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Tumor necrosis factor ╨Ю┬▒ acceleration of inflammatory responses by down-regulating heme oxygenase 1 in human peripheral monocytes.

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ARTHRITIS & RHEUMATISM
Vol. 56, No. 2, February 2007, pp 464–475
DOI 10.1002/art.22370
© 2007, American College of Rheumatology
Tumor Necrosis Factor ␣ Acceleration of Inflammatory
Responses by Down-Regulating Heme Oxygenase 1 in
Human Peripheral Monocytes
Yohei Kirino, Mitsuhiro Takeno, Shuji Murakami, Masayoshi Kobayashi, Hideo Kobayashi,
Kenji Miura, Haruko Ideguchi, Shigeru Ohno, Atsuhisa Ueda, and Yoshiaki Ishigatsubo
Objective. To examine the interaction between
heme oxygenase 1 (HO-1), a stress-induced antiinflammatory protein, and tumor necrosis factor ␣ (TNF␣) in
human peripheral blood monocytes.
Methods. Peripheral blood mononuclear cells
(PBMCs) were obtained from healthy donors or from
patients with rheumatoid arthritis (RA) receiving the
anti–tumor necrosis factor ␣ (anti-TNF␣) monoclonal
antibody infliximab. CD14ⴙ cells were isolated by magnetic cell sorting, cultured with TNF␣ or auranofin, and
transfected with a plasmid encoding HO-1 or an HO-1–
specific small interfering RNA vector. Protein and messenger RNA (mRNA) levels were examined by immunoblotting and real-time polymerase chain reaction.
Cytokine levels in culture supernatants were measured
by enzyme-linked immunosorbent assay. HO-1 gene
transcription was evaluated using a luciferase reporter
gene assay. Actinomycin D and cycloheximide were used
to monitor the stability of mRNA and protein.
Results. HO-1 is constitutively expressed by
CD14ⴙ PBMCs from healthy donors. TNF␣ suppressed
HO-1 expression by accelerating the decay of mRNA
without affecting gene transcription or protein stability.
Forced expression or selective knock-down of the HO-1
gene expression resulted in down-regulation or upregulation, respectively, of proinflammatory cytokine
synthesis by monocytes. Treatment with infliximab significantly increased HO-1 mRNA levels and reduced
TNF␣ synthesis by PBMCs from RA patients.
Conclusion. TNF␣ accelerated inflammatory responses by down-regulating HO-1 expression in human
monocytes. TNF antagonists may block this TNFdependent suppression of HO-1 expression, resulting in
an amelioration of inflammation.
Dr. Kirino’s work was supported by a 2005 grant from the
Yokohama Foundation for Advancement of Medical Science. Dr.
Takeno’s work was supported by a 2004–2005 grant-in-aid for scientific
research (project 16590991) from the Ministry of Education, Culture,
Sports, and Technology of Japan and a 2006 grant from the Yokohama
Foundation for Advancement of Medical Science. Dr. Ishigatsubo’s
work was supported by grants from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan (Yokohama City University
Center of Excellence Program), the Ministry of Health, Labor, and
Welfare (Health Science Research on Specific Disease), and Yokohama City University (2006 Strategic Research project K18006).
Yohei Kirino, MD, Mitsuhiro Takeno, MD, PhD, Shuji
Murakami, MD, Masayoshi Kobayashi, MD, Hideo Kobayashi, MD,
PhD, Kenji Miura, MD, Haruko Ideguchi, MD, PhD, Shigeru Ohno,
MD, PhD, Atsuhisa Ueda, MD, PhD, Yoshiaki Ishigatsubo, MD, PhD:
Yokohama City University, Graduate School of Medicine, Yokohama,
Japan.
Address correspondence and reprint requests to Yoshiaki
Ishigatsubo, MD, PhD, Department of Internal Medicine and Clinical
Immunology, Yokohama City University, Graduate School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama, Kanagawa, 236-0004,
Japan. E-mail: ishigats@med.yokohama-cu.ac.jp.
Submitted for publication May 19, 2006; accepted in revised
form October 23, 2006.
