of January 16, 2013.
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Lethal Murine Endotoxemia and Acute Lung
Endothelial MKK3 Is a Critical Mediator of
Amanda S. Shinn, Yitao Zhang and Patty J. Lee
Praveen Mannam, Xuchen Zhang, Peiying Shan, Yi Zhang,
published online 28 December 2012
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The Journal of Immunology
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The Journal of Immunology
at Yale University on January 16, 2013
The Journal of Immunology
Endothelial MKK3 Is a Critical Mediator of Lethal Murine
Endotoxemia and Acute Lung Injury
Praveen Mannam,* Xuchen Zhang,†Peiying Shan,* Yi Zhang,* Amanda S. Shinn,*
Yitao Zhang,* and Patty J. Lee*
Sepsis is a leading cause of intensive care unit admissions, with high mortality and morbidity. Although outcomes have improved
with better supportive care, specific therapies are limited. Endothelial activation and oxidant injury are key events in the path-
ogenesis of sepsis-induced lung injury. The signaling pathways leading to these events remain poorly defined. We sought to deter-
systemic inflammation to mimic sepsis. Lung injury parameters were assessed in lung tissue and bronchoalveolar lavage specimens.
Primary lung endothelial cells were cultured and assessed for mediators of inflammation and injury, such as ICAM-1, AP-1,
NF-kB, and mitochondrial reactive oxygen species. Our studies demonstrate that MKK3 deficiency confers virtually complete
protection against organ injury after i.p. LPS. Specifically, MKK32/2mice were protected against acute lung injury, as assessed by
reduced inflammation, mitochondrial reactive oxygen species generation, endothelial injury, and ICAM-1 expression after LPS
administration. Our results show that endothelial MKK3 is required for inflammatory cell recruitment to the lungs, mitochondrial
oxidant-mediated AP-1, NF-kB activation, and ICAM-1 expression during LPS challenge. Collectively, these studies identify
a novel role for MKK3 in lethal LPS responses and provide new therapeutic targets against sepsis and acute lung injury.
The Journal of Immunology, 2013, 190: 000–000.
every year (1). No curative therapy is available—only supportive
care. Furthermore, the incidence of sepsis is predicted to increase
with the aging of our expanding population. Targeted biologic
therapies are urgently needed. Our study offers new insights into
the critical role of endothelial MAPK kinase 3 (MKK3), a com-
ponent of the p38 MAPK pathway, in lethal sepsis.
MAPK pathways are core components of signal transduction in
the cell. They are intracellular signaling pathways that mediate cell
survival and death; proliferation; and differentiation in response to
a wide variety of signals, such as cytokines, growth factors, UV
light, osmotic stress, and LPS. Three distinct MAPK subfamilies
major tyrosine-phosphorylated protein induced by LPS and plays
an essential role in production of proinflammatory cytokines and
vascular adhesion molecules in response to LPS (2, 3). Upstream
epsis is the leading cause of acute lung injury and death in
sepsis develops in 750,000 people, of whom .210,000 die
kinases of p38 include MKK3 and MKK6 (4). MKK3 specifically
activates p38, but not JNK or ERK, in response to stress (5). A
targeted disruption in the MKK3 gene causes a selective defect in
p38 activation and TNF-a induction of cytokine gene expression
(6). MKK3-deficient macrophages have fundamental defects in
inflammatory response, as shown by reduced p38 phosphorylation
and production of inflammatory cytokines, such as IL-1a and
IL-1b, in response to LPS (7). MKK3-deficient macrophages also
have lower levels of TNF-a after LPS exposure (8). However, a
functional role for MKK3 in sepsis has not been reported.
Our laboratory has shown previously that MKK3 mediates anti-
apoptotic effects in lung injury induced by ischemia–reperfusion (9,
10). MKK3 also has protective effects in hyperoxia-induced lung
injury (11). In contrast, MKK3 mediates susceptibility to ventilator-
induced lung injury (12). MKK3 has also been shown to mediate
renal injury in ischemia–reperfusion, unilateral ureteric obstruction,
and diabetic models (13–15). MKK3 signaling has been reported to
play an essential role in pancreatic injury due to low-dose strepto-
zotocin (16). Therefore, it appears that the MKK3 pathway has
distinct roles, depending on the type of injury and organ or cell type.
A role for MKK3 in innate immune responses elicited by LPS has
not been explored. In this study, we show that MKK3-deficient
(MKK32/2) mice are protected from lung injury when challenged
with systemic LPS, a model of systemic inflammation. The mech-
anism of this protection is through reduced endothelial mitochon-
drial reactive oxygen species (ROS)–mediated AP-1, NF-kB acti-
vation, and ICAM-1 expression, leading to decreased inflammatory
cell recruitment and endothelial and tissue injury. Collectively, our
data identify MKK3 as an important upstream mediator of critical
processes in sepsis and may be a potential therapeutic target.
Materials and Methods
Generation of MKK32/2mice
MKK32/2mice were generated by deletion of exons 8 and 9, which encode
aa 217–221 of the murine MKK3 protein, as previously described (7).
*Department of Internal Medicine, Section of Pulmonary, Critical Care and Sleep
Medicine, Yale University School of Medicine, New Haven, CT 06520; and
†Department of Pathology, Yale University School of Medicine, New Haven, CT
Received for publication July 24, 2012. Accepted for publication November 27,
This work was supported by American Heart Association Grant AHA 09FTF2090019
(to P.M.) and by National Institutes of Health/National Heart, Lung, and Blood
Institute, Grants R01 HL090660 and R01 HL071595 (to P.J.L.).
Address correspondence and reprint requests to Dr. Patty J. Lee, Pulmonary and
Critical Care Medicine, Department of Internal Medicine, Yale University School
of Medicine, 333 Cedar Street, P.O. Box 208057, New Haven, CT 06520-8057.
E-mail address: firstname.lastname@example.org
Abbreviations used in this article: ALT, alanine aminotransferase; AST, aspartate
aminotransferase; BAL, bronchoalveoar lavage; BUN, blood urea nitrogen; CT,
threshold cycle; MDA, malondialdehyde; MKK3, MAPK kinase 3; MPO, myeloper-
oxidase; Nox, NADPH oxidase; ROS, reactive oxygen species; siRNA, small inter-
fering RNA; WT, wild-type.
Published December 28, 2012, doi:10.4049/jimmunol.1202012
at Yale University on January 16, 2013
MKK32/2mice expressed normal levels of MKK6, MKK4, JNK, and p38
MAP kinases, and, thus there were no compensatory changes in the ex-
pression of these other kinases as a consequence of MKK3 deficiency. The
MKK32/2mice were provided by R. Davis (University of Massachusetts
Medical School, Worcester, MA) and R. Flavell (Yale University, New
Haven, CT) and have been backcrossed onto a C57BL/6 background for
For survival and injury studies, the mice were given 40 mg/kg and 5 mg/kg
LPS i.p., respectively (Escherichia coli055:B5; Sigma-Aldrich). Body-
surface temperature was measured using an Infrascan infrared thermom-
eter (La Crosse Technologies). Bronchoalveolar lavage (BAL) was per-
formed by tracheal cannulation, and whole-lung lavage was performed
twice with a total volume of 1.8 ml ice-cold PBS. BAL was centrifuged at
3000 g, and the protein concentration of the supernatant was determined
using the BCA Protein Assay Reagent (Pierce Labs). Mouse serum tests
for transaminases [aspartate aminotransferase (AST), alanine aminotrans-
ferase (ALT)], blood urea nitrogen (BUN), and creatinine were performed
by Antech Diagnostics. Mouse serum troponin I was measured using an
ELISA kit (Life Diagnostics) according to the manufacturer’s protocol.
