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* Department of Surgery, University Health Network, and
Department of Medicine, St. Michael’s Hospital and University of Toronto, Toronto, Ontario, Canada;
Cardiovascular Division, Brigham and Women’s Hospital, Boston, MA 02115; and
Section of Immunobiology, Yale University School of Medicine and Howard Hughes Medical Institute, New Haven, CT 06510
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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69%. By contrast, while shock/resuscitation alone had
no effect on TLR4 mRNA levels, it markedly altered the response to LPS.
Specifically, antecedent shock prevented the LPS-induced reduction in
TLR4 mRNA levels. This reversal was explained by the ability of prior
resuscitated shock both to prevent the destabilization of TLR4 mRNA by
LPS and also to augment LPS-stimulated TLR4 gene transcription compared
with LPS alone. Oxidant stress related to shock/resuscitation appeared
to contribute to the regulation of TLR4 mRNA, because supplementation
of the resuscitation fluid with the antioxidant
N-acetylcysteine reversed the ability of
shock/resuscitation to preserve TLR4 mRNA levels following LPS. TLR4
protein levels in whole lung mirrored the changes seen for TLR4 mRNA.
Considered in aggregate, these data suggest that levels of
tlr4 expression are controlled both transcriptionally as
well as posttranscriptionally through altered mRNA stability and that
antecedent shock/resuscitation, a form of global ischemia/reperfusion,
might influence regulation of this gene. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B in response to LPS stimulation. Considered together, these
findings suggested that resuscitated hemorrhagic shock augments
LPS-induced lung injury by increasing the responsiveness of the
cellular signaling pathways to LPS challenge.
Recent studies have provided further insights into the signaling
pathways whereby LPS causes dissociation of I-B from the cytoplasmic
I-B/NF-B complex and leads to nuclear translocation of NF-B.
The Toll-like receptor 4 (TLR4), a member of the Toll-like receptor
family, has been shown to serve as the main upstream sensor for LPS
effect in vitro and in vivo. Cells derived from
tlr4-deficient mice or mice with a spontaneous mutant TLR4
are known to be resistant to LPS (5, 6), while
unresponsive HEK 293 cells are rendered responsive to LPS following
transfection with TLR4 cDNA (7). Two other cell surface
molecules, CD14 and MD-2, also appear necessary to optimize LPS
signaling. CD14, in its soluble form or as a GPI-anchored surface
protein, may act as a signaling bridge to promote surface recognition
of LPS by the cell (7, 8, 9). MD-2 is physically associated
with TLR4 on the cell surface and appears to both augment the
responsiveness of the cell to LPS and also regulate activation of
specific intracellular signaling pathways following LPS engagement
(10, 11, 12). Recent studies suggest that TLR4, CD14, and MD-2
may serve as a tripartite LPS receptor complex with LPS binding to each
of the component molecules (13).
Expression of tlr4 has been demonstrated in several tissues and is particularly pronounced among myelomonocytic cells (5, 14). However, tlr4 regulation in these cells remains largely unexplored. Muzio et al. (14) reported that LPS increased levels of TLR4 mRNA in human peripheral blood monocytes in an actinomycin D-dependent fashion, suggesting transcriptional regulation. By contrast, Poltorak et al. (5) demonstrated that TLR4 mRNA was constitutively present in RAW 264.7 cells and LPS rapidly and transiently suppressed TLR4 mRNA levels. Similarly, Nomura et al. (15) observed that LPS treatment of mouse peritoneal macrophages lowered both TLR4 mRNA levels and surface TLR4 expression. These studies did not examine whether alterations in TLR4 mRNA levels were due to altered transcription, mRNA stability, or both. The present studies were performed to examine the mechanisms regulating tlr4 gene expression in vivo and also to evaluate whether resuscitated hemorrhagic shock, a known predisposing event for the development of lung inflammation in man, might influence its regulation. In this paper, we show that tlr4 is regulated both transcriptionally and posttranscriptionally following exposure to LPS and that antecedent resuscitated hemorrhagic shock modulates the nature of the LPS response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male Sprague-Dawley rats (300–350 g; Charles River Breeding Laboratories, St. Constant, Quebec, Canada) were anesthetized with 80 mg/kg ketamine and 8 mg/kg xylazine administered i.p. The right carotid artery was cannulated with a 22-gauge angiocath (BD Biosciences, Franklin Lakes, NJ) for monitoring of mean arterial pressure (MAP), blood sampling, and resuscitation. Hemorrhagic shock was initiated by blood withdrawal and reduction of the MAP to 40 mmHg within 15 min, as previously described. This blood pressure was maintained by further blood withdrawal if the MAP was >45 mmHg, and by infusion of 0.5 ml of Ringer’s lactate (RL) if the MAP was <35 mmHg. Blood was collected into 0.1 ml citrate per milliliter of blood to prevent clotting. After a hypotensive period of 60 min, animals were resuscitated by transfusion of the shed blood and RL in a volume equal to that of shed blood, over a period of 2 h. In some studies, animals received N-acetylcysteine (NAC) (0.5 g/kg) via the artery before the infusion of RL. The catheter was then removed, the carotid artery was ligated, and the cervical incision was closed. Sham animals underwent the same surgical procedures, but hemorrhage was not induced. NAC delivery occurred in sham animals at an equivalent time to that received in shock animals (4).
