Signal Transduction Pathways of Tumor Necrosis
Factor–mediated Lung Injury Induced by Ozone in Mice
Hye-Youn Cho1, Daniel L. Morgan2, Alison K. Bauer1*, and Steven R. Kleeberger1
1Laboratory of Respiratory Biology,2Laboratory of Molecular Toxicology, National Institute of Environmental Health Sciences, National Institutes
of Health, Research Triangle Park, North Carolina
Rationale: Increasing evidence suggests that tumor necrosis factor
(TNF)-? plays a key role in pulmonary injury caused by environmen-
tal ozone (O3) in animal models and human subjects. We previously
determined that mice genetically deficient in TNF response are
protected from lung inflammation and epithelial injury after O3
Objectives: The present study was designed to determine the molec-
ular mechanisms of TNF receptor (TNF-R)–mediated lung injury
induced by O3.
Methods: TNF-R knockout (Tnfr?/?) and wild-type (Tnfr?/?) mice
were exposed to 0.3 ppm O3or air (for 6, 24, or 48 h), and lung
RNA and proteins were prepared. Mice deficient in p50 nuclear
factor (NF)-?B (Nfkb1?/?) or c-Jun–NH2terminal kinase 1 (Jnk1?/?)
and wild-type controls (Nfkb1?/?, Jnk1?/?) were exposed to O3
(48 h), and the role of NF-?B and mitogen-activated protein kinase
(MAPK) as downstream effectors of lung injury was analyzed by
bronchoalveolar lavage analyses.
Results: O3-induced early activation of TNF-R adaptor complex for-
mation was attenuated in Tnfr?/?mice compared with Tnfr?/?mice.
O3 significantly activated lung NF-?B in Tnfr?/?mice before the
development of lung injury. Basal and O3-induced NF-?B activity
and activator protein (AP)-1 were lower in Tnfr?/?mice basally and
after O3. Furthermore, inflammatory cytokines, including macro-
phage inflammatory protein-2, were differentially expressed in
Tnfr?/?and Tnfr?/?mice after O3. O3-induced lung injury was signifi-
cantly reduced in Nfkb1?/?and Jnk1?/?mice relative to respective
Conclusions: Results suggest that NF-?B and MAPK/AP-1 signaling
pathways are essential in TNF-R–mediated pulmonary toxicity
induced by O3.
Keywords: tumor necrosis factor receptor; knockout; nuclear factor-?B;
mitogen-activated protein kinase; activator protein-1
Ozone(O3) is aprincipaloxidant inair pollution.Elevatedambi-
ent O3levels have been associated with increased hospital visits
and respiratory symptoms in epidemiologic studies (1, 2). Sub-
jects with preexisting allergic/inflammatory airway disorders,
ble to O3and at risk of exacerbations (3). Recent evidence also
suggested that O3enhances the effect of inhaled allergen in
patients with asthma (4). Acute O3toxicity in rodent airways
(Received in original form September 28, 2005; accepted in final form January 25, 2007)
mental Health Sciences, National Institutes of Health, Department of Health and
tion, Center for Integrative Toxicology, Michigan State University, East Lansing,
Correspondence and requests for reprints should be addressed to Hye-Youn Cho,
Ph.D., Laboratory of Respiratory Biology, National Institute of Environmental
Health Sciences, National Institutes of Health, 111 TW Alexander Drive, Building
101, MD D-201, Research Triangle Park, NC 27709. E-mail: email@example.com
Am J Respir Crit Care Med
Originally Published in Press as DOI: 10.1164/rccm.200509-1527OC on January 25, 2007
Internet address: www.atsjournals.org
Vol 175. pp 829–839, 2007
AT A GLANCE COMMENTARY
Scientific Knowledge on the Subject
Nuclear factor-?B andMAPK/AP-1 signaling pathwaysare
essential in tumor necrosis factor receptor–mediated pul-
monary toxicity induced by ozone.
What This Study Adds to the Field
NF-?B and MAPK/AP-1 signaling pathways are essential
in TNF receptor–mediated lung injury induced by ozone.
includes predominant neutrophilic inflammation accompanied
mucus overproduction and hypersecretion, and cell death and
proliferation. However, the mechanisms of O3-induced effects
on the lung are not completely understood.
A number of investigations have focused on the potential
roles of inflammatory mediators, including tumor necrosis factor
(TNF)-?, in the pathogenesis of O3-induced lung inflammation
and injury. TNF-? is a member of the trimeric cytokine family
(5), which has diverse bioregulatory activities engaged in in-
flammation/immunity responses, cell proliferation/differentia-
tion, and apoptosis. TNF-? has a critical role in many acute
andchronic inflammatorydiseases, andanti-TNFstrategies have
proven to be clinically effective (6). TNF-? binds to two distinct
binding to TNF-R1 induces sequential recruitment of intracel-
protein (TRADD) and TNF-R–associated factor 2 (TRAF2) to
the membrane. TRAF2 is also a well-defined intracellular adap-
tor for TNF-R2. The interaction of TRAF2 in the TNF-R com-
plex with the inhibitor of ?B (I?B) kinase (IKK) and subsequent
phosphorylation of IKK and I?B eventually activates the tran-
scription factor, nuclear factor (NF)-?B (7). Another pathway
that becomes activated by the TRADD/TRAF2 complex is the
mitogen-activated protein kinase (MAPK) cascade, which in-
duces nuclear transactivation of activator protein (AP)-1 tran-
scription factors (7). TNF-? signaling directs transcriptional
regulation of inflammatory mediator genes, including early-
response cytokines (e.g., interleukin [IL]-1?), chemokines (e.g.,
macrophage inflammatoryprotein [MIP]-2), andadhesion mole-
cules (e.g., intercellular adhesionmolecule [ICAM]-1) invarious
airway cells (8, 9).
