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p53 Attenuates Lipopolysaccharide-Induced NF-
B Activation
and Acute Lung Injury
1
Gang Liu, Young-Jun Park, Yuko Tsuruta, Emmanuel Lorne, and Edward Abraham
2
The transcriptional factor p53 has primarily been characterized for its central role in the regulation of oncogenesis. A reciprocal
relationship between the activities of p53 and NF-
B has been demonstrated in cancer cells, but there is little information
concerning interactions between p53 and NF-
B in inflammatory processes. In this study, we found that neutrophils and mac-
rophages lacking p53, i.e., p53
ⴚ/ⴚ
, have elevated responses to LPS stimulation compared with p53
ⴙ/ⴙ
cells, producing greater
amounts of proinflammatory cytokines, including TNF-
␣
, IL-6, and MIP-2, and demonstrating enhanced NF-
B DNA-binding
activity. p53
ⴚ/ⴚ
mice are more susceptible than are p53
ⴙ/ⴙ
mice to LPS-induced acute lung injury (ALI). The enhanced response
of p53
ⴚ/ⴚ
cells to LPS does not involve alterations in intracellular signaling events associated with TLR4 engagement, such as
activation of MAPKs, phosphorylation of I
B-
␣
or the p65 subunit of NF-
B, or I
B-
␣
degradation. Culture of LPS-stimulated
neutrophils and macrophages with nutlin-3a, a specific inducer of p53 stabilization, attenuated NF-
B DNA-binding activity and
production of proinflammatory cytokines. Treatment of mice with nutlin-3a reduced the severity of LPS-induced ALI. These data
demonstrate that p53 regulates NF-
B activity in inflammatory cells and suggest that modulation of p53 may have potential
therapeutic benefits in acute inflammatory conditions, such as ALI. The Journal of Immunology, 2009, 182: 5063–5071.
Macrophages and neutrophils are among the first cells
that interact with invading microbial pathogens and
respond by producing proinflammatory mediators, in-
cluding cytokines and chemokines, reactive oxygen species, and
antimicrobial peptides, which participate in host defense mecha-
nisms aimed at eradicating bacterial or viral infection (1– 4). How-
ever, although inflammatory responses are crucial in mounting ef-
fective antimicrobial defense, overly exuberant inflammation can
be deleterious, resulting in organ dysfunction, including acute lung
injury (ALI)
3
(5, 6).
Inflammatory cells recognize distinct microbial products, which
exist as pathogen-associated molecular patterns (PAMPs), through
TLRs (7–10). Association of TLRs with PAMPs leads to recruit-
ment of adaptor proteins and kinases, such as MyD88, IL-1R-
associated kinase 1, IIL-1R-associated kinase 4, and TNF
receptor-associated factor 6, to the TLR intracellular domain
(7–10). The TLR-associated complex then activates down-
stream signaling cascades resulting in activation of MAPKs and
kinases, such as the I
B kinase (IKK), which are involved in
activating NF-
B (7–10). IKK phosphorylates I
B-
␣
and leads
to its degradation, allowing NF-
B to be translocated to the
nucleus, where it binds to the promoters of target genes, in-
cluding TNF-
␣
,MIP-2,I
B-
␣
, and cellular inhibitor of apopto-
sis, activating their transcription (7–10).
Increased activation of NF-
B is found in PBMC, neutrophils,
and alveolar macrophages after exposure to the TLR4 ligand, LPS,
and in patients with sepsis (11–14). In addition, greater or more
persistent nuclear accumulation of NF-
B is associated with
higher mortality and more severe organ dysfunction in such pa-
tients, probably due to excessive induction of proinflammatory cy-
tokines and delayed apoptosis of immune cells such as neutrophils
(11, 12, 15, 16).
p53 is a transcriptional factor that induces the expression of a num-
ber of downstream target genes involved in apoptosis, cell cycle ar-
rest, and DNA repair (17–19). In resting cells, p53 is maintained at
low levels, primarily through the actions of its negative regulator,
murine double minute (Mdm2), an E3 ubiquitin ligase (18). In re-
sponse to DNA damage and other cellular stresses, p53 is phosphor-
ylated by ATM (ataxia-telangiectasia, mutated), ATR (ATM and
Rad3 related), and/or ChK1/2 (checkpoint kinase 1/2) (18). p53 phos-
phorylation disrupts interactions between p53 and Mdm2, thus lead-
ing to p53 stabilization and increased transcriptional activity (18, 20).
Recently, a compound (nutlin-3a) that specifically blocks the interac-
tion between p53 and Mdm2 has been developed. Nutlin-3a stabilizes
p53 without inducing DNA damage and has been shown to enhance
p53 activity, such as induction of apoptosis and cell cycle arrest of
cancer cells (21–23).
Previous studies have demonstrated mutual regulation between
NF-
B and p53 in cancer cells (24 –26). For example, NF-
B was
shown to inhibit p53 transcriptional activity (25). This was sug-
gested to be a major mechanism by which NF-
B promotes on-
cogenesis (25). In addition, several p53 mutants were found to
induce the expression of NF-
B subunits, including p100 (27).
However, it is less clear how p53 itself regulates NF-
B activity,
especially in inflammatory cells after TLR engagement by PAMPs
or other ligands.