Altered gene expression in the lung after shock/resuscitation was studied using two protocols, one in vivo and the other ex vivo (16). For in vivo studies, animals underwent a tracheotomy using a 14-gauge catheter 1 h after the end of resuscitation (or sham), and then received either LPS (30 µg/kg E. coli O111:B4 in 200 µl saline) or saline alone (SAL) intratracheally followed by 20 mechanically ventilated breaths using a rodent ventilator. Therefore, the experimental protocol was described as having animals in one of four groups: sham/SAL, shock/SAL, sham/LPS, and shock/LPS. Using this protocol, we previously showed that animals subjected to shock/LPS exhibited increased lung permeability and neutrophil counts compared with all other groups, while sham/LPS group had a small increase in bronchoalveolar lavage (BAL) neutrophil count but no change in permeability (4). At various time points after LPS (or SAL) administration (t = 0), whole lung was recovered for examination by Western or Northern blot analysis. Ex vivo studies used alveolar macrophages retrieved by BAL following shock/resuscitation (or sham). At this time point, there was no difference between groups with respect to the total cell count in the BAL fluid or the absolute number of alveolar macrophages recovered. Macrophages obtained from shock/resuscitated or sham rats were then incubated for various times at 37°C in 5% CO2 either alone or in the presence of 0.1 µg/ml LPS. At the end of the incubation period, cells were pelleted by centrifugation at 300 x g for 10 min. Cells were then processed as indicated for analysis by flow cytometry, Western blot, or Northern blot.
Bronchoalveolar lavage
BAL was performed via the intratracheal catheter using cold PBS (8 mM disodium phosphate, 2 mM potassium phosphate, 0.14 M sodium chloride, and 0.01 M potassium chloride (pH 7.4), with 0.1 mM EDTA). BAL fluid was centrifuged at 300 x g for 10 min to pellet cells. The cell pellet was then suspended on NIM.2 neutrophil isolation medium (Cardinal Associates, Santa Fe, NM), and centrifuged at 750 x g, 20°C, for 45 min for macrophage isolation. The isolated macrophages were washed and resuspended in DMEM containing 10% FCS at a concentration of 1 x 106 cells/ml medium in polypropylene tissue culture tubes. Cell viability was >95%, as assessed by trypan blue exclusion, and consisted of a cell population of >95% macrophages, as assessed by Wright-Giemsa staining.
Northern blot analysis
Total RNA from lungs or alveolar macrophages was obtained using
the guanidium-isothiocyanate method (17). Briefly, lungs
or macrophages were harvested and immediately frozen in liquid
nitrogen. Tissue or cells were then thawed and homogenized in 4 M
guanidine-isothiocyanate containing 25 mM sodium citrate, 0.5%
sarcosyl, and 100 mM 2-ME. RNA was denatured, electrophoresed through a
1.2% formaldehyde-agarose gel, and transferred to nylon membrane.
Hybridization was conducted using a
[32P]dCTP-labeled TLR4 cDNA (kindly provided by
Dr. S. Frantz, Brigham and Women’s Hospital, Boston, MA; see Ref.
18), TNF-
cDNA (American Type Culture Collection,
Manassas, VA), and a [32P]ATP-end-labeled
30-base oligonucleotide probe for CINC, which is complementary to
nucleotides 134–164 of CINC cDNA (19). Blots were then
washed under conditions of high stringency and specific mRNA bands were
detected by autoradiography in the presence of intensifying screens, as
previously reported. Blots were stripped and reprobed for GAPDH to
control for loading. Expression of mRNA was quantitated using a
PhosphoImager and accompanying ImageQuant software (Molecular Dynamics,
Sunnyvale, CA) and was normalized to the GAPDH signal.