An essential role for TNF-? has been recently documented in
animal models of pulmonary inflammation and oxidative injury
responses caused by bleomycin and several environmental tox-
icants, including hyperoxia, endotoxin, and cigarette smoke
(10–14). Inhaled O3also enhances TNF-? release and TNF-R
expression in the airway cells or tissues (15, 16). Our positional
cloning studies in inbred mice identified Tnf as a candidate
830 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINEVOL 1752007
susceptibility gene for lung inflammation induced by subacute
exposures to 0.3 ppm O3 (17). In support of this hypothesis,
we and others have demonstrated that lack of TNF response
provided significant protection from O3-induced inflammation
and airway hyperreactivity in rodent lungs (16–20). Moreover,
ation of O3-induced lung functional changes with a TNF poly-
morphism haplotype including –308A, which is also known to
be involved in increased risk of asthma (23). In the present
study, we elucidated molecular mechanisms underlying TNF-
R–mediated pulmonary pathogenesis of subacute O3toxicity.
Some of the results of this study have been previously reported
in abstracts (24, 25).
Animals and Inhalation Exposure
Male Tnfr?/?(B6;129S-Tnfrsf1atm1ImxTnfrsf1btm1Imx/J), Nfkb1?/?(B6;129P2-
Nfkb1tm1Bal/J), and Jnk1?/?(B6.129-Mapk8tm1Flv/J) mice and their respec-
(Bar Harbor, ME). After acclimation, mice were placed in individual
stainless-steel wire cages within a Hazelton 1000 chamber (Lab Prod-
ulate air–filtered air supply. Mice had free access to water and pelleted
open-formula rodent diet NIH-07 (Zeigler Brothers, Gardners, PA.).
Mice were exposed continuously (for 6, 24, or 48 h) to 0.3 ppm O3. On
the basis of National Ambient Air Quality Standards for ambient O3
(0.12 ppm for 1 h and 0.08 ppm for 8 h) (26) and dosimetry studies in
which rodents require four- to fivefold higher doses of O3than humans
to create an equal deposition and pulmonary inflammatory response
(27), the O3concentration used in the current study is a reasonable
exposure level from which to make comparisons with humans. O3was
generated from ultra-high-purity air (? 1 ppm total hydrocarbons;
National Welders, Inc., Raleigh, NC) using a silent arc discharge O3
generator (model L-11; Pacific Ozone Technology, Benicia, CA). Con-
stant chamber air temperature (72 ? 3? F) and relative humidity (50 ?
15%) were maintained. O3concentration was continually monitored
(Dasibi model 1008-PC; Dasibi Environmental Corp., Glendale, CA).
Parallel exposure to filtered air was done in a separate chamber for
the same duration. Immediately after each exposure, mice were killed
by sodium pentobarbital overdose (104 mg/kg). All animal use was
approved by the National Institute of Environmental Health Sciences
Animal Care and Use Committee.
Left lung tissues were fixed by 10% neutral buffered formalin under
constant pressure (25 cm H2O) and sections were processed for histo-
pathology. Immunohistologic staining was done using an anti-TRAF2
antibody (sc-877; Santa Cruz Biotechnology, Inc., Santa Cruz, CA)
or a proliferating cell nuclear antigen (PCNA) staining kit (Zymed
Laboratories, Inc., South San Francisco, CA).
Bronchoalveolar Lavage Analyses
Whole lungs from mice exposed to O3 or air (48 h) were lavaged,
and lung cellular inflammation and hyperpermeability were assessed
as described previously (16).
Lung Nuclear Protein Isolation for Electrophoretic Mobility
Nuclear proteins were prepared from pulverized pieces of right lung.
Briefly, lung tissues were pulverized in liquid nitrogen using a mortar
and pestle, and were homogenized in a hypotonic buffer (10 mM N-2-
hydroxyethylpiperazine-N?-ethane [HEPES], pH 7.9; 0.5 M sucrose;
1.5 mM MgCl2; 10 mM KCl; 10% glycerol; 1 mM ethylenediaminetetra-
acetic acid [EDTA], pH 8.0; 1 mM dithiothreitol [DTT]; 1 mM phenyl-
methylsulfonyl fluoride [PMSF]) using a dounce homogenizer. Homog-
enates were treated with Nonidet P-40 (0.25%), incubated on ice for
15 minutes, and centrifuged (14,000 g, 20 min, 4?C). After collecting
pended in a hypertonic lysis buffer (20 mM HEPES, pH 7.9; 420 mM
NaCl; 1.5 mM MgCl2; 10% glycerol; 0.2 mM EDTA, pH 8.0; 0.5 mM
DTT; 0.5 mM PMSF; protease inhibitor cocktail), incubated in ice on
a rocking platform (150 rpm, 30 min), and centrifuged (14,000 g, 15
min, 4?C). Supernatants including nuclear proteins were collected and
stored at ?80?C. DNA binding activity of NF-?B or AP-1 was deter-
minedbyelectrophoreticmobility(gel) shiftanalysisofnuclear proteins
(5–10 ?g) as described previously (28). Specific binding activity was
p65NF-?B (sc-372X), anti–p50NF-?B (sc-1190X), or anti-pan Jun AP-1
(sc-44X) antibody followed by electrophoretic mobility shift analysis.