Irradiation and bone marrow transplantation
Whole-body irradiation of recipient mice and harvesting of donor bone
marrow were performed as described previously (17). Briefly, donor bone
marrow was flushed from the femora, tibiae, and humeri of mice. Cells
were pelleted at 300 g for 10 min at 4˚C before counting. Recipient mice at
6 wk of age underwent whole-body irradiation (1000 cGy), followed by i.v.
injection of whole bone marrow cells (9 3 106cells in 0.2 ml PBS). After
bone marrow transplantation, mice were maintained until 3 mo of age
under specific pathogen–free conditions at the Yale University School of
Medicine animal facility and fed acidic water.
Isolation of primary lung endothelial cells
Endothelial cells were isolated as described by Kuhlencordt et al. (18), with
some modifications. Briefly, lungs were extracted, minced, and digested
for 1 h with 0.1% collagenase (Roche Diagnostics). The digest was passed
through a 100-mm cell strainer and pelleted at 200 g for 5 min; resus-
pended in endothelial medium containing 20% FBS, 40% DMEM, and
40% F12 with 100 U/ml penicillin G and 100 mg/ml streptomycin; and
plated onto 0.1% gelatin-coated T75 flasks. Cells were cultured for 2–4 d
and resuspended in 2% FBS containing 10 ml biotin-labeled rat anti-mouse
CD31 (PECAM-1) Ab (BD Biosciences–Pharmingen). After incubation on
ice for 30 min, the cells were washed with streptavidin magnetic beads
(New England BioLabs). Cells were washed with 2% FBS, resuspended in
5 ml 2% FBS, and incubated on ice for 30 min. The cells were then placed
on the magnet for 5 min; unbound cells were removed, and bound cells
were resuspended in medium and plated onto a 0.1% gelatin-coated T25
flask. We confirmed with CD31 staining and flow cytometry that .95% of
the cells were endothelial cells.
Small interfering RNA knockdown of MKK3
ON-TARGETplus SMARTpool small interfering RNA (siRNA) against
MKK3andscrambledsiRNAwere obtainedfromThermoScientific (formerly
Dharmacon RNAi Technologies). Endothelial cells were seeded onto six-well
plates1 d prior to transfection, using 40% DMEM and 40% F12 tissue culture
medium supplemented with 20% FBS, without antibiotics. At the time of
transfection with the specific siRNA, the cells were 50–60% confluent.
Lipofectamine 2000 Reagent (Invitrogen) was used as the transfection agent.
After 48 h of incubation, the cells were collected and subjected to assays.
protocol (Roche Applied Science). Sections of formalin-fixed, paraffin-
embedded lung tissue were deparaffinized and rehydrated, rinsed with
PBS, and digested with proteinase K (Roche Applied Science) at a con-
centration of 20 mg/ml for 20 min. After PBS washes, sections were in-
cubated with TUNEL reaction mixture at 37˚C for 1 h, then incubated with
antifluorescein conjugated with alkaline phosphatase at 37˚C for 30 min.
Sections were washed twice with PBS and stained with NBT/5-bromo-4-
chloro-3-indolyl phosphate substrate solution before counterstaining with
nuclear fast-red. Apoptotic and normal cells were observed under a light
microscope. Normal cells exhibited red nuclear staining, whereas TUNEL-
positive cells, indicating cell death/apoptosis, exhibited purple nuclear
staining. A total of 500 cells were counted for each sample, and the number
of apoptotic cells is expressed as a percentage of the total counted.
Myeloperoxidase (MPO) levels were assessed as follows. Lung tissue was
homogenized in 50 mM phosphate buffer (pH 6.0). Then, after centrifu-
gation at 10,000 g for 15 min, the pellet was resuspended in 50 mM
hexadecyltrimethylammonium bromide (Sigma-Aldrich) in 50 mmol/l
potassium phosphate buffer, pH 6.0, before sonication for 20 s in an ice
bath. The samples were freeze thawed three times, after which sonication
was repeated. Suspensions were then centrifuged at 10,000 g for 10 min.
MPO activity was assayed spectrophotometrically by mixing 0.1 ml su-
pernatant with 2.9 ml 50 mmol/l phosphate buffer, pH 6.0, containing
0.167 mg/ml o-dianisidine dihydrochloride (Sigma-Aldrich) and 0.0005%
hydrogen peroxide (Sigma-Aldrich). The change in absorbance at 460 nm
was measured using a spectrophotometer (SmartSpec300; Bio-Rad) peri-
odically for 3 min. MPO activity was then derived from the observed
change in absorbance per minute.
Western blot analysis
Protein was extracted from cells using a radioimmunoprecipitation assay
buffer, electrotransferred, and immunoblotted with primary Abs. Detection
was performed with an HRP Western Blot Detection System (Cell Sig-
naling Technology). Equivalent sample loading was confirmed by stripping
membranes with Blot Restore Membrane Rejuvenation solution (Thermo
Scientific) and probing for actin, a-tubulin, or lamin A/C. All of the Abs
were obtained from Santa Cruz Biotechnology, except p-IKKa/b (Cell
Signaling Technology). Nuclear and cytoplasmic fractions of endothelial
cells were obtained using an NE-PER kit (Thermo Scientific) according to
instructions. Western blots of nuclear and cytoplasmic fractions were then
performed as detailed before.
Nuclear extracts were prepared using an NE-PER Nuclear and Cytoplasmic
Extraction Reagent Kit (Thermo Scientific) according to the manufacturer’s
protocol. The AP-1 site was synthesized as complementary oligodeoxyri-
bonucleotide strands. The sequence of AP-1 consensus oligonucleotides was
59-CGC TTG ATG ACT CAG CCG GAA-39 (Sigma-Aldrich). The DNA
binding ability of AP-1 in the nuclear extracts was assessed by EMSA with
biotin-labeled, double-stranded AP-1. EMSA was carried out using the
LightShift Chemiluminescent EMSA Kit (Pierce). Specific binding was
confirmed using a 200-fold excess of an unlabeled probe as a specific com-
petitor. Protein–DNA complexes were separated using a 6% non-denaturing
acrylamide gel electrophoresis and then transferred to positively charged
nylon membranes and cross-linked by UV irradiation. Gel shifts were vi-
sualized with streptavidin HRP according to standard protocols.