Heme oxygenase (HO) is an enzyme that converts heme into carbon monoxide, Fe2⫹, and biliverdin
(1,2). HO-1, an inducible isozyme of HO, is a 32-kd
heat-shock protein that is expressed in response to a
variety of noxious stimuli, including heavy metals, hyperoxia, hypoxia, endotoxins, and hydrogen peroxide
(1,2). Evidence suggests that increased expression of
HO-1 is beneficial in a variety of pathologic conditions
(1,2). For example, HO-1 gene therapy has been successfully used to treat various lung diseases in animals
(3–7), and chemical induction of HO-1 expression has
been shown to improve lupus nephritis in MRL/lpr mice
(8). In contrast, a deficiency of HO-1 expression is
associated with severe chronic inflammation, as shown in
studies of HO-1–knockout mice (9) and in a patient with
HO-1 deficiency (10). These findings are consistent with
a physiologic role of HO-1 in protection against inflammation.
464
TNF␣ DOWN-REGULATION OF HO-1 IN HUMAN PERIPHERAL MONOCYTES
Investigators in our laboratory have been studying the regulation of HO-1 expression in humans with
inflammatory and rheumatic diseases. Previous studies
have shown that serum HO-1 levels are increased in
patients with active hemophagocytic syndrome and
adult-onset Still’s disease and that HO-1 levels are
correlated with serum ferritin levels (11). Recent studies
indicate that HO-1 is abundantly expressed in the synovial tissues of patients with rheumatoid arthritis (RA),
although serum HO-1 levels are not elevated (11,12).
Up-regulating HO-1 expression in RA-derived synovial
cell lines resulted in suppressed inflammatory responses,
suggesting that endogenously expressed HO-1 plays a
regulatory role in the development of synovial inflammation and that induction of HO-1 may be of therapeutic utility (12).
Inflammation is mediated, at least in part, by
various cytokines that can alter HO-1 expression. For
example, there is a close association between
interleukin-10 (IL-10) and HO-1 (3,13,14). IL-10 induces the expression of HO-1, while HO-1 augments the
production of IL-10 and reduces the production of
proinflammatory cytokines. The interaction between
HO-1 and tumor necrosis factor ␣ (TNF␣), which plays
a pivotal role in the pathogenesis of chronic inflammatory diseases (15), is less clear. HO-1 expression is
reduced by TNF␣ in chondrocytes derived from osteoarthritis patients (16), whereas TNF␣-dependent enhancement of HO-1 expression was observed in human
endothelial cells (17,18), human monocytic tumorderived cell lines (19), and retinal pigment epithelial
cells (20).
The interplay between TNF␣ and HO-1 may be
clarified by studying monocyte/macrophage cell linage,
which might elucidate the mechanism underlying certain
inflammatory disorders. This is particularly relevant,
since TNF␣ antagonists are critical therapeutic agents
used for the treatment of chronic inflammatory diseases,
including RA (15,21,22).
In the present study, we investigated the effects
of TNF␣ on messenger RNA (mRNA) expression, gene
transcription, mRNA stability, and posttranslational regulation of HO-1 in human monocytes. The inflammatory
responses of monocytes transfected with HO-1 complementary DNA (cDNA) or HO-1–specific small interfering RNA (siRNA) were also examined. The results
indicated that TNF␣ significantly reduces HO-1 expression in peripheral monocytes, thereby augmenting inflammatory responses. In addition, a single injection of
infliximab resulted in the up-regulation of HO-1 mRNA
465
expression in peripheral blood mononuclear cells
(PBMCs) from RA patients.
PATIENTS AND METHODS
Patients and healthy donors. Twelve patients with RA
(10 women and 2 men), who met the American College of
Rheumatology (formerly, the American Rheumatism Association) 1987 criteria (23), were enrolled in the study (Table 1).
The mean ⫾ SD age of the patients was 53.6 ⫾ 13.1 years, and
they had a mean ⫾ SD disease duration of 8.8 ⫾ 7.6 years, a
mean ⫾ SD Steinbrocker stage of 2.4 ⫾ 1.2, and a mean ⫾ SD
global functional status of 1.8 ⫾ 0.6.