The gel was autoradiographed using an intensifying screen at ?70?C,
and autoradiograph images were scanned and quantified by a Bio-Rad
Gel Doc 2000 System (Hercules, PA).
Western Blot Analyses
Total lungproteins fromright lung tissueswere prepared in radioimmu-
noprecipitation (RIPA) buffer(phosphate-buffered saline, 1% Nonidet
P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 10 ?g
Lung cytosolic soluble fractions were acquired during nuclear protein
extraction as described above. Proteins (20–100 ?g) were analyzed
by Western blotting using specific antibodies against TRAF2 (sc-877),
TRADD (sc-7868), I?B-? (sc-371), phosphorylated I?B-? (p-I?B; sc-
8404), IKK (sc-7607), phosphorylated IKK (p-IKK; sc-21661-R), c-Jun–
NH2terminal kinase (JNK1; sc-571), p-JNK (sc-6254), extracellular signal-
regulated kinase (ERK; sc-154), p-ERK (sc-7383), and actin (sc-1615).
PCNA was detected in nuclear protein (20 ?g) using an anti-PCNA
antibody (sc-56). To determine TRADD-bound TRAF2 (an indicator of
TNF-R1 signaling complex formation) or TNF-R2–bound TRAF2 (an
indicator of TNF-R2 signaling complex formation), 200 ?g of total
lung protein were immunoprecipitated with anti-TRADD antibody (sc-
7868) or anti-TNF-R2 antibody (sc-1074), respectively, and each immu-
noprecipitate was processed for Western blot analysis with the anti-
TRAF2 antibody. Bands were scanned and quantified using the Bio-Rad
Gel Doc 2000 System.
Reverse Transcriptase–Polymerase Chain Reaction Analyses
Total RNA was isolated from right lung homogenate, and reverse
transcriptase–polymerase chain reaction was performed with mouse-
specific primers for TNF-?, lymphotoxin (LT)-?, MIP-2, and IL-1? (29).
Forward and reverse primers used for ICAM-1 (GI:194077) amplification
were 5?-atggcttcaacccgtgccaa-3? and 5?-gttacttggctcccttccga-3?, respec-
tively. Forward and reverse primers used for IL-6 (GI:198367) amplifi-
cation were 5?-aagaacgatagtcaattcca-3? and 5?-gatctcaaagtgacttttag-3?,
respectively. Each mRNA abundance was quantified by the Bio-Rad
Gel Doc 2000 System as described previously (28) using 18S ribosomal
RNA as an internal control.
Data were expressed as the group mean ? SEM. Two-way analysis of
variance (ANOVA) was used to evaluate the effects of exposure and
genotype in all experiments except p50 NF-?B binding activity in
Nfkb1?/?mice in which one-way ANOVA was used. The Student-
Newman-Keuls test was used for a posteriori comparisons of means
(p ? 0.05). All of the statistical analyses were performed using Sigma-
Stat 3.0 software program (SPSS, Inc., Chicago, IL).
Differential Activation of TNF-R Signal Pathways by O3in
Intracellular TNF-R complex formation. Intracellular TNF-R sig-
nal protein complex was measured as an indicator of TNF-R
activation after exposure to O3. TRAF2 is a common intracel-
lular signal transducer that mediates TNF-R1 and TNF-R2 re-
sponses, and it has recently been found to be essential for early
recruitment of downstream kinases for NF-?B and AP-1 activa-
protein indicated that TRADD-bound TRAF2 (an indicator of
intracellular TNF-R1 signal transducer complex formation) was
Cho, Morgan, Bauer, et al.: NF-?B and JNK Direct Ozone Toxicity 831
elevated after6 hours ofexposure, before the onsetof inflamma-
tion (Figure 1A). Complex formation was significantly attenu-
ated in Tnfr?/?mice compared with Tnfr?/?mice after O3expo-
sure (Figure 1A, Table 1). TRAF2 bound to TNF-R2 (an
indicator of TNF-R2 signaling complex formation) was signifi-
cantly increased by O3in Tnfr?/?mice, but not in Tnfr?/?mice
(Figure 1A, Table 1). Soluble TRAF2 was also relatively higher
in Tnfr?/?mice than in Tnfr?/?mice basally and after O3expo-
Figure 1. Intracellular tumor necrosis factor receptor (TNF-R) signal
transducers were suppressed in Tnfr-deficient mice. (A) Differential acti-
vation of TNF-R proximal signaling complex in Tnfr?/?and Tnfr?/?mice
after exposure to air and 0.3 ppm O3(6 or 24 h). Aliquots of total lung
homogenates were immunoprecipitated (IP) using anti–TNF-R1–
associated death domain protein (TRADD) or anti–TNF-R2 antibody
followed by Western blot (WB) with anti–TNF-R–associated factor 2
(TRAF2)antibody to determineTRADD-bound TRAF2 or TNF-R2–bound
TRAF2 levels, respectively. Soluble TRAF2 was determined by Western
blot of nonparticulate fractionsfrom lung homogenates. Representative
images from multiple analyses (n ? 3–4/group) are presented. Data
? SEM and resultsfrom statisticalanalyses (two-way analysis of variance
[ANOVA], p ? 0.05) are shown in Table 1. (B) Differential levels of
TRAF2 localized in terminal bronchioles of Tnfr?/?and Tnfr?/?mice after
48 hours of exposure to air and 0.3 ppm O3. TRAF2-positive lung cells
were immunohistologically stained with anti-TRAF2 antibody. High
magnification shows TRAF2 localized on the membrane and in the
cytoplasm. Bars indicate 100 ?m. Representative light photomicro-
graphs are presented.
sure, and O3reduced soluble TRAF2 levels in both genotypes
in a time-dependent manner (Figure 1A, Table 1). O3-induced
early increases in TRAF2–TRADD and TRAF2–TNF-R2 com-
plexes were concurrent with depletion of soluble TRAF2 be-
fore lung pathology developed in the wild-type mice, and sug-
gested the recruitment of “free” cytoplasmic TRAF2 to form
membrane complex in response to O3.
