| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Departments of
* Thoracic Surgery and
Molecular Genetics, Division of Gene Research, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan;
Department of Geriatric and Respiratory Medicine, Tohoku University School of Medicine, Sendai, Japan; and
Division of Infectious Genetics, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
LPS (endotoxin) is a component of the outer membrane of the cell wall
of Gram-negative bacteria. LPS induces a severe inflammatory response
by initiating multiple intracellular signaling events, including the
activation of NF-
B, which ultimately leads to the synthesis and
release of many proinflammatory mediators and adhesion molecules,
such as IL-1, IL-6, IL-8, TNF-
, and ICAM-1 (3, 4).
These events can lead to cell damage and enhance the inflammation. This
LPS-induced signaling occurs through recently discovered Toll-like
receptors (TLR).3 Toll
is a type I transmembrane receptor, first described in
Drosophila, that shares homology to cytoplasmic components
of the IL-1 signaling pathway (5). Recent studies have
suggested that TLR4 serves as the main mediator of responses to
LPS in vitro and in vivo (6, 7, 8, 9). LPS signaling through
TLR4 results in activation of the NF-B and subsequent signaling
events (10). Although changes in the TLR4 expression
directly alter the cellular responsiveness to LPS (11),
little knowledge about the mechanism of TLR4 regulation is available.
Rehli et al. (12) showed, using deletion analysis of the
TLR4 promoter, that potential binding sites for AP-1 and IFN response
factor (IRF)/PU.1 are present in the promoter region of human
TLR4 gene and that IRF/PU.1 participate in the basal
regulation of human TLR4 in myeloid cells.
The aim of this study was to evaluate the effect of hypoxia on TLR4 expression and LPS-induced cellular events in endothelial cells. Our data showed that TLR4 expression was decreased and LPS-induced ICAM-1 up-regulation was diminished under hypoxic conditions. Since several reports showed that the mitochondrial respiratory chain is one site of hypoxia sensing (13, 14, 15), the role of mitochondria-generated reactive oxygen species (ROS) was evaluated in this hypoxia-induced TLR4 down-regulation. We found that hypoxia increased the mitochondria-generated ROS in endothelial cells, and this ROS regulated hypoxia-induced TLR4 down-regulation. Furthermore, this TLR4 down-regulation was mediated by hypoxia-induced attenuation of AP-1 translocation to the nuclei.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
HUVEC, human pulmonary artery endothelial cells (HPAEC), and culture medium HuMedia EB2 were purchased from Kurabo (Osaka, Japan). Anti-human TLR4 mAb (HTA1216) was purified from BALB/c mice immunized with the Ba/F3 line expressing TLR4, as described previously (16). PE-conjugated mouse anti-human ICAM-1 mAb was purchased from BD PharMingen (San Diego, CA). LPS from Escherichia coli 055:B5 was purchased from Difco (Detroit, MI). Myxothiazol was purchased from ICN Biomedicals (Aurora, OH). 5-(and -6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) was purchased from Molecular Probes (Eugene, OR).
Cell culture
HUVEC or HPAEC were grown to confluence in HuMedia EB2 medium
supplemented with 2% FBS, 10 ng/ml human epidermal growth
factor, 1 mg/ml hydrocortisone, 50 mg/ml gentamicin, 50 ng/ml
amphotericin B, 5 ng/ml human fibroblast growth factor 
, and 10
mg/ml heparin at 37°C under 5% CO2 in 95%
air. The culture medium was changed to HuMedia EB2 without all
supplements except 2% FBS before study. The cultures were then placed
in a humidified airtight chamber. To expose to hypoxic conditions, the
air in the chamber was completely exchanged to a gas mixture containing
5% CO2 and 95% N2. Then
cells were incubated at 37°C within the chamber.
PO2, PCO2, and pH levels in
the culture medium were measured periodically. HPAEC were incubated in
either this normoxic or hypoxic environment for 48 h, then TLR4
mRNA expression was measured by RT-PCR, described below. HUVEC were
incubated in either a normoxic or hypoxic environment for 12, 24, 48,
or 72 h. Then the following experiments were performed.