Nuclear run-on analysis
LPS treated and untreated alveolar macrophages obtained from the
ex vivo experiments were washed with sterile 10 mM PBS and lysed in
situ with chilled lysis buffer (10 mM Tris-HCl (pH 7.9), 0.15 M NaCl, 1
mM EDTA, and 0.6% (v/v) Nonidet P-40) for 10 min on ice. Cell lysates
were centrifuged for 5 min at 500 x g at 4°C, and
the nuclear pellet was resuspended in chilled nuclear buffer containing
0.3 M
(NH4)2SO4,
100 mM Tris-HCl (pH 7.9), 4 mM MgCl2, 4 mM
MnCl2, 0.2 M NaCl, 0.4 mM EDTA, 0.1 mM PMSF, and
40% (v/v) glycerol. Cold transcription buffer (0.3 M
(NH4)2SO4,
100 mM Tris-HCl (pH 7.9), 4 mM MnCl2, 0.2 M NaCl,
0.4 mM EDTA, and 0.1 mM PMSF) containing 0.2 mM DTT, 40 U RNasin, 0.2
mM ATP, CTP, and GTP, and 150 µCi
[-32P]UTP (3000 Ci/mmol) was added to the
nuclear suspension and incubated for 30 min at 28°C. A total of 20 U
of RNase-free DNase I and 125 µg tRNA were added and incubated for 10
min at 37°C, followed by digestion with proteinase K at a final
concentration of 300 µg/ml in buffer (10 mM Tris-Cl (pH 7.9), 10 mM
EDTA, 0.5% SDS) for 30 min at 42°C. Nuclear transcripts were then
extracted as described above and resuspended at 2 x
106 cpm/ml in Northern hybridization buffer.
Equal amounts (1 µg) of gel-purified cDNA were denatured by boiling
in 0.4 M NaOH and 10 mM EDTA, neutralized with equal volumes of 2 M
ammonium acetate (pH 7), and slot-blotted onto nitrocellulose filters.
Hybridization was performed for 48 h at 42°C in Northern
hybridization buffer as detailed above (20). Hybridized
filters were washed and subjected to autoradiography and quantitation
as described above.
Actinomycin D chase experiments
To assess the effect of LPS and hemorrhage/resuscitation on the half-life of TLR4 mRNA transcripts, alveolar macrophages from shock/resuscitated and sham animals were treated with or without LPS for 0.5 h before addition of 10 µg/ml actinomycin D. Total cellular RNA was extracted at 0, 15, 30, 60, 120, 180, and 240 min after the addition of actinomycin D. Blots were reprobed with a GAPDH cDNA probe to ensure equal loading. TLR4 mRNA transcript levels were normalized for GAPDH mRNA using ImageQuant, and mRNA decay rates were determined according to the following formula: t1/2 = 0.693/k where k = ln (N0/Nt)/t, where N0 represents the density at t = 0 and Nt represents the density at time t. Results are the mean from three independent experiments.
Western blot analysis
The lung tissue homogenate was separated on an 8% SDS-PAGE under reducing conditions (21). Equivalent loading of the gel was determined by quantitation of protein as well as by Coomassie staining of the gel. Separated proteins were electroblotted onto polyvinylidene difluoride membrane and blocked for 1 h at room temperature with TBS containing 1% BSA. The membranes were then incubated with a 1/2500 dilution of antiserum against human TLR4 (Dr. R. Medzhitov, Brigham and Women’s Hospital) at room temperature for 1 h. Ag-Ab complexes were identified with goat anti-rabbit IgG tagged with HRP (Sigma-Aldrich, St. Louis, MO) and exposed to the ECL detection system (Amersham, Arlington Heights, IL) according to the manufacturer’s instructions.
Statistics
The data are presented as mean ± SE of n determinations as indicated in the figures. Data were analyzed by one-way analysis of variance; post hoc testing was performed using the Bonferroni modification of the t test. When individual studies are demonstrated, these are representative of at least three independent studies.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fig. 1
A shows levels of
TLR4 mRNA expression under the various experimental conditions. The
t = 0 time point represents tissues analyzed 1 h
after end resuscitation before intratracheal LPS challenge. As shown,
whole lungs from sham animals demonstrated constitutive expression of
TLR4 mRNA, and shock/resuscitation had no effect on these levels. Over
the 6-h experimental period, the levels of TLR4 mRNA in both sham/SAL
and shock-alone animals remained essentially stable compared with
t = 0. By contrast, sham/LPS animals showed a reduction
in TLR4 mRNA by 2 h after LPS, with near complete disappearance of
mRNA expression by 4 h. The response to LPS in the lungs of
animals exposed to prior resuscitated shock (shock/LPS) differed from
that seen for LPS-alone animals. The rapid early reduction noted for
LPS alone was less pronounced, with the levels tending to increase
toward control beginning at 4 h. Levels of the housekeeping gene
GAPDH remained constant throughout and did not differ between
groups.
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B illustrates that the pattern observed using isolated
alveolar macrophages was similar to that seen for whole lung.