Lung TRAF2 was detected constitutively by immunohisto-
chemical staining in cytoplasm and membranes of ciliated and
and in alveolar macrophages of Tnfr?/?mice and Tnfr?/?mice
(Figure 1B). TRAF2 was also detected in infiltrating inflamma-
tory cells and in terminal bronchiolar cells of the centriacinar
stitution in O3-exposed mice (Figure 1B) as demonstrated pre-
viously (16, 17). Consistent with immunoprecipitation/Western
blot data (Figure 1A), relatively fewer TRAF2-positive cells
with Tnfr?/?mice (Figure 1B).
NF-?B pathway. As a dimeric transcription factor, the activity
of NF-?B is regulated by its interaction with I?B, a family of
cytoplasmic NF-?B inhibitors. Activation of the NF-?B pathway
requires sequential phosphorylation of the upstream kinase
complex IKK and its substrate I?B, which leads to phosphoryla-
tional degradation of I?B and nuclear translocation of NF-?B
after having been liberated from NF-?B–I?B complexes. After
O3exposure, lung IKK(?/?) and I?B-? were enhanced similarly
in both genotypes (Figure 2A). However, p-IKK(?/?) level nor-
malized by IKK(?/?) was significantly lower in Tnfr?/?mice than
inTnfr?/?micebasally andafterO3(Table 1). Atime-dependent
increase of phosphorylated I?B-? (p-IkB-?/I?B-?) by O3was
evident in Tnfr?/?mice but marginal and significantly lower in
Tnfr?/?mice (Figure 2A, Table 1). Baseline DNA binding activi-
ties of total NF-?B and p50 ?B subunits were significantly sup-
pressed in Tnfr?/?mice compared with Tnfr?/?mice (Figure 2B,
Table 1). O3significantly enhanced the binding activity of total
NF-?B and specific p50 ?B over the constitutive level in both
genotypes (Figure 2B, Table 1). However, O3-induced total (6
and 24 h) and specific p65 (24 h) and p50 (24 h) NF-?B activity
was significantly lower in Tnfr?/?mice compared with Tnfr?/?
mice (Figure 2B, Table 1).
MAPK/AP-1 pathway. Phosphorylational activation of the
MAPK regulates nuclear AP-1 transactivation. Total JNK and
genotype (Figure 3A). Total activated levels of ERK and JNK
(determined by ratio of phosphorylated level to nonphosphory-
lated level) were significantly attenuated basally and after O3
in Tnfr?/?mice compared with Tnfr?/?mice, although O3also
(Figure 3A, Table 1). Basal and O3-induced (6 h) DNA binding
activity of nuclear total AP-1 was significantly suppressed in
Tnfr?/?mice, compared with wild-type mice (Figure 3B, Table 1).
Specific DNA–binding activity of AP-1 Jun proteins was consti-
tutively lower in Tnfr?/?than in Tnfr?/?mice, and was not sig-
nificantly enhanced by 24 hours after O3in Tnfr?/?mice (Figure
3B, Table 1).
Downstream inflammatory gene induction. Transcriptional in-
duction of several O3-inducible genes containing cis-acting ele-
was compared in Tnfr?/?and Tnfr?/?mice. Constitutive expres-
sion of TNF-? and IL-1? mRNA was significantly lower in
Tnfr?/?compared with Tnfr?/?mice (Figure 4). O3caused a
significant increase of IL-1? mRNA expression at 24 and 48
hours over the control level, whereas TNF-?, LT-?, MIP-2, and
ICAM-1 mRNA was significantly elevated above respective
baseline expression only at 48 hours when inflammation and
832AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 1752007
TABLE 1. QUANTIFIED RESULTS OF WESTERN BLOT ANALYSES AND ELECTROPHORETIC
MOBILITY SHIFT ASSAYS IN TNFR?/?AND TNFR?/?MICE
Proteins Detected GenotypeAir6 h O3
24 h O3
1 ? 0.01
0.59 ? 0.01
1 ? 0.01
0.03 ? 0.03†
1 ? 0.25
0.65 ? 0.09
1 ? 0.12
0.00 ? 0.00†
1 ? 0.21
0.29 ? 0.15
1 ? 0.19
0.68 ? 0.14†
1 ? 0.08
0.77 ? 0.07
1 ? 0.06
0.47 ? 0.11†
1 ? 0.02
0.36 ? 0.02†
1 ? 0.10
0.75 ? 0.22
1 (0.98, 1.02)
0.62 (0.65, 0.58)†
1 ? 0.07
0.63 ? 0.02†
3.37 ? 0.24*
2.07 ? 0.05*†
1.63 ? 0.10*
0.07 ? 0.00†
0.65 ? 0.25
0.32 ? 0.11†
1.38 ? 0.14*
0.00 ? 0.00†
2.02 ? 0.50
0.98 ? 0.23
1.73 ? 0.38*
0.96 ? 0.26†
2.31 ? 0.52*
1.24 ? 0.14
1.10 ? 0.01
0.68 ? 0.22
1.28 ? 0.06
1.25 ? 0.15*
2.07 ? 0.04*
1.34 ? 0.30†
1.37 (1.48, 1.26)
0.93 (0.95, 0.91)†
1.43 ? 0.01*
0.65 ? 0.13†
1.91 ? 0.37*
0.57 ? 0.23†
1.87 ? 0.19*
0.03 ? 0.03†
0.46 ? 0.16*
0.21 ? 0.13*†
1.45 ? 0.07*
0.81 ? 0.17*†