RT-PCR analysis and Northern blot analysis
Total cellular RNA was isolated with RNAzol B (Tel-Test,
Friendswood, TX) according to the manufacturer’s protocol. cDNA was
synthesized from 2 µg of total RNA by extension of random hexamers
with Superscript II (Life Technologies, Rockville, MD). PCR of the cDNA
was performed in a final volume of 20 µl containing 1.5 mmol/L
MgCl2, 0.5 U of DNA polymerase (AmpliTaq;
PerkinElmer, Branchburg, NJ), and 0.2 µM specific primers. PCR
amplification was performed for 32 cycles at 95°C for 40 s,
54°C for 40 s, and 72°C for 60 s. The oligonucleotide
primers used for RT-PCR were: human TLR4, 5'-TGG ATA CGT TTC CTT ATA
AG-3' and 5'-GAA ATG GAG GCA CCC CTT C-3'; human MD-2, 5'-TCA GAA GCA
GTA TTG GGT CT-3' and 5'- TTA GGT TGG TGT AGG ATG AC-3'; human
-actin, 5'-GCT GTG CTA TCC CTG TAC G-3' and 5'-TGC CTC AGG GCA GCG
GAA-3'. The synthesized PCR products were separated by electrophoresis
on a 1% agarose gel and visualized by ethidium bromide staining. PCR
bands were scanned and imported to Power Macintosh, and the intensities
of each band were analyzed using NIH Image software.
For Northern blot analysis, 20 µg of total RNA was electrophoresed, transferred to a positively charged nylon membrane (Hybond-N+; Amersham Pharmacia Biotech, Uppsala, Sweden), and hybridized with a 32P-labeled cDNA probe at 65°C. A cDNA probe specific for TLR4 was obtained by RT-PCR using the set of specific primers described above. The intensity of hybridization signals was quantified by densitometric scanning of autoradiographs using a Fujix BAS2000 Bio-image Analyzer (Fuji, Tokyo, Japan).
Immunostaining for flow cytometry
Surface expression of TLR4 on HUVEC was determined using mouse anti-human TLR4 mAb (HTA1216, 1 µg/105 cells). HTA1216 was detected with goat anti-mouse IgG-dichlorotriazinyl aminofluorescein (Chemicon International, Temecula, CA). Surface expression of IL-1R (type 1/P80) (IL-1R1) and TNF cell surface receptor 1 (TNF-R1) on HUVEC was determined using rat anti-human IL-1R1 mAb (BD PharMingen) and mouse anti-human TNF-R1 mAb (Genzyme/Thechne, Minneapolis, MN). Cells were analyzed by an EPICS XL-MCL (Beckman Coulter, Fullerton, CA).
Measurement of intracellular ROS
Intracellular ROS generation was assessed using CM-H2DCFDA, as described previously (17). HUVEC were seeded on 96-well tissue culture plates. After HUVEC were grown to confluence, cells were incubated at 37°C for 24 h under either normoxic or hypoxic conditions. Then CM-H2DCFDA (200 µM) was added and the incubation was continued for 45 min. CM-H2DCFDA is a compound that enters cells and fluoresces only when it is oxidized by ROS, particularly hydrogen peroxide and hydroxyl radicals. ROS generation was assessed by measuring the dichlorodihydrofluorescein (DCF) fluorescence on Fluoroskan Ascent FL (excitation 490 nm, emission 526 nm; Thermo Labsystems, Helsinki, Finland). To determine whether ROS generated from mitochondria affect TLR4 expression, myxothiazol (1 µM, mitochondrial site III electron transport inhibitor) was added before the incubation to block mitochondrial ROS generation.
EMSA
Dishes were gently scraped, and cells were collected by centrifugation at 300 x g for 5 min. Cells were lysed for 15 min at 4°C in a solution containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, and 0.5% Nonidet P-40. Nuclei were collected by centrifugation at 1500 x g for 30 s and then suspended in a solution of 20 mM HEPES, 0.4 M NaCl, 1 mM EDTA, 1 mM DTT, and 1 mM PMSF. The mixture was shaken vigorously for 15 min at 4°C, and the supernatant was collected after centrifugation for 5 min at 10,000 x g. Binding reactions were performed with 2 µg of nuclear protein in 20 mM HEPES (pH 7.9), 100 mM KCl, 0.5 mM DTT, 0.5 mM PMSF, 0.2 mM EDTA, 20% glycerol, 2 µg of salmon sperm DNA, 2 mM MgCl2, and 10,000 cpm of 32P-labeled oligonucleotide. DNA complexes were separated on a 4% polyacrylamide gel in Tris-borate-EDTA. The consensus oligonucleotide for the AP-1 binding site (sequence: 5'-CGC TTG ATG AGT CAG CCG GAA-3') or IRF/PU.1 binding site (5'-CGC TTT CAC TTC CTC TCA CCC TT-3') was labeled by standard procedures.