In this study, we investigated the involvement of p53 in the
responses of inflammatory cells to LPS. We found that p53 not
Department of Medicine, University of Alabama at Birmingham, AL 35294
Received for publication October 20, 2008. Accepted for publication February
5, 2009.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by a pilot grant to G.L. under National Institutes
of Health Grant 5P30DK072482 and National Institutes of Health Grant
5R01HL62221 to E.A.
2
Address correspondence and reprint requests to Dr. Edward Abraham, Department
of Medicine, University of Alabama at Birmingham, School of Medicine, 420 Boshell
Building, 1808 7th Avenue South, Birmingham, AL 35294. E-mail address:
eabraham@uab.edu
3
Abbreviations used in this paper: ALI, acute lung injury; PAMP, pathogenesis-
associated molecular patterns; IKK, I
B kinase; BAL, bronchoalveolar lavage; PI,
propidium iodide; MPO, myeloperoxidase; ChIP, chromatin immunoprecipitation.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
The Journal of Immunology
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803526
only negatively regulates activation of inflammatory cells, includ-
ing neutrophils and macrophages, by LPS, but also diminishes the
severity of LPS-induced ALI.
Materials and Methods
Mice
p53-deficient (p53
⫺/⫺
) mice on the C57BL/6 background were gifts from
Dr. K. Roth (University of Alabama at Birmingham). Age- and sex-
matched wild-type C57BL/6 (p53
⫹/⫹
) mice were purchased from the Na-
tional Cancer Institute-Frederick. Eight- to 10-wk-old male animals were
used for experiments. The mice were kept on a 12-h light/dark cycle with
free access to food and water. Animal protocols were reviewed and ap-
proved by the Institutional Animal Care and Use Committee of the Uni-
versity of Alabama at Birmingham.
Materials
Nutlin-3a was purchased from Cayman Chemical. LPS from Escherichia
coli 0111:B4 and rabbit anti-actin Abs were from Sigma-Aldrich.
PAM3CSK4 was from InvivoGen. Abs specific for phosphorylated JNK,
total JNK, phosphorylated ERK, total ERK, phosphorylated p38, total p38,
phosphorylated I
B-
␣
(S32/S36), I
B-
␣
, and phosphorylated p65 (S635)
were from Cell Signaling. Rabbit anti-p65, anti-GAPDH, and anti-p53 Abs
were from Santa Cruz Biotechnology. Custom mixture Abs and negative
selection columns for neutrophil isolation were purchased from StemCell
Technologies. Protein G-agarose beads were from Pierce.
In vivo ALI model
The murine ALI model was used as previously described (28, 29). Briefly,
mice were anesthetized with isoflurane. The tongue was then gently ex-
tended and the LPS solution (1 mg/kg LPS in 50
l) deposited into the
oropharyx. With this model, ALI, as characterized by neutrophil infiltration
into the pulmonary interstitium, development of interstitial edema, and
increased proinflammatory cytokine production, occurs after injection of
LPS, with the greatest accumulation of neutrophils into airways and his-
tologic injury being present 24 h after LPS exposure.
Harvest of bronchoalveolar lavage (BAL) fluid
BAL fluid samples were obtained from LPS-treated or control mice by
lavaging the lungs three times with 1 ml of iced PBS. Total cell counts
were measured in the BAL fluid with a hemocytometer and protein con-
centrations were measured with a Bio-Rad protein assay kit.
Isolation of neutrophils
Mouse neutrophils were purified from bone marrow cell suspensions as
described previously (28, 29). Briefly, the femur and tibia of a mouse were
FIGURE 1. p53
⫺/⫺
inflammatory cells demonstrate enhanced activa-
tion by LPS. A–C, p53
⫺/⫺
neutrophils produced significantly greater
amounts of proinflammatory cytokines than did p53
⫹/⫹
cells after LPS
stimulation. Two ⫻10
6
p53
⫹/⫹
or p53
⫺/⫺
neutrophils were treated with 10
ng/ml LPS for 6 h. Levels of TNF-
␣
(A), IL-6 (B), and MIP-2 (C) in culture
supernatants were measured by ELISA. ⴱ,p⬍0.05 and ⴱⴱⴱ,p⬍0.001
compared with p53
⫹/⫹
cells. D–F, p53
⫺/⫺
macrophages produced signif-
icantly greater amounts of proinflammatory cytokines than p53
⫹/⫹
cells
after LPS stimulation. In brief, 0.5 ⫻10
6
p53
⫹/⫹
or p53
⫺/⫺
peritoneal
macrophages were treated with 5 ng/ml LPS for 6 h. Levels of TNF-
␣
(D),
IL-6 (E), and MIP-2 (F) in culture supernatants were determined by
ELISA. ⴱⴱ,p⬍0.01 and ⴱⴱⴱ,p⬍0.001 compared with p53
⫹/⫹
cells.
Values are the mean ⫾SD of triplicate experiments.
FIGURE 2. Signaling events after TLR4 engagement are not altered in
p53
⫺/⫺
inflammatory cells. A, p53
⫹/⫹
or p53
⫺/⫺
peritoneal macrophages
were treated with 5 ng/ml LPS for the indicated times. The levels of total
and phosphorylated JNK, ERK1/2, and p38 were determined by Western
blotting. B, I
B-
␣
degradation, I
B-
␣
phosphorylation, and p65 phosphor-
ylation were comparable in p53
⫹/⫹
and p53
⫺/⫺
macrophages after LPS
stimulation. p53
⫹/⫹
or p53
⫺/⫺
peritoneal macrophages were treated as in
A.C, I
B-
␣
degradation was comparable in p53
⫹/⫹
and p53
⫺/⫺
neutro-
phils after LPS stimulation. p53
⫹/⫹
or p53
⫺/⫺
neutrophils were treated
with 10 ng/ml LPS for the indicated times and then cell extracts were
obtained for Western blotting. Data are representative of at least three
repeated experiments. WT, wild type.