5.31 ? 1.22*
1.28 ? 0.64†
3.59 ? 0.67*
1.88 ? 0.32*†
2.45 ? 0.46*
1.06 ? 0.33†
2.21 ? 0.19*
1.33 ? 0.07*†
3.32 ? 0.15*
2.01 ? 0.04*†
2.92 ? 0.41*
1.82 ? 0.11*†
1.57 (1.8, 1.34)*
1.15 (1.38, 0.93)*
1.45 ? 0.01*
0.86 ? 0.07†
(n ? 3)
(n ? 3)
(n ? 4)
(n ? 3)
(n ? 3)
(n ? 3)
(n ? 3)
(n ? 3)
(n ? 3)
(n ? 3)
(n ? 2)
(n ? 3)
Total NF-?B (EMSA)
p65 NF-?B (EMSA)
p50 NF-?B (EMSA)
Jun AP-1 (EMSA)
Definition of abbreviations: AP-1 ? activator protein-1; EMSA ? electrophoretic mobility shift assay; ERK ? extracellular signal-
regulated kinase; I?B ? inhibitor of ?B; IKK ? I?B kinase; IP ? immunoprecipitation; JNK ? c-Jun–NH2terminal kinase; NF-?B ?
nuclear factor-?B; p- ? phosphorylated; TNF-R2 ? tumor necrosis factor receptor-2; TRAF2 ? TNF-R–associated factor 2; TRADD ?
TNF-R1–associated death domain protein; WB ? Western blot.
Data are presented as mean ? SEM of relative ratio to air-exposed Tnfr?/?mice.
* Significantly different from genotype-matched air controls (p ? 0.05).
†Significantly different from exposure-matched Tnfr?/?mice (p ? 0.05).
‡Data presented as mean and individual normalized ratio to air-exposed Tnfr?/?mice.
injury by O3had reached a peak (Figure 4). Induced levels of
these genes were significantly lower in Tnfr?/?mice compared
with Tnfr?/?mice (Figure 4). In contrast, IL-6 transcript level
was significantly greater in Tnfr?/?mice than in Tnfr?/?mice
after 24- and 48-hour exposure (Figure 4).
Functional Role of NF-?B in O3-induced Pulmonary Toxicity
Lung inflammation, injury, and proliferation. Because NF-?B
binding activity was significantly lower in Tnfr?/?mice compared
with Tnfr?/?mice after O3exposure, we hypothesized that mice
deficient in a functional subunit of NF-?B (Nfkb1?/?) would be
less responsive to inflammatory effects of O3compared with
wild-type (Nfkb1?/?) control animals. No significant differences
in mean total protein concentration or cell differentials were
found in bronchoalveolar lavage (BAL) fluid between Nfkb1?/?
and Nfkb1?/?mice after air exposure (Figure 5A). O3-induced
increases in the mean total protein concentration and numbers
of neutrophils and epithelial cells were significantly attenuated
(70–50%) in Nfkb1?/?mice relative to the Nfkb1?/?control
on the numbers of BAL macrophages were found (Figure 5A).
Immunohistologic localization of PCNA indicated a few pro-
liferating cells throughout the lung sections in both genotypes
of mice exposed to air (Figure 5B). Cellular proliferation in the
injured regions (mainly in terminal bronchioles) caused by O3
mice (peak at 48 h, Figure 5B). Western blot analysis (Figure
5B) determined significant differences (twofold) in nuclear
PCNA (36 kD) between the two genotypes basally and after O3
Nuclear NF-?B–DNA binding activity. O3significantly stimu-
lated total NF-?B and specific p50 and p65 binding in the lungs
of Nfkb1?/?mice at 6 and/or 24 hours (Figure 6). In Nfkb1?/?
mice, total ?B activity (SB) was slightly enhanced by O3at 24
hours (Figures 6A–6D), whereas specific binding activity of
p50 ?B (SSB) was not detected (Figure 6B). Compared with
Nfkb1?/?mice, total (SB in Figure 6A) and p65 (SSB in Figure
6C) ?B binding activity was significantly depressed in Nfkb1?/?
mice, basally and after O3exposure (Figure 6D). This suggested
that absence of p50 subunit inhibited heterodimerization of
p65–p50 ?B for DNA binding.
Functional Role of MAPK8 (JNK1) in O3-induced
Because activated JNK (p-JNK) was also suppressed in Tnfr?/?
mice compared with Tnfr?/?mice after O3exposure, we com-
pared inflammatory responses to O3in Jnk?/?and Jnk?/?mice
to address functional relevance of this signaling pathway. No
significant differences in mean BAL total protein concentration
or numbers of neutrophils and epithelial cells were found be-
tween air- and O3-exposed Jnk1?/?mice (Figure 7). These lung
injury indices were significantly lower (60–75%) in Jnk1?/?mice
compared with those in Jnk1?/?mice after O3(Figure 7). O3did
notsignificantly change the meannumbers ofBALmacrophages
in either genotype (Figure 7).