Specifically, while cells from sham/SAL and shock/SAL exhibited steady
levels of TLR4 mRNA, LPS treatment of cells recovered from sham animals
resulted in rapid reduction in levels of expression, with a tendency
toward late restoration of control levels. By contrast, TLR4 mRNA
levels in alveolar macrophages recovered from shock/resuscitated
animals remain essentially unchanged following LPS treatment over the
course of the experimental period. A dose response examining the effect
of LPS alone on alveolar macrophages demonstrates that 0.01 µg/ml LPS
causes a slight reduction in levels of TLR4 mRNA, with progressive
diminution as the dose was increased up to 1 µg/ml (Fig. 1Ca). We also studied later time points following LPS
treatment. As shown in Fig. 1Cb, levels of TLR4 mRNA in
cells cultured in vitro for 18 h after being recovered from
shock/resuscitated animals did not differ from sham animals, while LPS
treatment for 18 h caused marked reduction in TLR4 mRNA levels
whether cells were recovered from sham or shock/resuscitated
animals.
The rapid reduction in mRNA levels in cells treated with LPS was not
observed when macrophages were treated with other inflammatory stimuli
in vitro. As shown in Fig. 1D, neither TNF- (Fig. 1D, upper panels) nor IL-1
(Fig. 1D, lower panels), given at concentrations shown
to up-regulate macrophage CD11b expression in vitro (data not shown),
altered TLR4 mRNA expression in alveolar macrophages. Considered
together, these findings suggest that shock/resuscitation per se has
little effect on TLR4 expression in lung macrophages but markedly
changes the responsiveness of these cells to LPS. In addition, the
events occurring in LPS (or vehicle)-stimulated alveolar macrophages
following their recovery from sham or shock/resuscitated animals appear
to accurately reflect in vivo events following intratracheal LPS (or
vehicle) and thus constitute a relevant model for studying regulation
of TLR4 in these cells.
Regulation of TLR4 mRNA expression
Steady state mRNA levels represent a balance between message
stability and rate of gene transcription. We addressed both
possibilities to investigate the mechanism of altered mRNA expression.
The rapid reduction in TLR4 mRNA levels following LPS treatment in
cells from sham animals suggested the possibility that message
stability might be altered. Consistent with this hypothesis, LPS
treatment of cells derived from sham animals shortened the half-life of
TLR4 mRNA compared with untreated cells by 69%, representing a
reduction from 168 ± 32 to 53 ± 5 min (mean ± SD;
n = 3–5 per group; p < 0.001) (Fig. 2). This effect of LPS on TLR4 mRNA
half-life was not observed in cells recovered from animals following
resuscitated shock. Shock alone caused a small increase in TLR4 mRNA
half-life (192 ± 51 min; n = 3), while shock/LPS
cells exhibited a TLR4 mRNA half-life of 139 ± 19 min
(n = 5; not significant compared with shock alone),
representing an 28% reduction compared with shock alone. Shock/LPS
did not differ from sham. These findings suggest that reduced TLR4 mRNA
stability following LPS in sham animals may account for the rapid
lowering in levels of TLR4 mRNA. In addition, the relative lack of
destabilization of TLR4 message in shock/resuscitated animals may
contribute to the differential effects of LPS on TLR4 mRNA levels
between sham and shock animals.
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A. In the
absence of treatment, cells recovered in the BAL fluid exhibited
constitutive transcription of TLR4, a finding consistent with the basal
levels of TLR4 mRNA evident in vivo and in vitro. The transcription
rate was similar in shock/resuscitated animals. In cells recovered from
sham animals and then exposed to LPS, there was an 1.4-fold increase
in the tlr4 transcription rate, while LPS treatment of cells
from shock/resuscitated animals resulted in an 9-fold rise in
transcription. Densitometry derived from three independent studies is
shown in Fig. 3B. Considered together with the mRNA
stability studies, these findings suggest that the preservation of TLR4
mRNA levels in shock/LPS animals is related to a combination of
enhanced mRNA stability plus increased mRNA transcription. In addition,
while sham/LPS caused a small increase in the tlr4
transcription rate, this effect was outweighed by the marked reduction
in the mRNA stability in terms of determining overall TLR4 mRNA
levels.
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Increased circulating xanthine oxidase released from the
gastrointestinal tract occurs following hemorrhagic shock
(23) and has been shown to contribute to subsequent lung
gene expression (24). We have previously shown that
supplementation of the resuscitation fluid with NAC prevented lung
injury and lessened the LPS-induced CINC and TNF mRNA and protein
following shock/resuscitation, suggesting a role for oxidant stress in
the priming process (4). To examine whether generated
oxidants might influence the regulation of tlr4 gene
expression following shock/LPS, we evaluated TLR4 expression either in
whole lung derived from NAC-resuscitated animals or in macrophages
recovered after shock/resuscitation by BAL (Fig. 4). In the absence of NAC, TLR4 mRNA
expression was again maintained in shock/LPS groups compared with LPS
alone, whether examined in whole lung (Fig. 4A) or in
recovered macrophages (Fig. 4B). However, NAC prevented the
effect of shock/LPS on preservation of TLR4 mRNA levels. Specifically,
animals resuscitated with NAC demonstrated markedly reduced levels of
TLR4 mRNA following LPS treatment compared with shock/LPS alone. This
effect appeared to be mainly due to an effect on tlr4
transcription. As demonstrated in Fig. 3A, NAC lowered the
tlr4 transcription rate to that observed in untreated cells.