5064 p53 REGULATES NF-
B ACTIVATION AND ACUTE LUNG INJURY
flushed with RPMI 1640 and the cells were passed through a 40-
m cell
strainer (BD Biosciences). The cell pellets were resuspended in PBS and
then incubated for 15 min, rotating at 4°C, with 20
l of primary Abs
specific for the cell surface markers F4/80, CD4, CD45R, CD5, and
TER119. This custom mixture is specific for T and B cells, RBC, mono-
cytes, and macrophages. Anti-biotin tetrameric Ab complexes (100
l)
were then added, and the cells were incubated for an additional 15 min at
4°C. Following this, 60
l of colloidal magnetic dextran iron particles were
added to the suspension and incubated for 15 min, rotating at 4°C. The cell
suspension was then placed into a column surrounded by a magnet. The T
cells, B cells, RBC, monocytes, and macrophages were captured in the
column, allowing the neutrophils to pass through by negative selection.
Isolation of peritoneal macrophages
Mice were injected i.p. with 1.5 ml of 4% thioglycolate solution. 4 days
after injection, mice were sacrificed and the peritoneal cavities were
flushed with 10 ml of DMEM. The peritoneal lavage fluids were centri-
fuged and the cells were resuspended with DMEM plus 10% FBS and
plated. After incubation for1hat37°C, the cells were washed three times
and nonadherent cells were removed by aspiration. The attached cells were
peritoneal macrophages.
Flow cytometry assays
Neutrophils or macrophages were treated with 0, 10, 20, or 40
M nut-
lin-3a for 6 h. The cells were then collected, washed once with cold PBS,
resuspended with binding buffer containing FITC-annexin V (Calbiochem)
and incubated at room temperature for 15 min. Propidium iodide (PI)
FIGURE 3. LPS-induced NF-
B DNA-binding activities are increased
in p53
⫺/⫺
cells. Aand B, p53
⫹/⫹
and p53
⫺/⫺
macrophages (A) or neutro-
phils (B) were treated with 5 or 10 ng/ml LPS for the indicated periods of
time. The cells were collected and nuclear extracts were prepared. EMSA
was performed as described in Materials and Methods. Data are from three
independent experiments.
FIGURE 4. Nutlin-3a up-regulates p53 levels, but down-regulates the response of inflammatory cells to LPS. A, Nutlin-3a induced p53 in macrophages.
p53
⫹/⫹
peritoneal macrophages were treated with the indicated concentrations of nutlin-3a for 6 h. Western blots were analyzed with densitometry. The
ratio of the levels of p53 to GAPDH in the cells treated without LPS or nutlin-3a was regarded as 1. The fold increase shown is the relative increase for
each indicated treatment compared with no treatment. Values are presented as mean ⫾SD of four independent experiments. ⴱⴱⴱ,p⬍0.001 compared with
no treatment. B–E, Nutlin-3a attenuates the activation of macrophages and neutrophils by LPS. Two ⫻10
6
p53
⫹/⫹
neutrophils (Band C)or2⫻10
6
peritoneal macrophages (Dand E) were pretreated with nutlin-3a at the indicated concentrations for 1 h. The neutrophils (Band C) or macrophages (Dand
E) were then treated without or with 5 (macrophages) or 10 (neutrophils) ng/ml LPS for 6 h. TNF-
␣
and IL-6 concentrations in the culture supernatants
were measured by ELISA. ⴱ,p⬍0.05; ⴱⴱ,p⬍0.01; and ⴱⴱⴱ,p⬍0.001 compared with the control group without nutlin-3a treatment. Values are presented
as mean ⫾SD of quadruplicate experiments. Data are representative of three independent experiments.
5065The Journal of Immunology
(Calbiochem) was then added into the buffers. The cells were then analyzed
by a BD Biosciences LSR II system. The x-axis is FITC and y-axis is PI
fluorescence. Cells with FITC-positive staining were regarded as apoptotic.
Western blotting assay
Western blotting assays were performed essentially as previously described (30).
Cell stimulation
Neutrophils or macrophages were pretreated without or with nutlin-3a at
various concentrations for 1 h. The cells were then stimulated with LPS or
PAM3CSK4 for various lengths of time. The supernatants or cells were
collected for the following analysis.
Cytokine and chemokine ELISA and protein assays
Immunoreactive TNF-
␣
, MIP-2, and IL-6 were quantified using DuoSet
ELISA Development kits (R&D Systems) according to the manufacturer’s
instructions.
Myeloperoxidase (MPO) assay
MPO was measured using a modification of a previously described method
(28, 29). In brief, lung tissue was homogenized using a Glas-Col homog-
enizer in 0.5 ml of 0.5% hexadecyltrimethyl ammonium bromide in 50 mM
potassium phosphate buffer (pH 6.0). The homogenate was centrifuged at
14,000 ⫻gfor 30 min at 4°C and the supernatant was collected for assay
of MPO activity as determined by measuring the H
2
O
2
-dependent oxida-
tion of o-dianisidine solution (3,3⬘-dimethoxybenzidine dihydrochloride in
potassium phosphate buffer, pH 6.0) at 450 nm.