Malondialdehyde (MDA) was measured using the Lipid Peroxidation Assay
Kit (Calbiochem; EMD Biosciences) according to the manufacturer’s
instructions. CM-H2DCFDA and MitoSOX Red (Invitrogen) were used
to determine levels of ROS in endothelial cells. Cells were seeded onto six-
well non–tissue culture plates 1 d before the experiment. On the next day
cells were stimulated with LPS (1 mg/ml, 6 h). Cells were washed and ex-
posed to CM-H2DCFDA (5 mM) or MitoSOX Red (2.5 mM) in regular
media and kept at 37˚C for 25 min. The cells were washed with PBS and
then detached gently with 0.4M EDTA and analyzed on a BD FACSCalibur
machine. CM-H2DCFDA was detected in the FL-1 channel, and MitoSOX
Red was detected in the FL-3 channel. Rotenone was purchased from Sigma-
Aldrich, and Mito-TEMPO was purchased from Santa Cruz Biotechnology.
Total RNA was extracted from one-half of one lung or cells using TRIzol
reagent according to the manufacturer’s protocol (Life Technologies BRL).
First-strand cDNA was synthesized using SuperScript II Reverse Tran-
scriptase (Invitrogen) with random hexamers; conditions were 10 min at
25˚C, 30 min at 48˚C, and 5 min at 95˚C. Real-time RT-PCR reactions
were carried out with the Power SYBR Green PCR Master Mix (Applied
Biosystems) and an ABI Prism 7000 Sequence Detection System (Applied
Biosystems). GAPDH was amplified as a control. Real-time PCR con-
ditions were 95˚C for 10 min and 40 cycles of 95˚C for 15 s, followed by
60˚C for 1 min. The relative quantification values for these gene expres-
sions were calculated from the accurate threshold cycle (CT), which is the
PCR cycle at which an increase in reporter fluorescence from SYBR green
dye can first be detected above a baseline signal. The CT values for
GAPDH were subtracted from the CT values for ICAM-1 and MKK3 in
each well, to calculate DCT. The DCT values for each sample were av-
eraged. To calculate the fold induction over controls (DDCT), the average
DCT values calculated for wild-type (WT) animals or cells were subtracted
2MKK3 MEDIATES SEPSIS AND LUNG INJURY
at Yale University on January 16, 2013
from DCT values calculated for MKK32/2animals or cells. Next, the fold
induction for each well was calculated using the 2–(DDCT)formula. The fold
inductionvalues for replicate wells were averaged, and data were presented
as the mean 6 SEM of triplicate wells.
Primers (59→ 39) used for RT-PCR were as follows. ICAM-1 for-
ward: 59-TCACCAGGAATGTGTACCTGA-39, reverse: 59-ATCACGAG-
GCCCACAATGAC-39; VCAM-1 forward: 59-CCCGGATGCGCTTGAC-
39, reverse: 59-CCGATTTGAGCGATCGTTTT-39; selectin E forward: 59-
was performed on lung sections. Arrows point to TUNEL-positive cells. The number of TUNEL-positive cells was quantified and expressed as a percentage
of the total number of lung cells counted on each section, mean 6 SEM. n = 3, *p , 0.05. (b) WTand MKK32/2mice were given IP LPS (40 mg/kg), and
TUNEL staining was performed on organ sections.
MKK32/2mice have less cell death after systemic LPS. (a) WT and MKK32/2mice were given i.p. LPS (40 mg/kg), and TUNEL staining
The Journal of Immunology3
at Yale University on January 16, 2013
GAACCAAAGACTCGGGCATGT-39, reverse: 59-TGACCACTGCAG-
GATGCATT-39; CXCL-1 forward: 59-CTGGATTCACCTCAAGAAC-
ATC-39, reverse: 59-CAGGGTCAAGGCAAGCCTC-39; CXCL-2 forward:
59-GCGCCCAGACAGAAGTCATAG-39, reverse: 59-AGCCTTGCCTTT-
GTTCAGTATC-39; MKK3 forward: 59-GTAGAGAAAGTGCGGCATG-
CT-39, reverse: 59-CCCGGATGCGCTTGAC-39; GADPH forward: 59-
TGTGTCCGTCGTGGATCTGA-39, reverse: 59-CCTGCTTCACCACCT-
MKK32/2mice are resistant to lung and organ injury after
We initially sought to determine whether MKK3-deficient mice are
resistant to organ injury after LPS exposure. We observed lower
levels of cell death in the lungs and vasculature of MKK32/2mice,
as shown by TUNEL staining (Fig. 1a). Other organs such as kidney,
MKK32/2mice after i.p. LPS (40 mg/kg). Original magnification 340. (B) BAL protein levels in WTand MKK32/2mice after i.p. LPS (40 mg/kg), mean 6
SEM. n = 5, *p , 0.01. (C) WTand MKK32/2micewere given i.p. LPS (40 mg/kg), and MPO levels were measured in organ lysates (mean 6 SEM, n = 5, p ,
0.05). (D) WTand MKK32/2mice were given i.p. LPS (40 mg/kg). Serum levels of ALTand AST (markers for hepatic damage) and BUN (measurement of
renal function) were assessed, mean 6 SEM, n = 7–8, *p , 0.05.
MKK32/2mice are resistant to lung and organ injury after systemic LPS. (A) Representative lung histopathologic findings of WT and
4 MKK3 MEDIATES SEPSIS AND LUNG INJURY
at Yale University on January 16, 2013
spleen, liver, and heart also showed substantially fewer TUNEL-
positive cells in MKK32/2mice (Fig. 1b). BAL cell counts were
not elevated in WT and MKK32/2mice, indicating absence of in-
flammatory cell influx into the alveolar space, typical of i.p. LPS,
as reported by others (19). However, parenchymal lung inflam-
mation was markedly increased in WT mice, as observed in rep-
resentative histopathologic sections of the lung (Fig. 2A). BAL
protein levels, a measure of lung endothelial barrier disruption,
were significantly elevated in WT mice compared with MKK32/2
mice (Fig. 2B). Lung MPO levels, a measure of neutrophil re-
cruitment, were significantly decreased in MKK32/2mice com-
pared with WT mice after i.p. LPS. We found similar differences
in MPO levels in kidney and liver, key target organs of systemic
LPS (Fig. 2C). We measured serum markers of organ injury in
mice given LPS. We found significantly higher levels of trans-
aminases (AST, ALT) and BUN in WT mice given LPS, compared
with MKK32/2mice, indicating higher liver and kidney damage,
respectively. We also checked levels of creatinine, another marker
of kidney injury, and troponin I, a marker of myocardial injury
(Fig. 2D). Although a trend toward higher creatinine and troponin
I levels in septic WT mice was noted, the differences did not reach
statistical significance (data not shown). Collectively, these data
showed that MKK3-deficient mice are resistant to lung and sys-
temic organ injury after LPS exposure.
Nonhematopoietic cells are important in MKK3-mediated
Next, we determined whether decreased organ injury in MKK32/2
mice correlated with changes in survival and the relative contri-
bution of MKK3 in cells of hematopoietic versus nonhema-
topoietic lineages to the responses. Survival studies showed that
MKK32/2mice transplanted with WT bone marrow were still
protected against lethal LPS, suggesting that the loss of MKK3 in
nonhematopoietic cells is sufficient for improved survival (Fig. 2A).