Altered mRNA stability may have also contributed to the effect of NAC
on TLR4 mRNA levels, because NAC supplementation of the resuscitation
fluid reduced TLR4 mRNA stability by 32% (Fig. 2C). NAC
did not influence the LPS-induced reduction in TLR4 mRNA stability
(Fig. 2C). These data suggest that oxidant stress resulting
from shock/resuscitation contributes to the altered TLR4 gene
regulation in response to LPS treatment.
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Having demonstrated that shock modulated TLR4 mRNA levels in
response to LPS exposure, we evaluated its effect on TLR4 protein
levels. Whole lung was recovered from animals at varying times after
LPS (or SAL) administration and evaluated for TLR4 protein by Western
blot analysis. As shown in Fig. 5, whole
lung TLR4 remained stable over the 4-h period after intratracheal SAL
in both sham and shock animals. By contrast, whole lung TLR4 levels
rapidly diminished in animals following LPS treatment. This reduction
was markedly attenuated in LPS-treated animals following antecedent
resuscitated shock.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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69%. 2)
Shock/resuscitation modulates the response to LPS. In both whole lung
and alveolar macrophages, antecedent-resuscitated shock prevents the
down-regulation of TLR4 in response to LPS so that TLR4 mRNA levels are
maintained near control levels. Two mechanisms appear to
contribute to this preservation. First, the reduction in mRNA stability
observed with LPS alone is reversed when cells are subjected to
antecedent-resuscitated shock. Second, shock/resuscitation primes for
increased transcription of tlr4 in response to LPS. 3)
Oxidant stress induced by hemorrhage/resuscitation contributes to the
maintenance of TLR4 mRNA levels. The use of the antioxidant NAC in the
resuscitation fluid prevents the priming for increased TLR4 mRNA
transcription and partially reverses the prolongation of TLR4 mRNA
half-life observed following shock/resuscitation. 4) Whole lung
expression of TLR4 protein mirrors changes in mRNA levels. Together,
these studies provide new insight into the regulation of
tlr4 gene and protein expression by LPS in vivo and its
modulation by antecedent-resuscitated shock, a form of global
ischemia/reperfusion.
In this rodent model, macrophages exhibited significant basal levels of
tlr4 gene expression and exposure to LPS exerted a net
effect of decreasing steady state mRNA and protein levels, both in
vitro and in vivo. Other investigators have reported the constitutive
expression of TLR4 mRNA in cells of myeloid lineage and its
stimulus-specific rapid reduction in mRNA levels (5, 15, 25). However, the present studies are the first to evaluate the
mechanism underlying this effect of LPS. Specifically, while LPS was
shown to cause both new gene transcription and destabilization of TLR4
mRNA transcripts, it appears that destabilization of TLR4 mRNA
transcripts predominated during the early time course of the studies,
thereby lowering steady state TLR4 mRNA levels. Other investigators
have reported different basal expression of TLR4 and altered
responsiveness of cells to LPS. For example, Frantz et al.
(18) reported that neither cardiac myocytes nor coronary
microvascular endothelial cells exhibited significant constitutive
expression of TLR4 mRNA, and that LPS treatment increased TLR4
expression in both. Differences between studies may be related to the
cell type studied as well as the species of origin. These may differ in
basal transcription rates of the tlr4 gene as well as the
relative effects of LPS on destabilization of the TLR4 mRNA transcript
and stimulation of tlr4 gene transcription. These studies
also underscore the fact that, in addition to new gene transcription,
the metabolic fate of mRNA transcripts following cellular activation
may influence steady state level of mRNA transcripts. That cell
stimulation can induce destabilization of mRNA transcripts has been
reported for inducible NO synthase, the -adrenergic receptor, and
the estrogen- receptor, among others (26, 27, 28). It
remains to be determined whether LPS also destabilizes the mRNA
transcript in man, as it does in the rat.
The maintenance of TLR4 mRNA levels in shock/resuscitated animals
following LPS treatment appears to be related to two mechanisms. First,
antecedent shock markedly increased LPS-induced tlr4 gene
transcription compared with the response to LPS in sham animals.