Wet:dry lung weight ratios
All mice used for lung wet:weight ratios were of identical ages. Lungs were
excised, rinsed briefly in PBS, blotted, and then weighed to obtain the
“wet” weight. Lungs were then dried in an oven at 80°C for 7 days to
obtain the “dry” weight.
EMSA
Nuclear extracts were prepared and assayed by EMSA as previously de-
scribed (28). For analysis of NF-
B, the
B DNA sequence of the Ig gene
was used. Synthetic double-stranded sequences (with enhancer motifs un-
derlined) were filled in and labeled with [
␥
-
32
P]dATP (PerkinElmer) using
T
4
polynucleotide kinase as follows:
B sequence, 5⬘-GCCATGGGGG
GATCCCCGAAGTCC-3⬘(Promega).
Chromatin immunoprecipitation (ChIP) assay
The ChIP assay was essentially performed as previously described (30).
Briefly, cells were fixed with 1% of formaldehyde for 10 min. Genomic DNA
was then sheared by sonication to lengths ranging from 200 to 1000 bp. For
FIGURE 5. Nutlin-3a does not induce apoptosis of macrophages or neutrophils. A–C, p53
⫹/⫹
neutrophils were treated with 0, 5, or 20
M nutlin-3a
for 6 h. Cells were then collected and flow cytometry was performed after staining with annexin V and PI to determine the levels of apoptosis. D–F, p53
⫹/⫹
macrophages were treated with 0, 10, or 40
M nutlin-3a for 6 h. The cells were then collected and stained with annexin V and PI, as above, to determine
the levels of apoptosis. Data are representative of three independent experiments.
5066 p53 REGULATES NF-
B ACTIVATION AND ACUTE LUNG INJURY
input determination, 5% of cell extract was taken and the rest of the extract was
incubated with rabbit anti-p65 polyclonal Abs overnight, followed by precip-
itation with protein G-agarose beads. Genomic DNA in the immuno-
complexes was purified by a Qiagen miniprep column, and the NF-
B-
responsive elements in the promoter of NF-
B target genes were
amplified by PCR. The primer sequence for amplification of NF-
B-responsive
elements in the promoter of mouse I
B-
␣
gene was sense 5⬘-TGGCGAGGTCT
GACTGTTGTGG-3⬘and antisense 5⬘-GCTCATCAAAAAGTTCCCTGTGC-
3⬘. The primer sequence for amplification of the mouse MIP2 promoter was sense
5⬘-CAGGAAAGGCAATCCCAGAAAGG-3⬘and antisense 5⬘-GGAGGGT
GCTGAACACTTGTAAGG-3⬘.
Statistical analysis
For each experimental condition, the entire group of animals was prepared
and studied at the same time. Data are presented as mean ⫾SD (in vitro
experiments) or ⫾SEM (in vivo experiments) for each experimental
group. ANOVA was used for comparisons between multiple groups. Stu-
dent’s ttest was used for comparisons between two groups. A value of p⬍
0.05 was considered significant.
Results
p53
⫺/⫺
inflammatory cells demonstrate enhanced activation by LPS
To examine the participation of p53 in the response of inflammatory
cells to LPS, bone marrow neutrophils were isolated from p53
⫹/⫹
and
p53
⫺/⫺
mice and treated with 10 ng/ml LPS. As shown in Fig. 1,
A–C, p53
⫺/⫺
neutrophils produced significantly more proinflamma-
tory cytokines, including TNF-
␣
, IL-6, and MIP-2, than did p53
⫹/⫹
neutrophils. To determine whether this is a cell type-specific effect, peri-
toneal macrophages were harvested from p53
⫹/⫹
and p53
⫺/⫺
mice and
treated with 5 ng/ml LPS. As was the case with p53
⫺/⫺
neutrophils,
p53
⫺/⫺
macrophages produced more TNF-
␣
, IL-6, and MIP-2 after LPS
stimulation than did p53
⫹/⫹
macrophages (Fig. 1, Dand E). These
data suggest that p53 negatively regulates the responses of inflamma-
tory cells, including neutrophils and macrophages, to LPS stimulation.
TLR4-associated signaling events are not affected in
p53
⫺/⫺
inflammatory cells
To determine why p53
⫺/⫺
inflammatory cells have enhanced re-
sponses to LPS, we examined cytoplasmic signaling events that
occur after TLR4 engagement. As shown in Fig. 2A, LPS induced
rapid phosphorylation of JNK, ERK, and p38 in macrophages.
However, there was no difference in MAPK activation between
p53
⫹/⫹
and p53
⫺/⫺
macrophages. Furthermore, I
B-
␣
degrada-
tion, I
B-
␣
phosphorylation, and p65 phosphorylation were sim-
ilar between p53
⫹/⫹
and p53
⫺/⫺
macrophages after LPS stimula-
tion (Fig. 2B). Like macrophages, p53
⫹/⫹
and p53
⫺/⫺
neutrophils
demonstrated no difference in LPS-induced I
B-
␣
degradation
(Fig. 2C). These data suggest that the enhanced responses of
p53
⫺/⫺
inflammatory cells are not the result of increased TLR4-
associated signaling events in response to LPS stimulation.