WT mice transplanted with MKK32/2bone marrow appeared to
have a trend toward improved survival after i.p. LPS, compared
with WT mice transplanted with WT bone marrow, but this trend
was not statistically significant (Fig. 3A). For the purposes of this
article, we focused on the role of endothelial MKK3, because of
the significant survival advantage that MKK3 deficiency in non-
hematopoietic lineages provided. Using the idea that the body
temperature of mice is an accurate marker of survival in various
models of sepsis (20), we found that MKK32/2mice transplanted
with WT bone marrow exhibited body temperatures similar to
those of MKK32/2mice transplanted with MKK32/2bone marrow
after LPS, indicating that MKK3 deficiency in nonhematopoietic
cells plays a more important role in protection than does MKK3
deficiency in hematopoietic cells. WT mice transplanted with
MKK32/2bone marrow did not have a statistically significant
recovery of body temperatures 6 h after LPS, consistent with the
survival data (Fig. 3B). Of note, the basal body temperatures were
similar in all groups. These data suggest that the loss of MKK3 in
primarily nonhematopoietic cells has sufficient protective effects
against lethal LPS to override any deleterious effects that MKK3
reconstitution, at least in bone marrow, may have. On the basis of
these data and the known critical role of endothelium in sepsis, we
chose to focus our subsequent in vitro studies on lung endothelial
ICAM-1 expression is lower in MKK32/2organs and
endothelial cells after LPS
Compared with WT lungs, MKK32/2lungs had decreased in-
flammatory influx after LPS (Fig. 2). Given that endothelial ad-
hesion and chemokine molecules are important in inflammatory
cell recruitment, we analyzed MKK32/2lungs for ICAM-1,
VCAM-1, selectin E, CXCL-1, and CXCL-2. In the molecule
profile we analyzed, ICAM-1 expression was notably different in
MKK32/2lungs after LPS, compared with WT lungs (Fig. 4A).
Levels of VCAM-1 and CXCL-2 were also lower in MKK32/2
lungs, but we decided to focus on ICAM-1, given the strong as-
sociation of ICAM-1 with septic responses in previous reports
(21). ICAM-1 mRNAwas decreased in the lungs, kidney, and liver
of MKK32/2mice after LPS (Fig. 4B). As ICAM-1 is expressed
by endothelial cells and function in inflammatory cell recruit-
ment (22), we proceeded to study ICAM-1 in primary mouse
lung endothelial cells. We found that ICAM-1 mRNA and protein
levels were decreased in MKK32/2cells at baseline and after LPS
stimulation (Fig. 4C, 4D). We examined surface expression of
ICAM-1 by flow cytometry and, consistent with our cell lysate data,
we found decreased surface expression of ICAM-1 in MKK32/2
endothelial cells (Fig. 4E). We confirmed specific regulation of
ICAM-1 by MKK3, using siRNA against MKK3. An ∼50% re-
duction in MKK3, using siRNA in WT endothelial cells, caused
a small but significant reduction of ICAM-1 after LPS exposure
(Fig. 4F). These studies show for the first time, to our knowledge,
that MKK3 is an upstream regulator of ICAM-1 in organs and
endothelial cells and a viable therapeutic target.
Activation of ICAM-1–related transcription factors was lower
in MKK32/2endothelial cells after LPS
To identify the mechanism whereby MKK3 regulates ICAM-1, we
explored the role of NF-kB and its upstream kinase, IKKa/b. NF-
kB has been demonstrated to regulate ICAM-1 via the IKK path-
determines survival after lethal LPS. Adoptive transfer of bone marrow
was performed from WT mice to WT mice (WT → WT), MKK32/2mice
to MKK32/2mice (MKK32/2→ MKK32/2), WT mice to MKK32/2mice
(WT → MKK32/2), and MKK32/2mice to WT mice (MKK32/2→ WT).
Survival (A) and body temperature in degrees centigrade (C) at 6 h (B)
were measured after i.p. LPS (40 mg/kg. n = 15, *p , 0.05 compared with
WT → WT.
MKK3 deficiency in nonhematopoietic cells, in part,
The Journal of Immunology5
at Yale University on January 16, 2013
(40 mg/kg), and ICAM-1, VCAM-1, selectin E, CXCL-1, and CXCL-2 mRNAwere measured by real-time PCR in lung lysates. The values are expressed
as mean fold induction over untreated mice 6 SEM. n = 5, *p , 0.05. (B) WT and MKK32/2mice were given i.p. LPS (40 mg/kg), and ICAM-1 mRNA
was measured by real-time PCR in lung, kidney, and liver lysates. The values are expressed as mean fold induction over untreated mice 6 SEM. n = 5, *p ,
0.05. (C) Primary cultures of lung endothelial cells were exposed to LPS (1 mg/ml), and mRNA levels of ICAM-1 were measured at different time points.
The values are expressed as mean fold induction over unstimulated cells 6 SEM. *p , 0.05. The results are representative of at least three independent
experiments. (D) Lung endothelial cells were exposed to LPS (1 mg/ml), and protein levels of ICAM-1 were assessed at different time points. Actin was
detected as a loading control. The results are representative of at least three independent experiments. (E) Lung endothelial cells were exposed to LPS (1
mg/ml), and surface expression of ICAM-1 was measured using flow cytometry. The values are expressed as mean fluorescent intensity 6 SEM. *p , 0.05.
The results are representative of at least three independent experiments. (F) Knockdown of MKK3 in WT lung endothelial cells was achieved using siRNA,
and MKK3 or ICAM-1 mRNA expression was detected by real-time PCR after LPS stimulation (1 mg/ml). The values are expressed as mean fold induction
over unstimulated cells 6 SEM. *p , 0.05. The results are representative of at least three independent experiments.
ICAM-1 expression is lower in MKK32/2organs and endothelial cells after LPS. (A) WT and MKK32/2mice were given i.p. LPS
6 MKK3 MEDIATES SEPSIS AND LUNG INJURY
at Yale University on January 16, 2013
way (23, 24). We found reduced translocation of NF-kB to the
nucleus in MKK32/2endothelial cells at baseline and after LPS,
as shown by Western blots of NF-kB in nuclear and cytoplasmic
fractions (Fig. 5A). The loss of NF-kB nuclear translocation is
known to be caused by the inability of the upstream kinase, IKKa/b,
to be phosphorylated and subsequently inactivated. Therefore,
we determined whether IKKa/b phosphorylation is altered in
MKK32/2cells. Consistent with our NF-kB Western blots, we
found reduced phosphorylation of IKKa/b in MKK32/2endo-
thelial cells compared with WT cells at baseline and in response to
LPS (Fig. 5B). ICAM-1 transcription is also dependent on the
transcription factor AP-1, which appears to cooperatively interact
cells were exposed to LPS (1 mg/ml), cell nuclear and cytoplasmic fractions were isolated and analyzed for the p65 subunit of NF-kB by Western blots.