The functional importance of TNF-? as a key modulator of O3-
studies. Anti–TNF-? antibody pretreatment decreased airway
Cho, Morgan, Bauer, et al.: NF-?B and JNK Direct Ozone Toxicity833
Figure 2. Nuclear factor (NF)-?B pathway was attenuated in Tnfr-
deficient mice. (A) Differential activation of IKK and I?B in the lungs
of Tnfr?/?and Tnfr?/?mice after exposure to air and 0.3 ppm O3(6 or
24 h) as assessed by Western blotting with phospho-specific antibodies.
Representative images from multiple analyses (n ? 3–4/group) are pre-
sented. Data are normalized to air-exposed Tnfr?/?mice, and normal-
ized group mean ? SEM and results from statistical analyses (two-way
ANOVA, p ? 0.05) are shown in Table 1. (B) Differential nuclear NF-
?B–DNA binding activity in the lungs of Tnfr?/?and Tnfr?/?mice after
exposure to air and 0.3 ppm O3(6 or 24 h). Aliquots of nuclear protein
isolated from pieces of right lung (n ? 3 mice/group) were incubated
with an end-labeled oligonucleotide probe containing NF-?B consensus
sequence. Total NF-?B–DNA binding was determined by gel shift analy-
sis (top panel). To detect specific binding activity of each NF-?B subunit
by gel supershift analyses, either anti-p65 (middle panel) or anti-p50
(bottom panel) subunit antibodies were added to the binding reactions.
SB indicates shifted bands of total bindings (NF-?B motif–protein com-
plex); SSB indicates super-shifted bands of specific bindings (NF-?B
motif–protein antibody complex). Representative images from multiple
analyses (n ? 3/group) are presented. Data are normalized to air-
exposed Tnfr?/?mice, and normalized group mean ? SEM and results
from statistical analyses (two-way ANOVA, p ? 0.05) are shown in
Figure 3. Mitogen-activated protein kinase/activator protein-1 (MAPK/
AP-1) pathway was attenuated in Tnfr-deficient mice. (A) Differential
phosphorylation of MAPK (p-JNK1, p-ERK) levels in the lungs of Tnfr?/?
and Tnfr?/?mice after exposure to air and 0.3 ppm O3(6 or 24 h)
as assessed by Western blotting. Representative images from multiple
analyses (n ? 3–4/group) are presented, and group mean ? SEM nor-
malized to air-exposed Tnfr?/?mice and statistical analyses are shown
in Table 1. (B) Differential AP-1–DNA binding activity in the lungs of
Tnfr?/?and Tnfr?/?mice after air and 0.3 ppm O3(6 or 24 h). Aliquots
of nuclear protein isolated from pieces of right lung (n ? 3 mice/group)
were incubated with an end-labeled oligonucleotide probe containing
AP-1 consensus sequence. Total AP-1–DNA binding was determined by
gel shift analysis (top panel). To detect specific binding activity of AP-1
Jun proteins by gel supershift analyses, anti-Jun antibody was added to
the binding reactions. SB indicates shifted bands of total bindings (AP-1
motif–protein complex); SSB indicates super-shifted bands of specific
bindings (AP-1 motif–protein–antibody complex). Representative im-
ages from multiple analyses are presented. Data are normalized to air-
exposed Tnfr?/?mice, and normalized group mean ? SEM (n ? 3/
group) or mean and individual values (total AP-1 binding, n ? 2/group)
are shown with results from statistical analyses (two-way ANOVA,
p ? 0.05) in Table 1.
inflammation, hyperpermeability, and cell proliferation after
acute or subacute O3exposure in rodents (17, 19, 20, 36). Lung
inflammation and epithelial injury were also reduced after sub-
acute O3 exposure in mice genetically deficient in TNF-R
O3-induced airway hyperreactivity was decreased in these
TNF-R knockout mice (16, 18). In the present study, we deter-
mined that relative to wild-type mice, activation of NF-?B and
834 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINEVOL 1752007
Figure 4. Tnfr deficiency reduced transcriptional induc-
tion of inflammatory mediators. Inflammatory gene ex-
pression wasdetected byreverse transcriptase–polymerase
chain reaction using total lung RNA isolated from Tnfr?/?
and Tnfr?/?mice after exposure to air and 0.3 ppm O3
(n ? 3/group). Representative cDNA band images for each
gene are shown in (A). Quantitated intensities of digitized
cDNA bands were normalized to the intensities of 18S
bands, and relative intensity to air-exposed Tnfr?/?mice
of each gene is shown in (B). Data are presented as group
means ? SEM. *Significantly higher than genotype-
matched air controls (two-way ANOVA, p ? 0.05);?signifi-
cantly higher than exposure-matched Tnfr?/?mice (two-
way ANOVA, p ? 0.05). ICAM-1 ? intercellular adhesion
molecule-1; LT-? ? lymphotoxin-?; MIP-2 ? macrophage
inflammatory protein-2; TNF-? ? tumor necrosis factor-?.
and significantly lower lung injury and inflammation were found
are the first to demonstrate that NF-?B and MAPK/AP-1 path-
ways are keysignaling components of TNF-mediated pulmonary
pathogenesis by inhaled O3(Figure 8).