LPS-induced NF-
B DNA-binding activity is elevated in p53
⫺/⫺
cells
After TLR4 engagement, NF-
B translocates to the nucleus where
it binds to the promoters of target genes and activates their tran-
scription (7–10). We thus investigated the DNA-binding activity of
NF-
B in nuclear extracts obtained from p53
⫹/⫹
and p53
⫺/⫺
in-
flammatory cells following LPS stimulation. As shown in Fig. 3A,
DNA-binding activity of NF-
B in nuclear extracts from LPS-
activated p53
⫺/⫺
macrophages was increased compared with that
found in p53
⫹/⫹
cells. Similarly, DNA-binding activity of NF-
B
from p53
⫺/⫺
neutrophils cultured with LPS were also higher than
that present in p53
⫹/⫹
macrophages (Fig. 3B).
Nutlin-3a up-regulates p53 levels, but down-regulates the
response of inflammatory cells to LPS
Our initial experiments demonstrated that p53
⫺/⫺
macrophages
and neutrophils have enhanced responses to LPS stimulation. We
next asked whether a further increase in p53 levels in p53
⫹/⫹
cells
can attenuate their responses to LPS. To examine this issue,
p53
⫹/⫹
neutrophils or macrophages were treated with nutlin-3a, a
specific inhibitor of p53 and Mdm2 interaction (21). As shown in
Fig. 4A, nutlin-3a increased p53 levels in a dose-dependent manner
in macrophages. Next, neutrophils or macrophages were pretreated
with nutlin-3a for 1 h and then subjected to LPS stimulation for
4 h. We found that nutlin-3a treatment resulted in diminished LPS-
induced TNF-
␣
and IL-6 production in both macrophages and neu-
trophils (Fig. 4, B–E). Of note, nutlin-3a alone did not activate
either neutrophils or macrophages to produce proinflammatory cy-
tokines (Fig. 4, B–E).
Since p53 appears to regulate NF-
B DNA-binding activity in
TLR4-stimulated inflammatory cells, we asked whether this was
a TLR4-specific response or might also occur after engagement of
TLR. To address this question, we examined the effects of nut-
lin-3a on neutrophils and macrophages treated with PAM3CSK4,
a TLR2 ligand. As shown in supplemental Fig. 1,
4
nutlin-3a sig-
nificantly decreased the activation of PAM3CSK4-treated neutro-
phils and macrophages. These data indicate that an increase in p53
levels from baseline in control p53
⫹/⫹
inflammatory cells attenu-
ates their responses not only to TLR4 ligands, such as LPS, but
also to activation through TLR2.
Exposure to nutlin-3a does not produce apoptosis of
macrophages or neutrophils
p53 induces cell cycle arrest and/or apoptosis, depending on the na-
ture of stimuli and the cell population (26). Because macrophages and
neutrophils are terminally differentiated cells, the effects of p53 on
these cells cannot involve its role in regulating cell cycle arrest. How-
ever, accelerated entry into apoptosis may result in an attenuated re-
sponse to LPS. To investigate whether the dampened responses of
4
The online version of this article contains supplemental material.
FIGURE 6. Nutlin-3a does not affect the TLR4-associated signaling
events in inflammatory cells. Aand B, Nutlin-3a does not affect the degree
of I
B-
␣
degradation or p38 phosphorylation in inflammatory cells after
LPS stimulation. p53
⫹/⫹
macrophages (A) or neutrophils (B) were pre-
treated with nutlin-3a for 60 min. The cells were then stimulated with LPS
for the indicated times. Cell extracts were prepared and the levels of p53,
I
B-
␣
, phosphorylated p38, total p38, and actin were determined by West-
ern blotting. Data are from three independent experiments.
5067The Journal of Immunology
nutlin-3a-treated inflammatory cells to LPS are results of enhanced
apoptosis, we performed annexin V-PI staining and found low levels
of apoptosis in nutlin-3a-treated macrophages and neutrophils, even
when the concentration of nutlin-3a was four times higher than that
needed to significantly decrease the responses of the cells to LPS (Fig.
5, A–F).
FIGURE 7. Nutlin-3a decreases LPS-induced NF-
B binding to DNA. A, Nutlin-3a diminishes NF-
B nuclear translocation in LPS-treated p53
⫹/⫹
, but
not p53
⫺/⫺
neutrophils. p53
⫹/⫹
or p53
⫺/⫺
neutrophils were pretreated with vehicle alone (DMSO) or 5
M nutlin-3a in DMSO for 1 h. The cells were
then left untreated or were cultured with 10 ng/ml LPS for the indicated times. Nuclear extracts were prepared and EMSAs were performed as described
in Materials and Methods.B, Densimetric quantitation of A. The densimetric values from p53
⫹/⫹
cells without treatment were used as controls. The
percentage increase for each treatment was calculated by determining the ratio of the indicated treatment—control values to control. Data are presented as
mean ⫾SD from triplicate experiments. ⴱ,p⬍0.05. C, Nutlin-3a diminishes NF-
B binding to the I
B-
␣
promoter. p53
⫹/⫹
macrophages were pretreated with
vehicle alone (DMSO) or 10
M nutlin-3a for 1 h. The cells were then left untreated or were cultured with 5 ng/ml LPS for the indicated times. The ChIP assay
was performed as described in Materials and Methods.D, Nutlin-3a diminishes NF-
B binding to the I
B-
␣
and MIP-2 promoters in LPS-stimulated neutrophils.
p53
⫹/⫹
neutrophils were pretreated with vehicle alone (DMSO) or with 5
M nutlin-3a in DMSO for 1 h. The cells were then left untreated or were cultured with
10 ng/ml LPS for the indicated times. ChIP assays were then performed. Data are from three independent experiments.