a-Tubulin was detected as cytoplasmic protein loading controls. Lamin A/C was detected as nuclear protein loading controls. The results are representative
of at least three independent experiments. (B) WT and MKK32/2lung endothelial cells were exposed to LPS (1 mg/ml), and lysates were analyzed for
phosphorylated (p)–IKKa/b by Western blots. a-Tubulin was detected as protein loading controls. The results are representative of at least three inde-
pendent experiments. (C) Nuclear extract prepared from the control or mouse lung endothelial cells transfected with MKK3 siRNA and followed by LPS
treatment (1 mg/ml) was mixed with biotin-labeled oligonucleotide containing AP-1 motif. Bound complexes were analyzed by electrophoresis. The results
are representative of at least three independent experiments. (D) Competitive inhibition of AP-1 binding with nonlabeled probe. Nuclear extract prepared
from the control or mouse lung endothelial cells transfected with MKK3 siRNA and followed by LPS treatment (1 mg/ml) was mixed with biotin-labeled
oligonucleotide containing AP-1 motif along with 2003 unlabeled probe. Bound complexes were analyzed by electrophoresis. The results are repre-
sentative of at least 3 independent experiments. (E) Control or WT mouse lung endothelial cells were transfected with MKK3 siRNA and treated with LPS
(1 mg/ml). Cell lysates were analyzed by Western blot to determine efficacy of MKK3 inhibition. The results are representative of at least three independent
Activation of ICAM-1 transcription factors was lower in MKK32/2endothelial cells after LPS. (A) After WTand MKK32/2lung endothelial
The Journal of Immunology7
at Yale University on January 16, 2013
with NF-kB in ICAM-1 gene regulation (25, 26). Hence, we ex-
amined whether AP-1 activity is altered in MKK32/2cells. We
found by EMSAs there was less AP-1 binding to the target se-
quence in MKK32/2cells compared with WT cells at baseline and
after LPS (Fig. 5C–E). These results show that MKK3 regulates
both basal and LPS-induced AP-1 and NF-kB–IKKa/b activation
and that a consequence of MKK3 deficiency is decreased AP-1,
NF-kB, and ICAM-1 expression. To our knowledge, our studies
are the first to identify a regulator of constitutive AP-1, NF-kB,
and ICAM-1 expression in endothelial cells.
ROS production was lower in MKK32/2mice and endothelial
cells after LPS
LPS is known to induce ROS and activate NF-kB in neutrophils
(27). To investigate whether MKK3 is involved in LPS-induced
ROS generation, we checked MDA levels in the serum of LPS-
exposed mice. MDA assay measures lipid peroxidation and is
a marker of ROS excess. We found that MDA levels were sig-
nificantly lower in the serum of MKK32/2mice compared with
that of WT mice after LPS (Fig. 6A). We also detected intracel-
lular ROS in endothelial cells by measuring CM-H2DCFDA, an
indicator of H2O2, a major component of ROS. We found that
CM-H2DCFDA levels were significantly lower in MKK32/2en-
dothelial cells at baseline and after LPS exposure (Fig. 6B), in-
dicating that the absence of MKK3 is associated with decreased
ROS. The major sources of ROS in cells are either the cytoplasmic
families of NADPH oxidases (Nox) or related family of dual
oxidases (Duox) or the mitochondria. To determine the source of
ROS, we initially checked mRNA expression of Nox 1–4 and
Duox 1,2 by PCR in lungs and endothelial cells and did not find
any difference between WT and MKK32/2mice and endothelial
cells. Furthermore, when we specifically inhibited Nox, using
diphenylene iodonium, we did not see any difference in ICAM-1
expression in WT endothelial cells (data not shown). We then
considered the mitochondria as a source and investigated the
levels of mitochondrial ROS, using MitoSOX Red, a fluorescent
and MDA levels were measured in serum. n = 5–8, mean 6 SEM shown, *p , 0.05. (B) WTand MKK32/2lung endothelial cells were exposed to LPS (1
mg/ml). Cells were stained with CM-H2DCFDA, which detects H2O2; levels were measured by flow cytometry. The values are expressed as mean fluo-
rescent intensity 6 SEM. *p , 0.05. The results are representative of at least three independent experiments. Representative fluorescence histograms of
LPS-exposed cells are shown. (C) WT and MKK32/2lung endothelial cells were exposed to LPS (1 mg/ml). Cells were stained with MitoSOX Red, which
detects mitochondrial ROS; levels were measured by flow cytometry. The values are expressed as mean fluorescent intensity 6 SEM. *p , 0.05. The results
are representative of at least three independent experiments. Representative fluorescence histograms of LPS-exposed cells are shown.
ROS production was lower in MKK32/2mice and endothelial cells after LPS. (A) WTand MKK32/2mice were given i.p. LPS (40 mg/ml),
8MKK3 MEDIATES SEPSIS AND LUNG INJURY
at Yale University on January 16, 2013
dye specific for mitochondrial ROS (28). We found that levels of
mitochondrial ROS were lower in MKK32/2than in WT endo-
thelial cells at baseline and in response to LPS (Fig. 6C).
Mitochondrial ROS is upstream of ICAM-1
Next, we determined whether causative links existed between
reduced mitochondrial ROS and ICAM-1 expression in MKK32/2
endothelial cells. We asked if specific induction of mitochondrial
ROS can restore ICAM-1 expression in MKK32/2endothelial
cells. Cells were exposed to rotenone, a mitochondrial respiratory
chain inhibitor that induces production of mitochondrial ROS. We
found that ICAM-1 mRNA was induced in WT and MKK32/2
endothelial cells after rotenone exposure, suggesting not only that
mitochondrial ROS is sufficient for ICAM-1 upregulation but that
depressed mitochondrial ROS can account for decreased ICAM-1
expression in MKK32/2endothelial cells (Fig. 7A). To confirm
these results, we used Mito-TEMPO, an antioxidant specific to the
mitochondria (29), to determine the contribution of mitochondrial
ROS to ICAM-1 expression. We found that in WT endothelial
cells Mito-TEMPO reduced significantly the expression of ICAM-
1 mRNA at baseline and after LPS exposure. In contrast, MKK32/2
endothelial cells showed no difference in ICAM-1 expression after
Mito-TEMPO exposure (Fig. 7B). The latter was expected be-
cause MKK32/2endothelial cells already have reduced mitochon-
drial ROS and addition of Mito-TEMPO has no added benefit. In
conclusion, we show that endothelial MKK3 is an important regu-
lator of mitochondrial redox status with subsequent regulation of
AP-1, NF-kB, and ICAM-1 during LPS exposure. Collectively,
these studies identify a novel role for MKK3 in lethal LPS re-
sponses and provide new therapeutic targets against sepsis and
acute lung injury.
Sepsis remains a critical problem, with significant mortality and
morbidity despite intense efforts to find effective therapies. The
prevailing theory of sepsis is an unregulated inflammatory response
chondrial ROS (5 mM, 5 h), and cell lysates were checked for ICAM-1 by real-time PCR. The results are representative of at least three independent
experiments. (B) WT and MKK32/2endothelial cells were pretreated with 50 mM Mito-Tempo for 2 h and then exposed to LPS (1 mg/ml, 2 h), and cell
lysates were checked for ICAM-1 by RT-PCR. The values are expressed as fold induction over unstimulated cells 6 SEM. *p , 0.05 compared with
samples untreated with Mito-TEMPO. The results are representative of at least three independent experiments.