Second, LPS-induced TLR4 mRNA destabilization was reduced in cells
exposed to prior shock/resuscitation compared with sham animals. The
latter of these two mechanisms seems to be the predominant one, at
least within the first 2 h after LPS treatment. Oxidants appear to
participate in the regulation of tlr4 gene expression. NAC
supplementation during resuscitation markedly reduced levels of TLR4
mRNA detected in shock/LPS animals at t = 4 h
following LPS administration. This appears to be primarily mediated via
an effect on the tlr4 transcription rate, which is
diminished to basal levels by NAC administration, although a lesser
effect on TLR4 mRNA stability was observed. The mechanism of the
NAC-sensitive augmentation in tlr4 gene transcription
remains to be defined. The most plausible explanation is that oxidants
enhance signaling through the TLR4-NF-B cascade and thus promote
augmented LPS-induced gene transcription. Evidence supporting an
oxidant effect on this signaling pathway is derived from two
observations made in our prior reports. First, NAC addition during
resuscitation prevented the augmented NF-B translocation in
macrophages following LPS treatment. Second, NAC supplementation
reversed the enhanced LPS-induced gene transcription of the
NF-B-dependent genes CINC and TNF in animals exposed to antecedent
shock/resuscitation. One alternate possibility is that NAC precluded
up-regulation of an NF-B-dependent gene whose product stimulates
TLR4 transcription through an NF-B-independent pathway. For example,
enhanced generation of an NF-B-dependent gene product such as
TNF- may synergize with the effects of LPS on TLR4 transcription and
lead to an augmented rise in TLR4 in shock/LPS animals. A recent report
by Frantz et al. (29) demonstrated that surface TLR2 was
required for oxidant-induced nuclear translocation of NF-B in
neonatal rat cardiac myocytes. These authors suggested that factor(s)
released from oxidant-treated cells might activate TLR2. As noted
above, further elucidation of the role of NF-B in tlr4
gene regulation is necessary to support these hypotheses.
Ischemia/reperfusion is known to activate stress-activated protein
kinase and ultimately promote AP-1-dependent reporter activity in rat
myocytes (30). Thus, the AP-1 consensus binding sequence
in the promoter of the tlr4 gene may also represent one
potential target for modulating TLR4 transcription in cells recovered
from shock/resuscitated animals and then exposed to LPS.
Prior studies by Nomura et al. (15) demonstrated that the
rapid reduction in TLR4 surface expression following LPS treatment
correlated with reduced cytokine release in response to a second
exposure to LPS. These investigators suggested that this
down-regulation of surface receptors might contribute to the
development of LPS tolerance. Based on the present studies, one might
speculate that the preservation of TLR4 following shock/resuscitation
and subsequent treatment with LPS might contribute to the enhanced
responsiveness of cells to LPS compared with cells isolated from sham
animals. This possibility remains to be tested. Shock/resuscitation may
have also altered the innate responsiveness of the TLR4 through
conformational changes or through altering its disposition on the cell
surface. Clustering of surface receptors by the oxidant stress related
to ischemia/reperfusion may contribute to cell signaling, as suggested
in studies by Rosette and Karin (31), wherein UV exposure
of mammalian cells induced clustering of surface receptors and their
activation. In this regard, one intriguing possibility is that oxidant
stress may enhance physical approximation of TLR4 with CD14 receptors,
an event which was recently shown to promote NF-B translocation
(32). The findings that prolonged exposure to LPS in both
sham-derived cells and shock-derived cells caused reduced levels of
TLR4 mRNA differ from those reported by others, where TLR4 mRNA
appeared to return to baseline levels over time (15).
While this may be a result of species differences, it is also possible
that prolonged exposure to LPS may have induced a loss of TLR4 from the
surface and thus rendered the cells unresponsive to long-term exposure
to LPS (15).
In summary, this study provides new insights into the regulation of TLR4 mRNA and protein expression in vivo and in vitro. The data suggest that steady state levels of TLR4 mRNA following exposure to LPS are subject to control by both transcriptional and posttranscriptional mechanisms. Furthermore, the balance of these two mechanisms in vivo may be influenced by antecedent events such as the local microenvironment of the cell. In the context of the trauma patient, the findings suggest a potential target for immunomodulation, even as early as the initiation of resuscitation.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ori D. Rotstein, Toronto General Hospital, 200 Elizabeth Street, Eaton Building North 9-232, Toronto, Ontario, Canada M5G 2C4. E-mail address: ori.rotstein{at}uhn.on.ca
3 Abbreviations used in this paper: CINC, cytokine-induced neutrophil chemoattractant; MAP, mean arterial pressure; NAC, N-acetylcysteine; TLR4, Toll-like receptor 4; RL, Ringer’s lactate; SAL, saline alone; BAL, bronchoalveolar lavage.