All of the patients had been treated with methotrexate
(mean ⫾ SD dosage 7.3 ⫾ 1.0 mg/week) and/or a combination
of the following agents: corticosteroids (10 patients), nonsteroidal antiinflammatory drugs (5 patients), and diseasemodifying antirheumatic drugs (DMARDs) (sulfasalazine in 1
patient, bucillamine in 1 patient, mizoribine in 1 patient, and
actarit in 1 patient). None of the patients received other
cytotoxic agents or gold preparations. RA disease activity was
evaluated with the Disease Activity Score in 28 joints (DAS28)
(24). Blood was drawn into heparinized tubes before, 14 days
after, and ⬎6 months after the patients received the initial
injection of infliximab.
PBMCs and monocytes were also derived from blood
samples obtained from healthy donors.
All experiments were performed after obtaining written informed consent. The study was approved by the local
Institutional Review Board.
Reagents. Recombinant human TNF␣ was obtained
from R&D Systems (Minneapolis, MN). Auranofin was obtained from Wako (Osaka, Japan), and IgG1␬ was obtained
from Serotec (Oxford, UK). Actinomycin D and cycloheximide
were obtained from Sigma-Aldrich (St. Louis, MO), and
infliximab was kindly provided by Tanabe Seiyaku (Osaka,
Japan).
Cell preparation and culture. PBMCs were isolated by
centrifugation over Ficoll-Hypaque (ICN, Aurora, OH). In
some experiments, PBMCs were further fractionated into
CD14⫹ and CD14– cells by magnetic-activated cell sorting
(Miltenyi Biotec, Gladbach, Germany) using human CD14
MicroBeads (Miltenyi Biotec). Flow cytometric analysis
showed that CD14⫹ cells were ⬎95% pure, while the negatively selected population contained ⬍1% CD14⫹ cells. The
resultant CD14⫹ cells were quiescent, as previously described
(25). These cells were incubated in HEPES-modified RPMI
1640 (Sigma-Aldrich) containing 10% fetal calf serum
(Equitech-Bio, Kerrville, TX), 2 mM L-glutamine (SigmaAldrich), 100 units/ml of penicillin plus 100 ␮g/ml of streptomycin
(Sigma-Aldrich) at 37°C in an atmosphere of 5% CO2 in air.
To determine HO-1 expression at the mRNA and
protein levels, cells were cultured in the presence or absence of
recombinant human TNF␣ (0.1–10 ng/ml) and/or auranofin
(0.1 ␮g/ml) for 6–24 hours. To evaluate the stability of HO-1
mRNA and protein, cells were incubated in the presence of 5
␮g/ml of actinomycin D and 10 ␮g/ml cycloheximide, respectively.
5.4
1.5
5.7
3.3
1.5
0.1
4.4
0.1
5.2
0.8
0.4
0.1
2.1
0.1
4.7
0.3
0.3
0.1
0.3
0.1
0.8
0.5
0.7
0
2.6 ⫾ 2.3 0.6 ⫾ 1.0‡
2 weeks
0.2
2.4
0.2
0.9
3.1
0.8
1.0
1.4
0.4
0.4
0.2
0
0.9 ⫾ 1.0‡
⬎6 months
2 weeks
⬎6 months
0
2 weeks
DAS28 score
⬎6 months
0
2 weeks
3.6
4.2
3.4
3.8
6.4
4.7
4.8
3.8
3.4
3.1
3.3
3.5
4.0 ⫾ 0.9‡
G
G
M
G
M†
N
G
M
N
M
M
G
–
EULAR
⬎6 months response
HO-1:CD14 ratio
52
29
10
6.47
3.9
1.78
1.4
3.6
69
52
37
6.57
6.33
2.65
1.1
0.8
29
19
19
5.66
4.88
3.85
1.0
1.1
64
24
39
5.43
4.31
2.79
0.1
4.1
88
40
ND
7.82
5.35
ND
1.7
3.1
51
37
35
6
5.21
5.12
0.6
1.2
41
15
25
5.9
4.04
2.55
0.5
1.2
68
40
99
8.22
5.97
4.53
0.8
1.0
49
19
45
4.79
3.81
5.32
1.2
1.6
12
2
3
3.43
1.58
2.38
0.7
0.9
40
12
13
4.27
3.57
3.48
0.6
1.8
7
3
5
3.93
2.79
2.2
0.5
0.6
48 ⫾ 24 24.3 ⫾ 15.6‡ 30 ⫾ 27.1§ 5.71 ⫾ 1.46 4.31 ⫾ 1.34‡ 3.33 ⫾ 1.22‡ 0.8 ⫾ 0.5 1.8 ⫾ 1.2‡
0
ESR, mm/hour
* The mean ⫾ SD age of the patients was 53.6 ⫾ 13.1 years. CRP ⫽ C-reactive protein; ESR ⫽ erythrocyte sedimentation rate; DAS28 ⫽ Disease Activity Score in 28 joints; HO-1
⫽ heme oxygenase 1; EULAR ⫽ European League Against Rheumatism; G ⫽ good response; M ⫽ moderate response; ND ⫽ no data; N ⫽ no response.