Mitochondrial ROS is upstream of ICAM-1. (A) WT and MKK32/2lung endothelial cells were exposed to rotenone, an inducer of mito-
The Journal of Immunology9
at Yale University on January 16, 2013
multiple trials of anti-inflammatory or anticytokine therapies have
been disappointing failures (31). Most of the unsuccessful thera-
pies try to inhibit mediators acting far downstream to the initial
stimulus that set in motion the inflammatory response. By iden-
tifying more proximal mediators, we may have a better chance
of halting the inflammatory response before deleterious conse-
quences ensue. Our study identifies MKK3, a proximal-activating
kinase in the p38 MAPK pathway, as a potential therapeutic target.
We demonstrate for the first time, to our knowledge, that MKK3
deficiency leads to protection against LPS-induced lung injury
through the reduction of endothelial mitochondrial ROS, AP-1,
NF-kB activation, and ICAM-1 expression, ultimately reducing
Mitochondria are ancient bacterial endosymbionts in eukary-
otic cells that are responsible for oxidative phosphorylation and
generation of ATP. Mitochondria are also a major source of ROS
and inducers of apoptosis. New evidence suggests that mitochon-
dria play a critical role in inflammatory responses (32, 33). Mi-
tochondrial dysfunction is associated with greater severity and
worse outcomes in patients with sepsis and lung injury (34, 35). In
this article, we show that MKK3-deficient mice have lower mi-
tochondrial ROS (at baseline and after LPS) and are protected
against endotoxemia and lung injury. ICAM-1 is predominantly
transcriptionally regulated, under the control of AP-1 and NF-kB
transcription factors, key players in the inflammatory responses of
sepsis (36). AP-1 and NF-kB are redox-sensitive transcription
factors and cooperatively influence the expression of ICAM-1 in
endothelial cells (37–40). The mechanism of regulation of AP-1
and NF-kB by oxidant signaling remains undetermined. IKK is
considered the major proximal redox-modulated regulatory kinase
for NF-kB signaling (41). Our studies indicate that MKK3 may
influence the phosphorylation of IKK through the mitochondrial
redox status in endothelial cells, in response to LPS. Our studies
establish novel links between MKK3, mitochondrial redox status,
AP-1, IKK–NF-kB signaling, and ICAM-1 expression in endo-
toxemia and lung injury (Fig. 8).
TLR4 is reported as the canonical LPS-responsive pathway.
Mice that are deficient in TLR4 function are highly resistant to
endotoxic shock (42–44). MKK3 appears to be an important
part of LPS responses and may function in tandem with TLR4 as
part of the innate immune response. The specific contribution of
MKK3 in innate immunity has not been well characterized, and
we show for the first time, to our knowledge, that MKK3 mediates
critical responses to LPS. We believe that MKK32/2mice are not
completely deficient in TLR4 responses, as the increase in ICAM-
1 in response to LPS was not completely abolished and MKK32/2
mice retained the ability to induce TLR4-induced cytokines, such
as IL-6 and TNF-a (not shown). There was a general reduction in
other adhesion and chemokine markers, such as VCAM-1 and
CXCL-2, in MKK3-deficient mice. Hence MKK3 likely has both
TLR4-dependent and TLR4-independent effects. Future studies
will help us delineate the specific contribution of MKK3 to TLR4
function. We show dramatic protection against injury in LPS-
challenged MKK32/2mice. Apart from TLR4-deficient mice,
only a few other studies show substantial protection against en-
dotoxic shock using knockout mice. Mice deficient in IL1-b–
converting enzyme (caspase-1), poly(ADP-ribose) polymerase 1,
and ICAM-1 were completely protected against death after LPS
exposure (21, 45, 46). Notably, we demonstrate that MKK32/2
endothelial cells and mice are deficient in ICAM-1 after LPS
exposure, which identifies MKK3 as a critical upstream regulator
of molecules that determine survival after LPS. Our identification
of MKK3 as a major mediator of LPS-induced injury provides
new insights into innate immune pathways and serves as a basis
for new therapies against sepsis.
Vascular inflammation is a sentinel event in sepsis. The endo-
thelium is central to the pathogenesis of sepsis through effects on
inflammation, leukocyte recruitment, vascular tone, coagulation,
and thrombosis. Key endothelial changes in sepsis are disruption of
tulate that endothelial MKK3 is required for LPS-in-
duced mitochondrial ROS generation, IKKa/b phos-
phorylation, NF-kB and AP-1 activation, and ICAM-1
expression, ultimately leading to inflammatory recruit-
Summary of MKK3 signaling. We pos-
10MKK3 MEDIATES SEPSIS AND LUNG INJURY
at Yale University on January 16, 2013
the endothelial barrier and promotion of leukocyte adhesion.
Leukocyte accumulation is orchestrated by coordinated expression
of chemokine and adhesion molecules. ICAM-1 on endothelial
cells attaches to b2 integrin receptors on leukocytes, leading to
firm binding and transmigration (47). We show that MKK3 defi-
ciency prevents the loss of endothelial barrier function and pre-
vents inflammatory influx by decreasing ICAM-1 expression.
MKK3 has not been reported to be involved in ICAM-1 regula-
tion, but our studies point to a pivotal role for MKK3 during LPS
challenge. Lung epithelial cells express ICAM-1 in response to
LPS (48). Reduced epithelial ICAM-1 expression in MKK3-
deficient mice may also contribute to the protection in sepsis, as
our bone marrow chimera mice suggest that MKK3 expression in
nonhematopoetic cells is important. In future studies, we will
examine the role of epithelial cells in determining septic responses
in MKK3-deficient mice.
Finally, it is notable that even the baseline levels of mito-
chondrial ROS, ICAM-1, AP-1, and NF-kB activation in MKK32/2
lung and endothelial cells are lower than those of WT. It is known
that ICAM-1 is expressed constitutively in specific vascular beds,
with the highest expression in the lung, and that the upregulation
of ICAM-1 expression in the lung increases after LPS exposure
(49). This observation suggests that the pulmonary vasculature
may serve as an active gateway for inflammatory cell recruitment
and is primed to adhere and recruit neutrophils in response to a
proinflammatory stimulus by rapidly upregulating ICAM-1 ex-
pression. Similar mechanisms of regulation may be in effect in
organs, such as liver and kidney, with high populations of endo-
thelial cells. We show that MKK3 plays an important role in this
regulation and demonstrate that MKK32/2mice have less ICAM-1
expression and tissue damage in lung, liver, and kidney. It is
noteworthy that the lung, kidney, and liver are the most common
organs to fail in sepsis and that multiorgan failure is the major cause
of death in sepsis-induced lung injury. Our studies have identified
MKK3 as a promising therapeutic target for sepsis-induced multi-
We thank Susan Ardito for administrative and editorial assistance and
Richard A. Flavell (Yale University, New Haven, CT) and Roger J. Davis
(University of Massachusetts Medical School, Worcester, MA) for the kind
gift of MKK3-deficient mice.
The authors have no financial conflicts of interest.