NF-?B and MAPK/AP-1 pathways are critical in develop-
sion of multiple genes involved in inflammation and immunity,
development, lymphoid differentiation, oncogenesis, and apo-
ptosis.Use ofNF-?B subunit–or Jnk-deficientmice has supplied
direct evidence for the role of NF-?B and MAPK in pulmonary
inflammation and allergy models. For example, Nfkb1?/?mice
were resistant to allergic airway eosinophilic inflammation and
mycobacterial infection (37, 38). c-Rel ?B deficiency also re-
duced airway hyperresponsiveness and chemokine induction
after allergen challenge (39). Lack of JNK (Jnk1 or Jnk2) inhib-
cal ventilation (40). These two redox-sensitive transcription fac-
tor signaling pathways have also been shown to be induced by
and colleagues (45) determined a functional role of pulmonary
NF-?B in the increase of inducible nitric oxide synthase and
Cho, Morgan, Bauer, et al.: NF-?B and JNK Direct Ozone Toxicity 835
Figure 5. Nuclear factor (NF)-?B was essential in O3-
induced pulmonary pathogenesis. (A) Effect of targeted
disruption of Nfkb1 was determined by bronchoalveolar
lavage (BAL) phenotypes after 48 hours of exposure to air
and 0.3 ppm O3. Data are presented as means ? SEM
matched air control mice (two-way ANOVA, p ? 0.05);
?significantly different from O3-exposed Nfkb1?/?mice
(two-way ANOVA, p ? 0.05). (B) Differential proliferation
of pulmonary cells in Nfkb1?/?and Nfkb1?/?mice after
48 hours of exposure to air or 0.3 ppm O3. S-phase cells
undergoing proliferation were detected by proliferating
cell nuclear antigen (PCNA) immunostaining. Represen-
tative light photomicrographs are shown. Bars indicate
100 ?m. Representative Western blot image demonstrates
NF-?B p50-dependent increase of nuclear PCNA in mouse
lungs.Graph depictsmean ?SEMfrom duplicatesnormal-
ized to air-exposed Nfkb?/?. *Significantly different from
genotype-matched air control mice (two-way ANOVA,
p ? 0.05);?significantly different from exposure-matched
Nfkb1?/?mice (two-way ANOVA, p ? 0.05).
TNF-? levels by inhaled O3. Our current observations support
NF-?B and MAPK as key mediators of TNF-R responses.
The present study, however, indicated that TNF signaling
does not account for all O3-induced NF-?B and MAPK/AP-1
activities. As depicted in Figures 2 and 3 (also see Table 1), O3
exposure significantly activated signal transducers of NF-?B and
MAPK/AP-1 pathways even in the absence of TNF-R. This
suggests that receptor-mediated signals other than TNF-R acti-
vate these pathways in response to O3. It is possible that greater
fold increases of certain NF-?B and MAPK signal proteins in
Tnfr?/?mice than in Tnfr?/?mice compared with genotype-
matched air-exposed control animals may be associated with
compensatory activation of these non–TNF-R signals in the ab-
sence of TNF-R. In support of this concept, Alcamo and associ-
ates (46) determined that TNF-R1/NF-?B p65-double deficient
mice were significantly more resistant to lung neutrophilic in-
flammation and chemokine/cytokine expression (e.g., ICAM-1,
MIP-2) than TNF-R1 single knockout mice during acute lung
injury induced by endotoxin. These studies thus indicated that
activation of pulmonary NF-?B may also occur independently
of TNF-R signaling after stimulation with exogenous stimuli. It
has become clear that proinflammatory responses by the pulmo-
nary innate immune system are partially mediated through pat-
familyof proteins (47–49).We previously determined that TLR4
contributes significantly to the pulmonary hyperpermeability re-
sponse to subacute O3exposure (50), and that mechanisms un-
derlying hyperpermeability are dissociated from those for TNF-
R–mediated cellular inflammation (16). Accumulating evidence
shows that MAPK and NF-?B signaling pathways are essential
836 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINEVOL 1752007
Figure 6. p50 Nuclear factor (NF)-?B deficiency
abolished total NF-?B activity in the lung. Nuclear
NF-?B–DNA binding activity in the lungs of
Nfkb1?/?and Nfkb1?/?mice after exposure to air
and 0.3 ppm O3(6 or 24 h). Aliquots of nuclear
protein isolated from pieces of right lung (n ? 3
mice/group) were incubated with an end-labeled
oligonucleotide probe containing NF-?B consensus
sequence. Total NF-?B–DNA binding was deter-
mined by gel shift analysis (A). To detect specific
binding activity of each NF-?B subunit by gel su-
pershift analyses, either anti-p50 (B) or anti-p65 (C)
subunit antibody was added to the binding reac-
tions. SB indicates shifted bands of total bindings
(NF-?B motif–protein complex); SSB indicates su-
per-shifted bands of specific bindings(NF-?B motif–
protein–antibody complex); FP indicates free probes.