† Evaluated by the DAS28 using the CRP level.
‡ P ⬍ 0.01 versus pretreatment level, by Wilcoxon’s signed rank test.
§ P ⬍ 0.05 versus pretreatment level, by Wilcoxon’s signed rank test.
1/45/F
2/73/F
3/59/F
4/68/F
5/53/M
6/55/F
7/37/F
8/50/F
9/71/F
10/53/M
11/50/F
12/29/F
Mean ⫾ SD
0
CRP, mg/dl
Characteristics of the rheumatoid arthritis study patients*
Patient/age/sex
Table 1.
466
KIRINO ET AL
TNF␣ DOWN-REGULATION OF HO-1 IN HUMAN PERIPHERAL MONOCYTES
Immunoblot analysis. The expression of HO-1 protein
was determined by immunoblotting as described previously
(12). Briefly, cells were treated for 30 minutes on ice with lysis
buffer (137 mM NaCl, 20 mM Tris HCl, 50 mM NaF, 1 mM
EDTA, and Triton X-100) supplemented with a protease
inhibitor (Sigma-Aldrich), and the supernatants were recovered by centrifugation for 30 minutes at 15,000 revolutions per
minute. The samples were resolved electrophoretically on a
4–20% gradient of polyacrylamide gel (Daiichi Kagaku, Tokyo, Japan) and transferred onto a polyvinylidene difluoride
membrane (Millipore, Billerica, MA). After blocking overnight at 4°C with 5% skim milk–Tris buffered saline, the
membrane was incubated for 1 hour at room temperature or
overnight at 4°C with optimally diluted anti–HO-1 murine
monoclonal antibody (Stressgen, Victoria, British Columbia,
Canada), anti–inducible nitric oxide synthase (anti-iNOS) rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa
Cruz, CA), or antiactin goat polyclonal antibody (Santa Cruz
Biotechnology), and subsequently for 45 minutes with horseradish peroxidase (HRP)–conjugated anti-mouse secondary
antibody (Amersham Life Sciences, Piscataway, NJ), HRPconjugated donkey anti-rabbit IgG (Amersham Life Sciences),
or rabbit anti-goat IgG HRP conjugate (Zymed, South San
Francisco, CA).
The signals were developed with the enhanced chemiluminescence detection system (Amersham Life Sciences).
The amount of blotted protein was measured densitometrically
by using Scion image analysis software (Scion, Frederick, MD)
and an image processing software (National Institutes of
Health Image Engineering, Bethesda, MD).
Reverse-transcription–polymerase chain reaction
(RT-PCR) and real-time PCR. Total RNA was isolated from
cells with TRIzol reagent (Invitrogen, Carlsbad, CA) (8,11,12).