Luciferase assays
HUVEC were seeded in 24-well plates at a density of 1 x 105 cells/well 1 day before transfection. An equal amount of luciferase reporter plasmid pAP1-Luc (PathDetect AP-1 cis-Reporting System; Stratagene, La Jolla, CA) was transfected using Lipofectin (Invitrogen, Gaithersburg, MD) according to the manufacturer’s protocol. After a 48-h incubation under either normoxic or hypoxic conditions, HUVEC were harvested, washed, and lysed in 100 µl of lysis buffer, and luciferase activity was measured using 20 µl of lysate and 100 µl of luciferase substrate (Nippon Gene, Toyama, Japan). The luminescence was quantitated as a relative light unit on a luminometer (Lumat LB 9507; Berthold Japan, Tokyo, Japan). Results are presented as the average of four experiments where background has been subtracted.
ICAM-1 immunostaining for flow cytometry
Either myxothiazol (1 µM) or vehicle was added before the incubation, then HUVEC were incubated for 48 h under either normoxic or hypoxic conditions. After the exposure, HUVEC were incubated with LPS (50 ng/ml) at 37°C for 4 h. Then cells were stained with PE-conjugated mouse anti-human ICAM-1 mAb and analyzed by EPICS XL-MCL.
Statistics
Data were expressed as the mean ± SEM. Data were compared using ANOVA. When overall differences were identified, multiple contrasts with a Bonferroni adjustment were used to identify which groups were significantly different. Statistical significance was defined as p < 0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To determine whether hypoxia changed TLR4 expression, HUVEC were
incubated under hypoxic conditions and TLR4 expression was measured by
Northern blot analysis, RT-PCR, and immunostaining. The oxygen pressure
of the culture medium was 38.1 ± 2.1 torr under hypoxic
conditions and 148.3 ± 1.8 torr in normoxic conditions. There was
no significant difference in either pH or PCO2 in
the culture media (data not shown). These conditions were reached by 30
min after starting the exposure and were constant throughout the
experiment. There was no loss of viability after the exposure (data not
shown). TLR4 mRNA expression decreased after hypoxia for 48 h
(Fig. 1
A). In contrast,
hypoxia did not change mRNA expression of MD-2, an adaptor protein
required for TLR4-induced signaling (Fig. 1A). This decrease
in TLR4 mRNA expression was also observed in HPAEC after hypoxia for
48 h (Fig. 1B). TLR4 mRNA expression decreased to
70% of the normoxic group in HUVEC (Fig. 1C). This TLR4
mRNA down-regulation occurred in a time-dependent manner (Fig. 1D). Immunostaining and flow cytometry for TLR4 protein
expression on the cell surface demonstrated that TLR4 expression
decreased to 60% of the normoxia group by 48 h of exposure to
hypoxia. This decrease was not apparent by 24 h (Fig. 2). Additionally, both IL-1R1 and TNF-R1
protein expression on HUVEC were also decreased by hypoxia (Fig. 3).
|
|
|
Recently, mitochondria have been described as a major source of
ROS in response to hypoxia, and hypoxia increases ROS generation as a
result of electron flux through mitochondrial complex III
(18). To determine whether mitochondria-generated ROS play
a role in hypoxia-induced TLR4 down-regulation, we measured ROS
generation by hypoxia exposure in the presence and absence of
myxothiazol, a mitochondrial site III electron transport inhibitor that
blocks mitochondrial ROS generation. Hypoxia induced a significant
increase in DCF fluorescence within HUVEC (Fig. 4A), indicating that
intracellular ROS were generated in HUVEC by hypoxia. Furthermore,
myxothiazol abolished the ROS generation induced by hypoxia (Fig. 4A). To further study the importance of
mitochondria-generated ROS on the changes in TLR4 expression,
myxothiazol was added to the culture medium before exposure to either
normoxia or hypoxia and TLR4 expression was measured. As seen in Fig. 4, B and C, myxothiazol completely prevented the
hypoxia-induced down-regulation of TLR4 at both the mRNA and protein
level.
|
There are potential binding sites for AP-1 and IRF/PU.1 within the
proximal promoter region of TLR4 (12). To
determine whether AP-1 or IRF/PU.1 contributes to hypoxia-induced TLR4
down-regulation, the translocation of AP-1 and IRF/PU.1 to nuclei and
their ability to bind consensus oligonucleotides were examined by EMSA.