1. Angus, D. C., W. T. Linde-Zwirble, J. Lidicker, G. Clermont, J. Carcillo, and
M. R. Pinsky. 2001. Epidemiology of severe sepsis in the United States: analysis
of incidence, outcome, and associated costs of care. Crit. Care Med. 29: 1303–
2. Ono, K., and J. Han. 2000. The p38 signal transduction pathway: activation and
function. Cell. Signal. 12: 1–13.
3. Han, J., J. D. Lee, L. Bibbs, and R. J. Ulevitch. 1994. A MAP kinase targeted by
endotoxin and hyperosmolarity in mammalian cells. Science 265: 808–811.
4. Han, J., X. Wang, Y. Jiang, R. J. Ulevitch, and S. Lin. 1997. Identification and
characterization of a predominant isoform of human MKK3. FEBS Lett.403: 19–22.
5. De ´rijard, B., J. Raingeaud, T. Barrett, I. H. Wu, J. Han, R. J. Ulevitch, and
R. J. Davis. 1995. Independent human MAP-kinase signal transduction pathways
defined by MEK and MKK isoforms. Science 267: 682–685.
6. Wysk, M., D. D. Yang, H. T. Lu, R. A. Flavell, and R. J. Davis. 1999. Re-
quirement of mitogen-activated protein kinase kinase 3 (MKK3) for tumor ne-
crosis factor-induced cytokine expression. Proc. Natl. Acad. Sci. USA 96: 3763–
7. Lu, H. T., D. D. Yang, M. Wysk, E. Gatti, I. Mellman, R. J. Davis, and
R. A. Flavell. 1999. Defective IL-12 production in mitogen-activated protein
(MAP) kinase kinase 3 (Mkk3)-deficient mice. EMBO J. 18: 1845–1857.
8. Pellizzari, R., C. Guidi-Rontani, G. Vitale, M. Mock, and C. Montecucco. 1999.
Anthrax lethal factor cleaves MKK3 in macrophages and inhibits the LPS/
IFNgamma-induced release of NO and TNFalpha. FEBS Lett. 462: 199–204.
9. Zhang, X., P. Shan, J. Alam, R. J. Davis, R. A. Flavell, and P. J. Lee. 2003.
Carbon monoxide modulates Fas/Fas ligand, caspases, and Bcl-2 family proteins
via the p38alpha mitogen-activated protein kinase pathway during ischemia-
reperfusion lung injury. J. Biol. Chem. 278: 22061–22070.
10. Zhang, X., P. Shan, J. Alam, X. Y. Fu, and P. J. Lee. 2005. Carbon monoxide
differentially modulates STAT1 and STAT3 and inhibits apoptosis via a phos-
phatidylinositol 3-kinase/Akt and p38 kinase-dependent STAT3 pathway during
anoxia-reoxygenation injury. J. Biol. Chem. 280: 8714–8721.
11. Otterbein, L. E., S. L. Otterbein, E. Ifedigbo, F. Liu, D. E. Morse, C. Fearns,
R. J. Ulevitch, R. Knickelbein, R. A. Flavell, and A. M. Choi. 2003. MKK3
mitogen-activated protein kinase pathway mediates carbon monoxide-induced
protection against oxidant-induced lung injury. Am. J. Pathol. 163: 2555–
12. Dolinay, T., W. Wu, N. Kaminski, E. Ifedigbo, A. M. Kaynar, M. Szilasi,
S. C. Watkins, S. W. Ryter, A. Hoetzel, and A. M. Choi. 2008. Mitogen-activated
protein kinases regulate susceptibility to ventilator-induced lung injury. PLoS
One 3: e1601.
13. Lim, A. K., D. J. Nikolic-Paterson, F. Y. Ma, E. Ozols, M. C. Thomas,
R. A. Flavell, R. J. Davis, and G. H. Tesch. 2009. Role of MKK3-p38 MAPK
signalling in the development of type 2 diabetes and renal injury in obese db/db
mice. Diabetologia 52: 347–358.
14. Ma, F. Y., G. H. Tesch, R. A. Flavell, R. J. Davis, and D. J. Nikolic-Paterson.
2007. MKK3-p38 signaling promotes apoptosis and the early inflammatory re-
sponse in the obstructed mouse kidney. Am. J. Physiol. Renal Physiol. 293:
15. Wang, Y., H. X. Ji, J. N. Zheng, D. S. Pei, S. Q. Hu, and S. L. Qiu. 2009.
Protective effect of selenite on renal ischemia/reperfusion injury through
inhibiting ASK1-MKK3-p38 signal pathway. Redox Rep. 14: 243–250.
16. Fukuda, K., G. H. Tesch, F. Y. Yap, J. M. Forbes, R. A. Flavell, R. J. Davis, and
D. J. Nikolic-Paterson. 2008. MKK3 signalling plays an essential role in
leukocyte-mediated pancreatic injury in the multiple low-dose streptozotocin
model. Lab. Invest. 88: 398–407.
17. Zhang, X., P. Shan, G. Jiang, L. Cohn, and P. J. Lee. 2006. Toll-like receptor 4
deficiency causes pulmonary emphysema. J. Clin. Invest. 116: 3050–3059.
18. Kuhlencordt, P. J., E. Rosel, R. E. Gerszten, M. Morales-Ruiz, D. Dombkowski,
W. J. Atkinson, F. Han, F. Preffer, A. Rosenzweig, W. C. Sessa, et al. 2004. Role
of endothelial nitric oxide synthase in endothelial activation: insights from eNOS
knockout endothelial cells. Am. J. Physiol. Cell Physiol. 286: C1195–C1202.
19. Matute-Bello, G., C. W. Frevert, and T. R. Martin. 2008. Animal models of acute
lung injury. Am. J. Physiol. Lung Cell. Mol. Physiol. 295: L379–L399.
20. Warn, P. A., M. W. Brampton, A. Sharp, G. Morrissey, N. Steel, D. W. Denning,
and T. Priest. 2003. Infrared body temperature measurement of mice as an early
predictor of death in experimental fungal infections. Lab. Anim. 37: 126–131.
21. Xu, H., J. A. Gonzalo, Y. St Pierre, I. R. Williams, T. S. Kupper, R. S. Cotran,
T. A. Springer, and J. C. Gutierrez-Ramos. 1994. Leukocytosis and resistance to
septic shock in intercellular adhesion molecule 1-deficient mice. J. Exp. Med.
22. McDonald, B., K. Pittman, G. B. Menezes, S. A. Hirota, I. Slaba,
C. C. Waterhouse, P. L. Beck, D. A. Muruve, and P. Kubes. 2010. Intravascular
danger signals guide neutrophils to sites of sterile inflammation. Science 330:
23. De Plaen, I. G., X. B. Han, X. Liu, W. Hsueh, S. Ghosh, and M. J. May. 2006.
Lipopolysaccharide induces CXCL2/macrophage inflammatory protein-2 gene
expression in enterocytes via NF-kappaB activation: independence from en-
dogenous TNF-alpha and platelet-activating factor. Immunology 118: 153–163.
24. Kisseleva, T., L. Song, M. Vorontchikhina, N. Feirt, J. Kitajewski, and
C. Schindler. 2006. NF-kappaB regulation of endothelial cell function during
LPS-induced toxemia and cancer. J. Clin. Invest. 116: 2955–2963.