One microgram of total RNA served as template for singlestrand cDNA synthesis in a reaction using oligo(dT) primers
and SuperScript II (Invitrogen). For the PCR, 1 ␮l of cDNA
was incubated with 9.375 ␮l of deionized distilled water, 2 ␮l of
dNTP, 2.5 ␮l of 10⫻ PCR buffer, and 0.125 ␮l of Taq
polymerase (Takara, Otsu, Japan) and a primer pair for
HO-1 (sense 5⬘-CAGGCAGAGAATGCTGAG-3⬘ and antisense 5⬘-GCTTCACATAGCGCTGCA-3⬘), CD14 (sense
5⬘-CGGCCGAAGAGTTCACAAGT-3⬘ and antisense 5⬘AGTGCAGTCCTGTGGCTTC-3⬘), GAPDH (sense 5⬘ACAGTCAGCCGCATC-3⬘ and antisense 5⬘-AGGTGCGGCTCCCTA-3⬘), and ␤-actin (sense 5⬘-TCCTGTGGCATCCACGAAACT-3⬘ and antisense 5⬘-GAAGCATTTGCGGTGGACGAT-3⬘). Cycling conditions included 27
cycles of amplification for 30 seconds at 94°C, 30 seconds at
55°C, 1 minute at 72°C, and a final extension phase consisting
of 1 cycle of 10 minutes at 72°C. PCR products were run on a
1.5% agarose gel stained with ethidium bromide.
The primers and probes for human HO-1, CD14,
TNF␣, and GAPDH used in the real-time PCR were purchased from PE Applied Biosystems (Foster City, CA). Realtime PCR was performed using a BD QTaq DNA Polymerase
(BD Biosciences Clontech, Mountain View, CA), and the data
were analyzed with the ABI Prism 7700 sequence detection
system (PE Applied Biosystems, Foster City, CA). Briefly,
one-fiftieth of the cDNA derived from 1 ␮g of total RNA, 200
nmoles/liter of probe, and 800 nmoles/liter of primers were
467
incubated in 25 ␮l of total reaction buffer including 0.75 units
of the QTaq DNA Polymerase at 50°C for 2 minutes and 95°C
for 10 minutes, followed by 40 cycles of 95°C for 15 seconds
and 60°C for 1 minute.
The analysis system (PE Applied Biosystems) determined the number of cycles at which the amplified DNA in the
sample exceeded the threshold (Ct) during the PCR. Gene
expression levels in the individual samples were calculated on
standard curves of each cDNA generated by serial dilutions of
the PCR amplified products. The data on HO-1, CD14, and
TNF␣ were standardized to the expression of GAPDH in the
same samples, using a multiplex PCR technique. The level of
HO-1 mRNA expression in each sample was reported as
arbitrary units. The ⌬⌬Ct method was used to semiquantify
TNF␣ mRNA levels, according to manufacturer’s protocol
(PE Applied Biosystems).
Plasmid construction. The human HO-1 cDNA–
expressing plasmid pcDNAHO-1 was constructed as previously described (12). We amplified 4.5 kb and 4.0 kb of the
HO-1 promoter regions by using KOD plus DNA polymerase
(Takara) from human genomic DNA with panels of the
following primers: for 4.5 kb sense, 5⬘-TTGGGCTTGTCTTCCTTGCT-3⬘; for 4.0 kb sense, 5⬘-CCTCAGCTTCTCTTTAGGTG-3⬘, and for the common antisense, 5⬘CATCCGGCCGGTGCTGGGCTCGT-3⬘. The PCR products
were cloned using pcR-Blunt II-TOPO (Invitrogen) and then
subcloned into the pGL3 Basic Vector (Promega, Madison,
WI) at the Kpn I and Xho I restriction sites. The resultant
constructs were designated as pHO-1(⫺4.5k) and pHO-1
(–4.0k), respectively. We used pSilencer neo (Ambion, Austin,
TX) as the siRNA expression vector.
As previously described (12), the sequences of human
HO-1–specific siRNA were determined according to the
AA(N19) rule (where N represents any nucleotide) (26). Two
complementary oligonucleotides were synthesized (Takara):
5⬘-GATCCGTGCTGAGTTCATGAGGAACTTCAAGAGAGTTCCTCATGAACTCAGCATTTTTTGGAAA-3⬘
(sense) and 5⬘-AGCTTTTCCAAAAAATGCTGAGTTCATGAGGAACTCTCTTGAAGTTCCTCATGAACTCAGCACG-3⬘ (antisense). These oligonucleotides were annealed followed by ligation into the linearized plasmid at Bam HI and
Hind III restriction sites. The pSilencer neo Negative Control
(Ambion), encoding a hairpin siRNA whose sequence is not
found in humans, was used as the negative control. The HO-1
siRNA expression vector was named psHO-1, and the scrambled siRNA expression vector was named psCont.