Nuclear AP-1 activity using consensus oligonucleotides was
significantly decreased by hypoxia (Fig. 5A). AP-1 activity measured
using the unique sequence of human TLR4 promoter region
(5'-GAG GTC AGA TGA CTA ATT GGG A-3') as the binding oligonucleotide
also demonstrated that the AP-1 activity was similarly decreased by
hypoxia (data not shown). In contrast, nuclear IRF/PU.1 activities did
not change under hypoxic conditions (Fig. 5B).
Administration of myxothiazol tended to prevent this decrease in AP-1
activity (Fig. 6, p =
0.054).
|
|
HUVEC transiently transfected with AP-1 reporter plasmid exhibited
decreased luciferase activity after hypoxia exposure. Administration of
myxothiazol prevented a hypoxia-induced decrease in luciferase activity
(Fig. 7).
|
To determine whether hypoxia-induced down-regulation of TLR4 had
an effect on the LPS response in endothelial cells, we examined the
changes in LPS-induced ICAM-1 expression. Flow cytometric analysis
showed that LPS induced ICAM-1 up-regulation under normoxic conditions
(Fig. 8A), but this increase
was less under hypoxic conditions (Fig. 8B). PE-fluorescent
intensity increased 4.7-fold above the baseline under normoxic
conditions but only 2.3-fold under hypoxic conditions. This effect of
hypoxia on cellular hyporesponsiveness to LPS was prevented by
myxothiazol (Fig. 8D).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
illustrates the postulated pathway
through which this down-regulation occurs. Other investigators have
demonstrated that changes in TLR4 expression directly alter cellular
responsiveness to LPS, and LPS tolerance has been clearly associated
with the down-regulation of surface TLR4 expression (11).
Our data show that the increase in ICAM-1 expression induced by LPS was
diminished by hypoxia, indicating that hypoxia-induced down-regulation
of TLR4 does alter cellular responsiveness to LPS. Taken together, our
data suggest that hypoxia induces alterations in the inflammatory
response of endothelial cells to LPS and thus to many Gram-negative
pathogens.
|
A). CD14 is another regulator of
LPS signaling, but endothelial cells do not express this molecule
(19). Thus, the effects of hypoxia on cellular
responsiveness to LPS appear to be directly and specifically on TLR4
expression. The net effect of TLR4 down-regulation to the patient is not clear. Diminished responsiveness to circulating LPS may have important protective effects (20, 21). However, the reduction in TLR4 expression may enhance susceptibility to bacterial infection. A destructive mutation in the TLR4 gene that results in null expression predisposes mice to develop Gram-negative sepsis (6), suggesting that decreased expression of functional TLR4 on immune cells prevents clearance of Gram-negative bacteria. The effect of hypoxia on TLR4 expression on immune cells is not known. Whether the observed decrease in TLR4 expression on endothelial cells or on other nonimmune cells increases susceptibility of bacterial infection is also not yet clear.
How do endothelial cells sense hypoxic conditions? Our data indicate
that mitochondria-generated ROS are involved in this hypoxia-induced
TLR4 down-regulation. Recently, several reports showed that hypoxia
increases ROS generation from mitochondria and these oxidant signals
appear to act as second messengers in adaptive responses to hypoxia.
Thus, the mitochondrial respiratory chain is known to be one site of
hypoxia sensing (13, 14, 15). Our data show that hypoxia
induces ROS generation in HUVEC. Myxothiazol, a mitochondrial site III
electron transport inhibitor, attenuated the hypoxia-induced ROS
generation (Fig. 4A), suggesting that mitochondria-generated
ROS play a role in hypoxia sensing in endothelial cells. Furthermore,
myxothiazol completely prevented the hypoxia-induced TLR4
down-regulation (Fig. 4, B and C). Taken
together, mitochondria-generated ROS are required for hypoxia-induced
TLR4 regulation.