Received for publication June 6, 2001. Accepted for publication March 5, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 Y. Liu, Y. Yuan, Y. Li, J. Zhang, G. Xiao, Y. Vodovotz, T. R. Billiar, M. A. Wilson, and J. Fan Interacting Neuroendocrine and Innate and Acquired Immune Pathways Regulate Neutrophil Mobilization from Bone Marrow following Hemorrhagic Shock J. Immunol., January 1, 2009; 182(1): 572 - 580. [Abstract] [Full Text] [PDF]
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 A. Rodrigues, D. B.C. Queiroz, L. Honda, E. J. R. Silva, S. H. Hall, and M. C. W. Avellar Activation of Toll-Like Receptor 4 (TLR4) by In Vivo and In Vitro Exposure of Rat Epididymis to Lipopolysaccharide from Escherichia Coli Biol Reprod, December 1, 2008; 79(6): 1135 - 1147. [Abstract] [Full Text] [PDF]
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 M. Q. Nicol, J.-M. Mathys, A. Pereira, K. Ollington, M. H. Ieong, and P. R. Skolnik Human Immunodeficiency Virus Infection Alters Tumor Necrosis Factor Alpha Production via Toll-Like Receptor-Dependent Pathways in Alveolar Macrophages and U1 Cells J. Virol., August 15, 2008; 82(16): 7790 - 7798. [Abstract] [Full Text] [PDF]
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A. A. Maung, S. Fujimi, M. P. MacConmara, G. Tajima, A. M. McKenna, A. J. Delisle, C. Stallwood, A. B. Onderdonk, J. A. Mannick, and J. A. Lederer Injury Enhances Resistance to Escherichia coli Infection by Boosting Innate Immune System Function J. Immunol., February 15, 2008; 180(4): 2450 - 2458. [Abstract] [Full Text] [PDF]
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C. L. Leaphart, J. Cavallo, S. C. Gribar, S. Cetin, J. Li, M. F. Branca, T. D. Dubowski, C. P. Sodhi, and D. J. Hackam A Critical Role for TLR4 in the Pathogenesis of Necrotizing Enterocolitis by Modulating Intestinal Injury and Repair J. Immunol., October 1, 2007; 179(7): 4808 - 4820. [Abstract] [Full Text] [PDF]
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 G. Ramesh, B. Zhang, S. Uematsu, S. Akira, and W. B. Reeves Endotoxin and cisplatin synergistically induce renal dysfunction and cytokine production in mice Am J Physiol Renal Physiol, July 1, 2007; 293(1): F325 - F332. [Abstract] [Full Text] [PDF]
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 S. J. Skerrett, C. B. Wilson, H. D. Liggitt, and A. M. Hajjar Redundant Toll-like receptor signaling in the pulmonary host response to Pseudomonas aeruginosa Am J Physiol Lung Cell Mol Physiol, January 1, 2007; 292(1): L312 - L322. [Abstract] [Full Text] [PDF]
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 F.-Y. Lin, Y.-H. Chen, Y.-W. Lin, J.-S. Tsai, J.-W. Chen, H.-J. Wang, Y.-L. Chen, C.-Y. Li, and S.-J. Lin The Role of Human Antigen R, an RNA-binding Protein, in Mediating the Stabilization of Toll-Like Receptor 4 mRNA Induced by Endotoxin: A Novel Mechanism Involved in Vascular Inflammation Arterioscler Thromb Vasc Biol, December 1, 2006; 26(12): 2622 - 2629. [Abstract] [Full Text] [PDF]
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F.-Y. Lin, Y.-H. Chen, J.-S. Tasi, J.-W. Chen, T.-L. Yang, H.-J. Wang, C.-Y. Li, Y.-L. Chen, and S.-J. Lin Endotoxin Induces Toll-Like Receptor 4 Expression in Vascular Smooth Muscle Cells via NADPH Oxidase Activation and Mitogen-Activated Protein Kinase Signaling Pathways Arterioscler Thromb Vasc Biol, December 1, 2006; 26(12): 2630 - 2637. [Abstract] [Full Text] [PDF]
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 N. A. Maris, M. C. Dessing, A. F. de Vos, P. Bresser, J. S. van der Zee, H. M. Jansen, C. A. Spek, and T. van der Poll Toll-like receptor mRNA levels in alveolar macrophages after inhalation of endotoxin. Eur. Respir. J., September 1, 2006; 28(3): 622 - 626. [Abstract] [Full Text] [PDF]
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 K. A. Powers, K. Szaszi, R. G. Khadaroo, P. S. Tawadros, J. C. Marshall, A. Kapus, and O. D. Rotstein Oxidative stress generated by hemorrhagic shock recruits Toll-like receptor 4 to the plasma membrane in macrophages J. Exp. Med., August 7, 2006; 203(8): 1951 - 1961. [Abstract] [Full Text] [PDF]
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D. Okutani, M. Lodyga, B. Han, and M. Liu Src protein tyrosine kinase family and acute inflammatory responses Am J Physiol Lung Cell Mol Physiol, August 1, 2006; 291(2): L129 - L141. [Abstract] [Full Text] [PDF]