Transfections. CD14⫹ cells (2 ⫻ 105 cells/well) were
transfected for 24 hours with 1 ␮g of plasmid in the presence
of FuGene 6 transfection reagent (Roche Diagnostics, Indianapolis, IN) in a 6-well plate (Sumitomo, Tokyo, Japan),
according to manufacturer’s protocol. Thereafter, transfected
cells were used for experiments. The transfection efficacy of
the green fluorescent protein–expressing plasmid pMAX-GFP
(Amaxa, Cologne, Germany) to human primary monocytes in
the same procedures was evaluated by flow cytometric analysis.
The results showed that the efficacy was consistently ⬎40%
(data not shown).
Reporter gene assay. CD14⫹ cells (2 ⫻ 105 /well) were
transfected with 1 ␮g of pHO-1(–4.0k) or pHO-1(–4.5k) in the
presence of 50 ng/ml of pRL-CMV (Promega) expressing
Renilla luciferase. After 16 hours, cells were divided into 3
468
KIRINO ET AL
Figure 1. Endogenous expression of heme oxygenase 1 (HO-1) protein and mRNA in peripheral blood
mononuclear cells (PBMCs) from healthy donors. A, Levels of HO-1 and actin protein in freshly isolated
PBMCs, CD14⫹, and CD14– cells were examined by immunoblotting (top) and by densitometric scanning
(bottom). Densitometric values are the mean and SEM level of HO-1 protein relative to actin in 7
independent experiments. B and C, Expression of mRNA for HO-1, CD14, and ␤-actin in freshly isolated
CD14⫹ and CD14– cells was determined by reverse transcription–polymerase chain reaction (RT-PCR)
(B) and was estimated semiquantitatively by real-time PCR (C). Semiquantitative values are the mean and
SEM expression of mRNA for HO-1 and CD14 relative to GAPDH in 5 healthy donors. ⴱ ⫽ P ⬍ 0.05;
ⴱⴱ ⫽ P ⬍ 0.001, by paired t-test.
separate wells of a 24-well plate (Sumitomo) and stimulated
with auranofin (1 ␮g/ml) or TNF␣ (10 ng/ml) for another 12
hours. Firefly and Renilla luciferase activities were measured
with a TD20/20 luminometer (Turner Designs, Sunnyvale, CA)
using a Dual Luciferase Reporter Assay system (Promega).
Firefly luciferase activities were standardized against Renilla
luciferase activity and were expressed as the relative luciferase
activity.
Enzyme-linked immunosorbent assay (ELISA). Concentrations of IL-6, IL-8, and TNF␣ in the culture supernatants were determined by ELISA using optimal pairs of
capture and detection biotinylated antibodies. Antibodies
against IL-6 and TNF␣ were obtained from R&D Systems, and
IL-8 was obtained from BD Biosciences. Recombinant human
IL-6, IL-8, and TNF␣ (R&D Systems) were used for standards.
Statistical analysis. Paired t-test, Wilcoxon’s signed
rank test, and Spearman’s rank correlation were used to test
for differences. P values less than 0.05 were considered statistically significant.
RESULTS
Preferential expression of HO-1 in CD14-positive
cells in PBMCs from healthy donors. HO-1 expression
by PBMCs from normal healthy donors was examined.
Freshly isolated PBMCs expressed low, but detectable,
amounts of HO-1 protein (Figure 1A). When the cells
were fractionated according to CD14 expression, the
CD14⫹ population preferentially expressed HO-1 protein and mRNA (Figures 1A–C). Thus, HO-1 was
constitutively expressed by circulating CD14⫹ monocytes, but was rarely expressed by lymphocytes from
healthy donors.