It is not clear how mitochondria-generated ROS transduce the signal for
TLR4 down-regulation. Potential binding sites for AP-1 and IRF/PU.1 are
present in the promoter region of the human TLR4 gene, and
IRF/PU.1 participate in the basal regulation of human TLR4 in myeloid
cells by deletion analysis of the TLR4 promoter
(12). Additionally, IFN-
up-regulates TLR4 expression
in HUVEC (22). These data suggest that IFN- and
IRF/PU.1 contribute to the TLR4 up-regulation. However, IFN- is not
detectable in HUVEC culture (23) and participation of IRF
in this TLR4 down-regulation in HUVEC seems unlikely. Interestingly,
our data indicate that hypoxia decreases AP-1 but not IRF/PU.1
activity, suggesting that AP-1 but not IRF/PU.1 plays a major role in
TLR4 down-regulation. How hypoxia attenuates the AP-1-binding activity
is not clear. Oxidation of a conserved cysteine residue in the
DNA-binding domains of the AP-1 protein decreases DNA binding and
transactivation of AP-1 (24, 25). Thus, oxidation of AP-1
by hypoxia-induced ROS is the most likely cause of decreased AP-1
activity, although alterations in the translocation of AP-1 cannot be
excluded. We therefore conclude that a decrease in AP-1-binding
activity by hypoxia may contribute to the hypoxia-induced TLR4
down-regulation (Fig. 9).
Hypoxia also diminished the receptor expression of inflammatory
cytokines, IL-1R1 and TNF-R1 (Fig. 7). Although both IL-1R1 and TNF-R1
have an AP-1 binding site in their promoter region (26, 27), interestingly IL-1R1, which initiates the same
intracellular signaling pathway as TLR4, showed a similar degree of
down-regulation in TLR4 expression after hypoxia exposure. Therefore,
to understand the net effect of hypoxia on the whole inflammatory
process and intracellular signaling pathways is an interesting next
question.
In summary, this study demonstrates that TLR4 expression on HUVEC was down-regulated by hypoxia at both the mRNA and protein levels. This decrease was mediated by ROS generated from mitochondria. These ROS may be acting by decreasing the AP-1-binding activity. This decrease in TLR4 expression was associated with a decrease in ICAM-1 up-regulation induced by LPS, suggesting that the reduction in TLR4 expression has important physiologic sequelae. Our data suggesting that hypoxia induces hyporesponsiveness to LPS lead to important questions about the effects of hypoxia in patients with sepsis or endotoxemia. Since both LPS and hypoxia are potent inducers of cellular apoptosis (28, 29), the decrease in responsiveness to LPS under hypoxic conditions may have a cell-protective effect.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Hiroshi Kubo, Department of Geriatric and Respiratory Medicine, Tohoku University School of Medicine, 1-1 Seiryoumachi Aobaku, Sendai, Miyagi 980-8574 Japan. E-mail address: hkubo{at}geriat.med.tohoku.ac.jp
3 Abbreviations used in this paper: TLR, Toll-like receptor; ROS, reactive oxygen species; IRF, IFN response factor; HPAEC, human pulmonary artery endothelial cell; CM-H2-DCFDA, 5-(and -6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate; DCF, dichlorodihydrofluorescein.
Received for publication January 8, 2002. Accepted for publication June 12, 2002.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
during hypoxia: a mechanism of O2 sensing J. Biol. Chem. 275:25130.
induce Toll-like receptor 2 and Toll-like receptor 4 expression in human endothelial cells: role of NF-B activation. J. Immunol. 166:2018.B and AP-1 in intact cells: AP-1 as secondary antioxidant-responsive factor. EMBO J. 12:2005.[Medline]
This article has been cited by other articles:
|
|
 |
|
  P. F.-Y. Cheung, C.-K. Wong, W.-K. Ip, and C. W.-K. Lam FAK-mediated activation of ERK for eosinophil migration: a novel mechanism for infection-induced allergic inflammation Int. Immunol., March 1, 2008; 20(3): 353 - 363. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Waki, B. Liu, M. Miyake, K. Katahira, D. Murphy, S. Kasparov, and J. F.R. Paton Junctional Adhesion Molecule-1 Is Upregulated in Spontaneously Hypertensive Rats: Evidence for a Prohypertensive Role Within the Brain Stem Hypertension, June 1, 2007; 49(6): 1321 - 1327. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
 |
|
 D. Y. Jung, H. Lee, B.-Y. Jung, J. Ock, M.-S. Lee, W.-H. Lee, and K. Suk TLR4, but Not TLR2, Signals Autoregulatory Apoptosis of Cultured Microglia: A Critical Role of IFN-{beta} as a Decision Maker J. Immunol., May 15, 2005; 174(10): 6467 - 6476. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