FIGURE 8. Increased severity of LPS-induced ALI
in p53
⫺/⫺
mice. A–D, Concentrations of proinflamma-
tory cytokines in lung homogenates and BAL fluid of
p53
⫺/⫺
mice were significantly higher than those in
p53
⫹/⫹
mice. p53
⫹/⫹
or p53
⫺/⫺
mice (n⫽5 in each
group) were intratracheally injected with 1 mg/kg LPS
dissolved in 50
l of PBS. At 24 h after LPS instillation,
the mice were sacrificed and BAL fluid and lung ho-
mogenates were collected as described in Materials and
Methods. Levels of IL-6 and MIP-2 in lung homoge-
nates (Aand B) and BAL fluid (Cand D) were deter-
mined by ELISA. ⴱ,p⬍0.05 compared with p53
⫹/⫹
mice. E, Total protein levels in the BAL fluid of
p53
⫺/⫺
mice were significantly higher than those
present in p53
⫹/⫹
mice. ⴱ,p⬍0.05 compared with
p53
⫹/⫹
mice. F, Neutrophil counts in the BAL fluid
of p53
⫺/⫺
mice were significantly higher than those
in p53
⫹/⫹
mice. Values are presented as mean ⫾
SEM. Data are representative of two independent
experiments.
5068 p53 REGULATES NF-
B ACTIVATION AND ACUTE LUNG INJURY
Nutlin-3a does not affect TLR4-associated signaling pathways in
inflammatory cells
To determine whether nutlin-3a affects the inflammatory responses
of macrophages and neutrophils through modulation of signaling
cascades induced by TLR4 engagement, p53
⫹/⫹
macrophages
were pretreated with 10
M nutlin-3a for 1 h and then stimulated
with LPS for differing lengths of time. As shown in Fig. 6A, the
kinetics of p38 phosphorylation and I
B-
␣
degradation were com-
parable in macrophages pretreated with or without nutlin-3a. Sim-
ilarly, LPS-induced p38 phosphorylation and I
B-
␣
degradation
were not affected in neutrophils pretreated with nutlin-3a (Fig. 6B).
These data suggest that p53 induction does not affect signaling
events, including MAPK and IKK activation, induced by TLR4
engagement in inflammatory cells.
Nutlin-3a decreases LPS-induced NF-
B DNA-binding activity
Since nutlin-3a does not affect TLR4-associated signaling events,
we next determined whether nutlin-3a regulates NF-
B activity in
the nucleus. As shown in Fig. 7, Aand B, NF-
B DNA-binding
activity in neutrophils was increased by LPS stimulation. LPS-
treated p53
⫺/⫺
neutrophils demonstrated increased NF-
B- bind-
ing activity compared with p53
⫹/⫹
cells (Fig. 7, Aand B). Culture
with nutlin-3a decreased LPS-induced NF-
B binding to DNA in
p53
⫹/⫹
neutrophils (Fig. 7, Aand B). Furthermore, nutlin-3a treat-
ment did not affect NF-
B DNA-binding activity in LPS-stimu-
lated p53
⫺/⫺
neutrophils, indicating that the effects of nutlin-3a on
the NF-
B DNA-binding activities are specifically dependent on
p53 induction (Fig. 7, Aand B).
To examine whether nutlin-3a regulates NF-
B binding to the
promoters of target genes, we performed ChIP assays in LPS-stim-
ulated cells. As shown in Fig. 7C, LPS stimulation increased p65
binding to the I
B-
␣
promoter in macrophages. However, nut-
lin-3a pretreatment decreased LPS-induced p65 binding to the
I
B-
␣
promoter (Fig. 7C). Similarly, nutlin-3a pretreatment also
down-regulated p65 binding to the promoters of I
B-
␣
and MIP2
in LPS-activated neutrophils (Fig. 7D).
p53
⫺/⫺
mice demonstrate increased severity of LPS-induced ALI
Neutrophils and macrophages play a central role in the pathogenesis of
ALI (1, 12, 31, 32). Because our in vitro experiments demonstrated en-
hanced NF-
B DNA- binding activity and increased production of proin-
flammatory cytokines in LPS-activated p53
⫺/⫺
neutrophils and macro-
phages, we hypothesized that LPS-induced ALI would be more severe in
p53
⫺/⫺
mice. As shown in Fig. 8,A–D, there were significantly higher
FIGURE 9. Nutlin-3a treatment attenuates LPS-induced ALI. A–C, Nutlin-3a decreases cytokine levels in the lungs after LPS instillation. p53
⫹/⫹
mice
were injected i.p. with 25 mg/kg nutlin-3a every 24 h for 2 days in 50
l of DMSO, while control mice were injected i.p. with 50
l of vehicle (DMSO;
n⫽5 mice in each group). Intratracheal LPS at 1 mg/kg dissolved in 50
l of PBS or PBS alone was administered 4 h after the second injection of nutlin-3a
or vehicle. At 24 h after LPS administration, the mice were sacrificed and lung homogenates were prepared. TNF-
␣
(A), IL-6 (B), and KC (C) levels were
measured by ELISA. D, Nutlin-3a treatment diminished LPS-induced lung MPO activity. p53
⫹/⫹
mice were treated as in A–C. MPO activity was
determined as described in Materials and Methods.ⴱⴱⴱ,p⬍0.001 when compared with the PBS ⫹vehicle group; ###, p⬍0.001 when compared with
the LPS ⫹vehicle group. E, Nutlin-3a treatment diminished LPS-induced increases in lung wet:dry ratios. p53
⫹/⫹
mice were treated as in A–C. Lung
wet:dry ratios were determined as described in Materials and Methods.ⴱⴱ,p⬍0.01 when compared with the PBS ⫹vehicle or LPS ⫹nutlin group. F,
Nutlin-3a increased p53 levels in lung tissues. p53
⫹/⫹
mice (n⫽3) were pretreated every 24 h for 2 days with 25 mg/kg nutlin-3a i.p. in 50
l of DMSO
or vehicle (DMSO) alone. At 4 h after the second injection of nutlin-3a or vehicle, LPS (1 mg/kg) dissolved in 50
l of saline or saline alone was
administered intratracheally. Lung homogenates were collected 24 h after LPS injection and p53 levels were determined by Western blotting. GAPDH was
used as a loading control. Values are presented as mean ⫾SEM. Data are representative of two independent experiments.