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J. Fan, Y. Li, Y. Vodovotz, T. R. Billiar, and M. A. Wilson Hemorrhagic shock-activated neutrophils augment TLR4 signaling-induced TLR2 upregulation in alveolar macrophages: role in hemorrhage-primed lung inflammation Am J Physiol Lung Cell Mol Physiol, April 1, 2006; 290(4): L738 - L746. [Abstract] [Full Text] [PDF]
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J.-S. Chang, J. F. Huggett, K. Dheda, L. U. Kim, A. Zumla, and G. A. W. Rook Myobacterium tuberculosis Induces Selective Up-Regulation of TLRs in the Mononuclear Leukocytes of Patients with Active Pulmonary Tuberculosis. J. Immunol., March 1, 2006; 176(5): 3010 - 3018. [Abstract] [Full Text] [PDF]
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C. Tsatsanis, A. Androulidaki, T. Alissafi, I. Charalampopoulos, E. Dermitzaki, T. Roger, A. Gravanis, and A. N. Margioris Corticotropin-Releasing Factor and the Urocortins Induce the Expression of TLR4 in Macrophages via Activation of the Transcription Factors PU.1 and AP-1 J. Immunol., February 1, 2006; 176(3): 1869 - 1877. [Abstract] [Full Text] [PDF]
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Z. Guo, S. Garg, K. M. Hill, L. Jayashankar, M. R. Mooney, M. Hoelscher, J. M. Katz, J. M. Boss, and S. Sambhara A Distal Regulatory Region Is Required for Constitutive and IFN-{beta}-Induced Expression of Murine TLR9 Gene J. Immunol., December 1, 2005; 175(11): 7407 - 7418. [Abstract] [Full Text] [PDF]
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T. V. Pedchenko, G. Y. Park, M. Joo, T. S. Blackwell, and J. W. Christman Inducible binding of PU.1 and interacting proteins to the Toll-like receptor 4 promoter during endotoxemia Am J Physiol Lung Cell Mol Physiol, September 1, 2005; 289(3): L429 - L437. [Abstract] [Full Text] [PDF]
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 H.-Y. Cho, A. E. Jedlicka, R. Clarke, and S. R. Kleeberger Role of Toll-like receptor-4 in genetic susceptibility to lung injury induced by residual oil fly ash Physiol Genomics, June 16, 2005; 22(1): 108 - 117. [Abstract] [Full Text] [PDF]
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 K. A. Barsness, J. Arcaroli, A. H. Harken, E. Abraham, A. Banerjee, L. Reznikov, and R. C. McIntyre Hemorrhage-induced acute lung injury is TLR-4 dependent Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2004; 287(3): R592 - R599. [Abstract] [Full Text] [PDF]
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 L. Armstrong, A. R. L. Medford, K. M. Uppington, J. Robertson, I. R. Witherden, T. D. Tetley, and A. B. Millar Expression of Functional Toll-Like Receptor-2 and -4 on Alveolar Epithelial Cells Am. J. Respir. Cell Mol. Biol., August 1, 2004; 31(2): 241 - 245. [Abstract] [Full Text] [PDF]
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T. J. Murphy, H. M. Paterson, J. A. Mannick, and J. A. Lederer Injury, sepsis, and the regulation of Toll-like receptor responses J. Leukoc. Biol., March 1, 2004; 75(3): 400 - 407. [Abstract] [Full Text] [PDF]
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H. M. Paterson, T. J. Murphy, E. J. Purcell, O. Shelley, S. J. Kriynovich, E. Lien, J. A. Mannick, and J. A. Lederer Injury Primes the Innate Immune System for Enhanced Toll-Like Receptor Reactivity J. Immunol., August 1, 2003; 171(3): 1473 - 1483. [Abstract] [Full Text] [PDF]
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 M. A. Matthay, G. A. Zimmerman, C. Esmon, J. Bhattacharya, B. Coller, C. M. Doerschuk, J. Floros, M. A. Gimbrone Jr, E. Hoffman, R. D. Hubmayr, et al. Future Research Directions in Acute Lung Injury: Summary of a National Heart, Lung, and Blood Institute Working Group Am. J. Respir. Crit. Care Med., April 1, 2003; 167(7): 1027 - 1035. [Abstract] [Full Text] [PDF]
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A. E. Medvedev, A. Lentschat, L. M. Wahl, D. T. Golenbock, and S. N. Vogel Dysregulation of LPS-Induced Toll-Like Receptor 4-MyD88 Complex Formation and IL-1 Receptor-Associated Kinase 1 Activation in Endotoxin-Tolerant Cells J. Immunol., November 1, 2002; 169(9): 5209 - 5216. [Abstract] [Full Text] [PDF]
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