25. Roebuck, K. A. 1999. Oxidant stress regulation of IL-8 and ICAM-1 gene ex-
pression: differential activation and binding of the transcription factors AP-1 and
NF-kappaB (Review). [Review] Int. J. Mol. Med. 4: 223–230.
26. Fujioka, S., J. Niu, C. Schmidt, G. M. Sclabas, B. Peng, T. Uwagawa, Z. Li,
D. B. Evans, J. L. Abbruzzese, and P. J. Chiao. 2004. NF-kappaB and AP-1
connection: mechanism of NF-kappaB-dependent regulation of AP-1 activity.
Mol. Cell. Biol. 24: 7806–7819.
27. Asehnoune, K., D. Strassheim, S. Mitra, J. Y. Kim, and E. Abraham. 2004. In-
volvement of reactive oxygen species in Toll-like receptor 4-dependent activa-
tion of NF-kappa B. J. Immunol. 172: 2522–2529.
28. Mukhopadhyay, P., M. Rajesh, K. Yoshihiro, G. Hasko ´, and P. Pacher. 2007.
Simple quantitative detection of mitochondrial superoxide production in live
cells. Biochem. Biophys. Res. Commun. 358: 203–208.
29. Dikalova, A. E., A. T. Bikineyeva, K. Budzyn, R. R. Nazarewicz, L. McCann,
W. Lewis, D. G. Harrison, and S. I. Dikalov. 2010. Therapeutic targeting of
mitochondrial superoxide in hypertension. Circ. Res. 107: 106–116.
30. Hotchkiss, R. S., and I. E. Karl. 2003. The pathophysiology and treatment of
sepsis. N. Engl. J. Med. 348: 138–150.
31. Deans, K. J., M. Haley, C. Natanson, P. Q. Eichacker, and P. C. Minneci. 2005.
Novel therapies for sepsis: a review. J. Trauma 58: 867–874.
32. Kepp, O., L. Galluzzi, and G. Kroemer. 2011. Mitochondrial control of the
NLRP3 inflammasome. Nat. Immunol. 12: 199–200.
33. Zhang, Q., M. Raoof, Y. Chen, Y. Sumi, T. Sursal, W. Junger, K. Brohi,
K. Itagaki, and C. J. Hauser. 2010. Circulating mitochondrial DAMPs cause
inflammatory responses to injury. Nature 464: 104–107.
The Journal of Immunology 11
at Yale University on January 16, 2013
34. Carre ´, J. E., J. C. Orban, L. Re, K. Felsmann, W. Iffert, M. Bauer, H. B. Suliman,
C. A. Piantadosi, T. M. Mayhew, P. Breen, et al. 2010. Survival in critical illness
is associated with early activation of mitochondrial biogenesis. Am. J. Respir.
Crit. Care Med. 182: 745–751.
35. Brealey, D., M. Brand, I. Hargreaves, S. Heales, J. Land, R. Smolenski,
N. A. Davies, C. E. Cooper, and M. Singer. 2002. Association between mito-
chondrial dysfunction and severity and outcome of septic shock. Lancet 360:
36. Tak, P. P., and G. S. Firestein. 2001. NF-kappaB: a key role in inflammatory
diseases. J. Clin. Invest. 107: 7–11.
37. Sen, C. K., S. Khanna, A. Z. Reznick, S. Roy, and L. Packer. 1997. Glutathione
regulation of tumor necrosis factor-alpha-induced NF-kappa B activation in skel-
etal muscle-derived L6 cells. Biochem. Biophys. Res. Commun. 237: 645–649.
38. Janssen-Heininger, Y. M., M. E. Poynter, and P. A. Baeuerle. 2000. Recent
advances towards understanding redox mechanisms in the activation of nuclear
factor kappaB. Free Radic. Biol. Med. 28: 1317–1327.
39. Schmidt, K. N., P. Amstad, P. Cerutti, and P. A. Baeuerle. 1995. The roles of
hydrogen peroxide and superoxide as messengers in the activation of tran-
scription factor NF-kappa B. Chem. Biol. 2: 13–22.
40. Fan, H., B. Sun, Q. Gu, A. Lafond-Walker, S. Cao, and L. C. Becker. 2002.
Oxygen radicals trigger activation of NF-kappaB and AP-1 and upregulation of
ICAM-1 in reperfused canine heart. Am. J. Physiol. Heart Circ. Physiol. 282:
41. Pantano, C., N. L. Reynaert, A. van der Vliet, and Y. M. Janssen-Heininger.
2006. Redox-sensitive kinases of the nuclear factor-kappaB signaling pathway.
Antioxid. Redox Signal. 8: 1791–1806.
42. Poltorak, A., X. He, I. Smirnova, M. Y. Liu, C. Van Huffel, X. Du, D. Birdwell,
E. Alejos, M. Silva, C. Galanos, et al. 1998. Defective LPS signaling in C3H/HeJ
and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282: 2085–2088.
43. Kawai, T., O. Adachi, T. Ogawa, K. Takeda, and S. Akira. 1999. Unrespon-
siveness of MyD88-deficient mice to endotoxin. Immunity 11: 115–122.
44. Haziot, A., E. Ferrero, F. Ko ¨ntgen, N. Hijiya, S. Yamamoto, J. Silver, C. L. Stewart,
and S. M. Goyert. 1996. Resistance to endotoxin shock and reduced dissemination
of gram-negative bacteria in CD14-deficient mice. Immunity 4: 407–414.
45. Li, P., H. Allen, S. Banerjee, S. Franklin, L. Herzog, C. Johnston, J. McDowell,
M. Paskind, L. Rodman, J. Salfeld, et al. 1995. Mice deficient in IL-1 beta-
converting enzyme are defective in production of mature IL-1 beta and resis-
tant to endotoxic shock. Cell 80: 401–411.
46. Oliver, F. J., J. Me ´nissier-de Murcia, C. Nacci, P. Decker, R. Andriantsitohaina,
S. Muller, G. de la Rubia, J. C. Stoclet, and G. de Murcia. 1999. Resistance to
endotoxic shock as a consequence of defective NF-kappaB activation in poly
(ADP-ribose) polymerase-1 deficient mice. EMBO J. 18: 4446–4454.
47. Rahman, A., and F. Fazal. 2009. Hug tightly and say goodbye: role of endothelial
ICAM-1 in leukocyte transmigration. Antioxid. Redox Signal. 11: 823–839.
48. Lee, J. H., L. Del Sorbo, S. Uhlig, G. A. Porro, T. Whitehead, S. Voglis, M. Liu,
A. S. Slutsky, and H. Zhang. 2004. Intercellular adhesion molecule-1 mediates
cellular cross-talk between parenchymal and immune cells after lipopolysac-
charide neutralization. J. Immunol. 172: 608–616.
49. Pane ´s, J., M. A. Perry, D. C. Anderson, A. Manning, B. Leone, G. Cepinskas,
C. L. Rosenbloom, M. Miyasaka, P. R. Kvietys, and D. N. Granger. 1995. Re-
gional differences in constitutive and induced ICAM-1 expression in vivo. Am. J.
Physiol. 269: H1955–H1964.
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at Yale University on January 16, 2013