The total (indicated as SB in A) and specific (indi-
cated as SSB) p50 (B) or p65 (C) NF-?B–DNA bind-
ing activity was quantified using a Bio-Rad Gel
Doc 2000 System, and mean ? SEM (n ? 3/group)
or mean and individual values (n ? 2/group) nor-
malized to air-exposed Nfkb1?/?mice were pre-
sented (D). *Significantly different from genotype-
matched air control mice(two-way ANOVA for total
andp65 ?B,one-way ANOVAfor p50?B,p ?0.05);
?significantly different from exposure-matched
Nfkb1?/?mice (two-way ANOVA, p ? 0.05).
in TLR4/MyD88-dependent cell signaling (51, 52). In addition,
lung injury induced by a particle (residual oil fly ash) was sig-
nificantly attenuated in mice with dominant mutant Tlr4 (C3H/
HeJ) compared with Tlr4 normal mice (C3H/HeOuJ), and
this resistance was shown to be mediated through suppressed
Figure 7. c-Jun–NH2 terminal kinase (JNK) was essential
in O3-induced pulmonary pathogenesis. Effect of targeted
disruption of Jnk1 was determined by bronchoalveolar
lavage (BAL) phenotypes after 48 hours of exposure to
0.3 ppm O3. Data are presented as means ? SEM (n ?
3–5 mice/group). *Significantly different from genotype-
matched air control mice (two-way ANOVA, p ? 0.05);
?significantly different from O3-exposed Jnk1?/?mice
(two-way ANOVA, p ? 0.05).
activation of downstream MAPK/AP-1 and NF-?B pathways
(29). Collectively, these investigations suggest that interaction
exists between TNF and TLR4 signaling mechanisms through
NF-?B and MAPK pathways during the pathogenesis of pul-
monary oxidative injury. In the current study, abolishment of
Cho, Morgan, Bauer, et al.: NF-?B and JNK Direct Ozone Toxicity837
Figure 8. A hypothetical molecular mechanism underlying
inhaled O3–induced pulmonary inflammation and injury.
O3may cause ligand binding to tumor necrosis factor re-
ceptor (TNF-R) on pulmonary cells to elicit trimerization
of TNF-R and receptor complex formation by recruitment
of accessory proteins, including TNF-R1–associated death
domain protein (TRADD) and TNF-R–associated factor 2
(TRAF2). This event will trigger phosphorylation of down-
stream signal transducers, including mitogen-activated
protein kinase (MAPK) kinase (MEK) and inhibitor of ?B
tion of MAPK, including c-Jun–NH2terminal kinase (JNK)
and phosphorylational degradation of I?B, respectively.
Activator protein (AP)-1 proteins activated by phosphory-
lated MAPK and nuclear factor (NF)-?B subunits (e.g., p50,
p65) liberated from I?B–NF-?B complex would be subse-
quently translocalized into nuclei for DNA binding to mod-
ulate inflammatory effector gene expression. These signal-
ing pathways and possibly feedback regulation by TNF-?
(dashed arrows) and/or by other cytokines and receptors
(dotted arrow) may be essential to propagate airway
inflammation and injury caused by O3, and exacerbate
symptoms in subjects with preexisting respiratory disease
O3-induced hyperpermeability in Jnk1?/?mice (see Figure 7) and
Nfkb1?/?mice (see Figure 5A) supports this possibility.
The current study also identified multiple proinflammatory
genes that were differentially regulated in Tnfr?/?and Tnfr?/?
mice during O3-induced lung inflammation. Included among
these is the potent neutrophil chemoattractant MIP-2, which is
also TNF dependent in murine pulmonary models of silica and
cigarette smoke toxicity (10, 13). We also observed TNF-R–
mediated induction of TNF-? (autoregulation) in O3-exposed
lungs. A similar observation was reported in the lungs after
cigarette smoke exposure, and mice deficient in TNF-R had
decreased expression of TNF-?, whereas TNF-? was induced in
wild-type mice after exposure (13). Presence of functional AP-1
and NF-?B binding sites in mouse MIP-2 (34, 53) and TNF-?
(54, 55) gene promoters further supports TNF-mediated MIP-2
and TNF-? regulation via these transcription factors. The injuri-
ous effects of TNF-dependent IL-1? have also recently been
determined after acute O3exposure (56). Interestingly, in the
present study, IL-6 mRNA was overexpressed in O3-resistant
Tnfr?/?mice, which may suggest a protective role for this cyto-
kine. IL-6 has been shown to have antiinflammatory properties.
For example, IL-6 deficiency augmented hydrogen peroxide–
induced murine alveolar epithelial cell death (57), and anti–IL-6
antibody treatment significantly increased neutrophilic inflam-
mation caused by O3exposure in rats (58). However, converse
effects have also been reported, and IL-6 was determined to be
proinflammatory during the early phase of O3exposure in mice
Figure 8 depicts a schematic representation of the molecular
mechanisms that we have investigated and identified as putative
signal transduction pathways leading to pulmonary toxicity
caused by inhaled O3. In summary, we uncovered that NF-?B
and MAPK/AP-1 play key roles in subacute O3-induced lung
inflammation and injury mediated through TNF-R. Although
further investigation is required to clarify the complex link be-
tor networks, the current study provided details of molecular
events underlying pulmonary O3toxicity. Our observations may
have important implications for understanding the pathogenesis
of inflammatory sequelae after environmental O3exposure in
normal subjects and individuals with preexisting lung disease.
Conflict of Interest Statement: None of the authors has a financial relationship
with a commercial entity that has an interest in the subject of this manuscript.
Acknowledgment: Ozone exposures were conducted at the National Institute of
Environmental Health Sciences (NIEHS) Inhalation Facility under contract to Alion
the inhalation exposures. Drs. Farhad Imani and Donald Cook at the NIEHS pro-
vided excellent critical review of the manuscript.
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