TNF␣ suppression of HO-1 in peripheral monocytes. Proinflammatory cytokines affect HO-1 expression (16–20). When PBMCs were treated with TNF␣,
HO-1 protein and mRNA levels decreased in a dosedependent manner (Figures 2A and B). Auranofin induced HO-1 expression in PBMCs, a finding consistent
with earlier results involving RA-derived synovial and
monocytic cell lines (12,27) (Figure 2A). This auranofin-induced HO-1 expression was also suppressed by
TNF␣, whereas the gold compound partly restored the
TNF-dependent down-regulation of HO-1 (Figure 2A).
Irrespective of the presence or absence of auranofin, the
TNF␣-dependent suppression of HO-1 was abrogated
by infliximab (Figure 2C). However, infliximab did not
alter HO-1 expression in the absence of TNF␣, which
indicates that it did not directly modulate HO-1 expression
through binding to membrane-type TNF␣ (Figure 2C).
Since PBMCs consist of mixed populations, it is
uncertain whether TNF␣ directly acts on CD14⫹ monocytes, which preferentially express HO-1. Otherwise, the
effect can be mediated by CD14– cells in response to
TNF␣. To this end, we examined the effects of TNF␣ on
HO-1 in fractionated CD14⫹ and CD14– cells (Figure
TNF␣ DOWN-REGULATION OF HO-1 IN HUMAN PERIPHERAL MONOCYTES
469
Figure 2. Effects of recombinant human tumor necrosis factor ␣ (TNF␣) on the expression of heme
oxygenase 1 (HO-1) protein and mRNA in peripheral blood mononuclear cells (PBMCs) from healthy
donors. A, Effect of TNF␣ on endogenous or auranofin (0.1 ␮g/ml)–induced HO-1 protein expression in
PBMCs was examined by immunoblotting (top) and by densitometric scanning (bottom). Densitometric
values are the mean and SEM relative change in 7 healthy controls; a value of 1 was assigned to the
untreated sample. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01; # ⫽ P ⬍ 0.001; § ⫽ P ⬍ 0.0001, by paired t-test. B, Effect
of TNF␣ (1 ng/ml) on HO-1 mRNA expression in PBMCs was determined by reverse transcription–
polymerase chain reaction (RT-PCR) (top) and was estimated semiquantitatively by real-time PCR
(bottom). Semiquantitative values are the mean and SEM relative change in 9 healthy controls. # ⫽ P ⬍
0.001, by paired t-test. C, Effect of infliximab (10 ␮g/ml) or isotype-matched IgG1␬ on TNF␣ (1
ng/ml)–induced suppression of HO-1 in PBMCs. Results are representative of 7 independent experiments.
D, Effect of TNF␣ (1 ng/ml) on freshly isolated CD14⫹ and CD14– cells. Results are representative of
7 independent experiments.
2D). HO-1 expression was decreased by treatment with
TNF␣ in CD14⫹ cells, whereas HO-1 protein was
undetected in CD14– cells (Figure 2D). These data
indicate that TNF␣ directly attenuates HO-1 expression at the mRNA and protein levels in CD14⫹ monocytes.
No effect of TNF␣ on transcription of the HO-1
gene by human monocytes. Expression of HO-1 protein
is regulated by NF-E2–related factor 2 (Nrf2) and
other transcription factors, such as NF-␬B, activator
protein 1 (AP-1), AP-2, and hypoxia inducible factor 1␣
(2,19,28–31). To determine whether TNF is involved
in HO-1 gene transcription, a luciferase-expressing
plasmid containing 4.5 kb of the human HO-1 promoter region, designated pHO-1(⫺4.5k), was used. In
addition, a pHO-1(⫺4.0k) plasmid lacking the antioxidant response element where the activator Nrf2
and the BACH-1 repressor are involved in HO-1 gene
regulation through competition for interactions with
small Maf proteins (32), was used (Figure 3A). Auranofin increased luciferase activity ⬃10-fold in monocytes transfected with pHO-1(⫺4.5k), but not pHO-1
(⫺4.0k), suggesting that the effect of auranofin depends
on the antioxidant response element (Figure 3B). In
contrast, TNF␣ did not affect the luciferase activity
of monocytes transfected with either pHO-1(⫺4.5k)
or pHO-1(⫺4.0k) (Figure 3B). The data indicate
that TNF␣ suppresses HO-1 mRNA without affecting
HO-1 gene transcription in human peripheral monocytes.
