A
, a prominent ISRE binding activity could be detected in nuclear
extracts from LPS-treated wild-type macrophages only after 4 h of
stimulation. The LPS-induced ISRE binding complex appeared to be ISGF3
[43
]; the mobility of the complex was indistinguishable
from that of the IFN-ß-induced ISGF3 (Fig. 3B
, lane 12), and the
LPS-induced complex was specifically reduced by inclusion of
excess unlabeled homologous ISRE oligonucleotide but not GAS or
irrelevant oligonucleotide in the reaction (lanes 9–11). Furthermore,
antibody supershift assays showed that antibodies against STAT1, STAT2,
or ISGF3- (p48) diminished complexes activated by both LPS and
IFN-ß (Fig. 3C)
. LPS treatment also activated a GAS motif binding
activity within 4 h of treatment in wild-type macrophages
(Fig. 4A
). Antibody reactivity indicated that the LPS-induced GAS binding
complex was likely composed of STAT1 homodimers (Fig. 4B)
. The ability
of LPS to activate both ISRE and GAS binding activities was relatively
slow (requiring 4 h) and suggested that this effect of LPS might
be indirect.
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B. Several experimental results supported this possibility.
First, LPS was a potent stimulus of IFN-ß mRNA expression in
peritoneal macrophages from wild-type mice (Fig. 5A
). Second, expression of LPS-induced IP-10, IRF-1, and iNOS
mRNAs was ablated in macrophages from
IFN-/ßR-/- mice [28
], while expression
of TNF- mRNA was comparable in macrophages of both wild-type and
type I IFN receptor-deficient phenotypes (Fig. 5B)
. Finally, antibodies
against type I IFN were able to block LPS-induced IP-10, IRF-1, and
iNOS mRNA expression, further supporting the autocrine/paracrine action
of LPS-induced type I IFN (Fig. 5C)
.
|
mRNA expression
were essentially unaffected in STAT1-deficient mice. These results
indicate that STAT1 is also necessary for the optimal induction of
LPS-induced gene expression in vivo.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B activation was not impaired in LPS-stimulated
STAT1-deficient macrophages; (4) LPS induced expression of IFNß mRNA
in wild-type macrophages; (5) LPS-induced IP-10, IRF-1, and iNOS mRNA
expression was lost in macrophages from IFN/ßR-/-
mice and in macrophages treated with antibodies against type I IFN. Although there have been reports of LPS-stimulated STAT activation, the direct action of LPS on the JAK/STAT-signaling pathway has not been clearly demonstrated. A previous report showed that LPS can directly activate a STAT-like factor in THP-1 human monocytic cells [21 ], although other studies failed to detect direct activation of known STATs in LPS-stimulated mouse macrophages and human monocytes [15 , 23 , 24 ]. In contrast, LPS has been reported to induce indirect activation of certain STAT family members [22 , 24 , 25 ]. Although LPS induces many cytokines which are capable of activating STAT family members [17 , 26 , 27 ], the observed STAT activation in LPS-stimulated macrophages is relatively limited. In human monocytes, LPS has been shown to activate STAT5 through intermediate expression of granulocyte-macrophage colony-stimulating factor [24 ] while STAT1 appears to be the predominant LPS-inducible STAT in mouse peritoneal macrophages via intermediate production of type I IFNs. It is interesting that LPS has also been shown to induce STAT3 activation in livers from mice injected with LPS [22 ]. In that study, LPS-induced IL-6 was considered the most likely direct activator of STAT3. Although we have not measured IL-6 or STAT3 activation, the induction of the IP-10, IRF-1, and iNOS mRNAs in livers from LPS-treated mice is STAT1 dependent.
LPS is well known to stimulate type I IFN production by monocytes and macrophages, and this is believed to be important for LPS-induced iNOS gene expression via type I IFN activation of STAT1 [16 , 25 ]. The present study directly demonstrated the requisite role of STAT1 in LPS-induced iNOS gene expression as well as in IRF-1 and IP-10 gene expression both in vitro and in vivo. Furthermore, since type I IFNs have been shown to activate several members of the STAT family [44 45 46 ], the present result clearly indicates that STAT1 is the critical determinant for certain LPS-inducible genes in mouse macrophages.
STAT1 and NF-B have been shown to cooperate for transcriptional
activation of many inflammatory genes which contain the cognate binding
sites in their promoters [13
, 39
,
42
, 47
]. Indeed, all three genes examined
here exhibited this characteristic. Although LPS is a potent inducer of
NF-B, which may activate transcription of IP-10, IRF-1, or iNOS soon
after stimulation, this signal alone is insufficient for the optimal
induction of these genes. Indeed, LPS-induced type I IFN appears to be
required for the maximum transcriptional activation of all three genes.
LPS-induced type I IFNs induced both ISGF3 and STAT1 homodimers, which
bind respectively to the ISRE and GAS motifs in the promoter of these
genes. These different forms of STAT1 could then cooperate with NF-B
to induce full transcriptional activation of these two genes.
Previous studies on IRF-1 knockout mice have revealed an absolute
requirement for IRF-1 in the induction of iNOS gene expression in
response to IFN- and LPS [48
]. The present study
suggests that LPS-induced NF-B and IRF-1 alone might be insufficient
to induce expression of the iNOS gene. The LPS-induced iNOS expression
is almost completely abolished in STAT1-deficient peritoneal
macrophages despite the fact that LPS activates NF-B and induces
modest IRF-1 expression (Fig. 1
; 
20% of wild type). Thus, although
STAT1 induces IRF-1 expression, coordinate activation of STAT1,
NF-B, and IRF-1 may be required for the transcriptional control of
the iNOS gene [41
, 48
, 49
].
Positive and negative regulatory roles for type I IFNs in immune
responses have been reported previously. Although type I IFN is
essential for antiviral activities [28
], type I IFNs
have been shown to negatively regulate IL-12 expression and the
associated IFN- production in vivo and in vitro [50
].
In some experimental circumstances, the exogenous addition of type I
IFN has been shown to suppress IFN--induced iNOS expression by
inhibiting IRF-1 expression or NF-B activation [51
,
52
]. Thus, the biological activity of type I IFN in
immune and inflammatory responses appears to depend on the time when
the IFNs are expressed and the tissue microenvironment.
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ACKNOWLEDGEMENTS |
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Received September 21, 2000; accepted November 27, 2000.
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and IFN- is mediated by p65/p50 and p65/c-Rel and interferon-responsive factor Stat1- (p91) that can be activated by both IFN- and IFN- FEBS Lett 354,220-226[Medline]
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