5069The Journal of Immunology
levels of the proinflammatory cytokines IL-6 and MIP-2 in lung tissue
and BAL fluid from p53
⫺/⫺
mice compared with p53
⫹/⫹
mice. Further-
more, protein concentrations in BAL fluid, an indicator of lung leak, were
significantly higher in LPS-treated p53
⫺/⫺
mice than those found in
p53
⫹/⫹
mice (Fig. 8E). In addition, neutrophil counts in BAL fluids from
p53
⫺/⫺
mice were increased (Fig. 8F). These data suggest that the en-
hanced responses of p53
⫺/⫺
macrophages and neutrophils to LPS, and
perhaps of other pulmonary cell populations as well, contribute to more
severe LPS-induced lung injury in p53
⫺/⫺
mice.
Nutlin-3a attenuates LPS-induced ALI
The ability of nutlin-3a to attenuate inflammatory responses of neu-
trophils and macrophages prompted us to ask whether nutlin-3a can
also reduce the severity of LPS-induced ALI. As shown in Fig. 9,
A–C, treatment with nutlin-3a before LPS administration significantly
decreased levels of proinflammatory cytokines, including TNF-
␣
,
IL-6, and KC, in the lungs. Pulmonary MPO activity, a marker of
neutrophil infiltration into the lungs (28), was also reduced in nutlin-
3a-treated mice (Fig. 9D). Furthermore, lung wet:dry ratios, a mea-
sure of interstitial pulmonary edema and severity of ALI, was dimin-
ished in nutlin-3a-treated mice compared with those found in mice
treated with vehicle (Fig. 9E). As expected, p53 expression was in-
creased in the lung tissues of nutlin-3a-treated mice (Fig. 9F).
Discussion
The reciprocal regulation of the activities of NF-
B and p53 has
been the focus of numerous studies (33, 34). In cancer cells, these
transcriptional factors appear to have opposite roles in modulating
cellular functions (35). For example, NF-
B promotes inflamma-
tion and inhibits apoptosis, while p53 induces cell cycle arrest and
promotes apoptosis (36, 37). The evidence that NF-
B signaling
regulates p53 activity is ample. For example, one of the NF-
B
family molecules, Bcl-3, up-regulates Mdm2 expression, thereby
inhibiting p53 transcriptional activity (38). Another NF-
B sub-
unit, p52, can specifically bind to the promoter of the p53 target
gene, p21, and inhibits p53-dependent p21 basal expression (39).
How p53 modulates NF-
B activity remains less clear. p53 me-
diates increased I
B-
␣
expression and resultant decreases in
NF-
B activation (35). It has also been shown that p53 suppresses
IKK activity and subsequent activation of NF-
B (40). In addition,
reciprocal sequestration of coactivators, including CBP and p300,
has been thought to be a major mechanism underlying the mutual
suppression that exists between p53 and NF-
B (26, 41).
In this study, we found that p53, even at basal levels, is involved in
regulation of NF-
B activity since p53
⫺/⫺
inflammatory cells dem-
onstrated enhanced responses to LPS. These findings, as well as stud-
ies from other groups demonstrating that significantly more proin-
flammatory cytokines are produced in the thymus of LPS-treated
p53
⫺/⫺
mice than in wild-type mice (42), suggest that overtly exu-
berate responses of p53
⫺/⫺
inflammatory cells to LPS stimulation could
be one of the mechanisms explaining the enhanced LPS-induced lung
injury and increased susceptibility to LPS-associated mortality in p53
⫺/⫺
mice. However, elevation of NF-
B-dependent inflammatory cytokines
in p53
⫺/⫺
mice may not be the sole explanation for their sensitivity to
endotoxemia. Komarova et al. (42) also found that p53
⫺/⫺
macro-
phages had decreased phagocytic activity. Defects in the ingestion of
apoptotic cells, and specifically of apoptotic neutrophils, are associ-
ated with unfavorable outcomes from inflammatory diseases (43– 45).
p53 has become a novel therapeutic target for treatment of inflam-
matory diseases and modulation of p53 by a natural product,
genistein, was shown to be able to attenuate TLR4-induced activation
of monocytes (46).
Intracellular events induced by TLR4 engagement, such as activa-
tion of MAPK and IKK, were comparable in p53 wild-type and
knockout cells. These findings indicate that p53 negatively regulates
NF-
B activity downstream of IKK activation and I
B-
␣
degrada-
tion. Indeed, we observed increases in NF-
B DNA-binding activity
in LPS-treated p53
⫺/⫺
macrophages and neutrophils compared with
that found in p53
⫹/⫹
cells. Nevertheless, we were unable to detect
p53 binding to the I
B-
␣
promoter in LPS-stimulated p53
⫹/⫹
mac-
rophages (data not shown), suggesting that there is no direct and phys-
ical involvement of p53 in modulating binding of NF-
B to the pro-
moters of its target genes. Since enhanced interaction with the
coactivator p300/CBP increases NF-
B DNA-binding activity and
subsequent transcriptional activity, it may be a mechanism by which
p53
⫺/⫺
cells demonstrate increased responses to LPS stimulation.
Exposure of LPS-stimulated macrophages and neutrophils to nut-
lin-3a reduced their production of proinflammatory cytokines. Treat-
ment with nutlin-3a also decreased the severity of LPS-induced ALI.
Nutlin-3a is a specific inhibitor of the interaction between p53 and
Mdm2 and thereby enhances the activity of p53 (47). Compared with
other physiological and nonphysiological inducers of p53, such as
chemotherapeutic drugs and ion irradiation, nutlin-3a creates no DNA
damage, but does specifically stabilize and activate p53. Nutlin-3a
induces cell cycle arrest and apoptosis of a variety of cancer cells both in
vitro and in vivo (48). The decrease in the proinflammatory responses of
nutlin-3a-treated cells to LPS was not caused by cell death since nutlin-3a
did not induce any apparent alteration in apoptosis among either neutro-
phils or macrophages after4hoftreatment. Furthermore, nutlin-3a ex-
posure did not alter proximal signaling events, such as activation of
MAPK and IKK, after TLR4 engagement, which is consistent with the
findings by Dey et al. (49) that nutlin-3a does not affect TNF-
␣
-or
IL-1-induced I
B-
␣
phosphorylation and degradation or p65 phos-
phorylation in cancer cells. However, nutlin-3a treatment did inhibit
the binding of NF-
B to promoters of target genes in LPS-treated
macrophages and neutrophils. These inhibitory effects of nutlin-3a on
NF-
B activation appear to be specific for nutlin-3a-induced alter-
ations in p53 as nutlin-3a failed to attenuate NF-
B binding to DNA
in p53
⫺/⫺
cells.
One potential mechanism by which nutlin-3a-induced p53 activa-
tion negatively regulates NF-
B activity is through sequestration of
NF-
B-binding coactivators such as p300/CBP (26, 41). Although a
recent study found that p53 is involved in the regulation of IKK kinase
activity (40), we found no alterations in I
B-
␣
phosphorylation or
degradation in LPS-treated p53
⫺/⫺
neutrophils or macrophages or in
p53
⫹/⫹
cells treated with nutlin-3a. Although these results indicate
that p53 does not participate in regulating the activation of IKK

,
which is the primary IKK isoform responsible for I
B-
␣
phosphorylation
after TLR4 engagement (50), they do not rule out a role for interactions
between p53 and other IKK isoforms, such as IKK
␣
, in modulating
NF-
B activity. IKK
␣
does not participate in I
B-
␣
degradation (50).
However, it is translocated to the nucleus where it phosphorylates histone
H3, among other activities. Phosphorylation of histone H3 promotes the
accessibility of NF-
B as well as cofactors to promoter regions, thereby
facilitating NF-
B-dependent transcription (51, 52). Whether inhibition
of IKK
␣
-induced histone H3 phosphorylation is one of the mecha-
nisms by which p53 modulates NF-
B binding to promoters and tran-
scription of NF-
B-regulated genes needs further exploration.
Although we only found minimal apoptosis of neutrophils and
macrophages 4 h after nutlin-3a treatment that was not different from
the degree of apoptosis present among untreated cells, there is still a
possibility that nutlin-3a enhances apoptosis of these inflammatory
cell populations over more extended periods of time. Neutrophils are
short-lived cells and play a central role in development and perpetu-
ation of LPS-induced lung injury by accumulating in the lungs and
producing high levels of proinflammatory mediators, including cyto-
kines, chemokines, and reactive oxygen species (53). Increased neu-
trophil apoptosis and resultant clearance from the lungs during sepsis
5070 p53 REGULATES NF-
B ACTIVATION AND ACUTE LUNG INJURY
has been shown to be beneficial (53, 54). Thus, some of the beneficial
effects of nutlin-3a and p53 activation in ALI could come from en-
hanced neutrophil apoptosis induced by p53.
In conclusion, we found that p53 negatively regulates NF-
B
activity by decreasing binding of NF-
B to the promoters of genes
for proinflammatory cytokines, thereby contributing to the in-
creased response of p53
⫺/⫺
inflammatory cells to LPS stimulation
and enhancing lung injury in LPS-treated p53
⫺/⫺
mice. Modula-
tion of p53 by nutlin-3a diminished the response of neutrophils and
macrophages to stimulation through TLR2 or TLR4 and also at-
tenuated LPS-induced ALI.
Disclosures
The authors have no financial conflict of interest.
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