470
KIRINO ET AL
Figure 3. Effects of tumor necrosis factor ␣ (TNF␣) on gene transcription, mRNA decay, and protein degradation of heme oxygenase
1 (HO-1) in human peripheral monocytes. A, Map of the HO-1 promoter region, showing the positions of ⫺4.5 kb and ⫺4.0 kb from
which plasmids were constructed, as well as the antioxidant response element (ARE) and a luciferase (Luc)–coding region. B,
Monocytes were transfected with plasmid pHO-1(⫺4.0k) or pHO-1(⫺4.5k) for 16 hours and then stimulated with TNF␣ (10 ng/ml)
or auranofin (1 ␮g/ml) for another 12 hours. Values are the mean and SEM relative change in luciferase activity in 5 healthy donors;
a value of 10 was assigned to auranofin-treated samples from pHO-1(⫺4.5k)–transfected cells. # ⫽ P ⬍ 0.001; NS ⫽ not significant,
by paired t-test. C, Effect of TNF␣ on HO-1 mRNA stability. Peripheral blood mononuclear cells were treated with TNF␣ (1 ng/ml)
in the presence of the transcription inhibitor actinomycin D (Act D; 5 ␮g/ml). HO-1 mRNA levels were corrected to those of
GAPDH based on real-time polymerase chain reaction results. Values are the mean and SEM relative change in 5 independent
experiments; a value of 1 was assigned to the control sample at 0 hours. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01, by paired t-test. D, Effect of
the TNF␣ on HO-1 protein stability. CD14⫹ cells were treated with TNF␣ (10 ng/ml) in the presence of the translation inhibitor
cycloheximide (CHX; 10 ␮g/ml). Shown are the results of HO-1 and actin protein expression (from 1 of 7 experiments) (top) and of
densitometric scans (all 7 experiments) (bottom). Densitometric values are the mean and SEM relative change in cycloheximide
activity.
TNF␣ acceleration of HO-1 mRNA decay in
PBMCs. TNF can modulate mRNA stability (33,34).
The absence of TNF␣ activity in HO-1 gene transcription suggested that the observed effects involved posttranscriptional regulation. To monitor mRNA degradation, PBMCs were incubated with TNF␣ plus
actinomycin D, which blocks gene transcription. Realtime PCR analysis showed that TNF␣ treatment significantly shortened the half-life of HO-1 mRNA
(Figure 3C).
No effect of TNF␣ on HO-1 protein stability.
TNF␣ can also be involved in posttranslational regulation, since TNF␣ down-regulates the inhibitor of DNA
binding 1 (ID-1) protein through activation of the
ubiquitin/proteasome degradation pathway (35). Moreover, HO-1 degradation is partially suppressed by
MG132, an ubiquitin/proteasome inhibitor, indicating
that this pathway may affect HO-1 protein stability (36).
To examine the effects of TNF␣ at the posttranslational
level, HO-1 protein levels in CD14⫹ monocytes were
evaluated in the presence of 10 ␮g/ml of cycloheximide,
a protein translation inhibitor. TNF␣ had no effect on
the degradation of endogenously expressed HO-1 protein (Figure 3D). These results indicate that TNF␣
suppresses HO-1 expression by enhancing mRNA decay,
rather than by affecting gene transcription and protein
stability.
HO-1 regulation of inflammatory responses in
human monocytes. Previous studies established that
the absence of HO-1 is associated with vigorous inflammatory responses (9,10). Thus, TNF␣-dependent
reduction in HO-1 expression may further enhance
the development of inflammation. To examine this
possibility, pcDNAHO-1 was transfected into CD14⫹
monocytes (Figures 4A and B). These transfected
cells secreted significantly lower amounts of TNF␣, IL-6,
and IL-8 than did the mock-transfected controls
(Figure 4C).
TNF␣ DOWN-REGULATION OF HO-1 IN HUMAN PERIPHERAL MONOCYTES
Figure 4. Effects of transfecting monocytes with a heme oxygenase 1
(HO-1)–encoding plasmid. A, HO-1 protein levels were monitored in
mock-transfected monocytes or in monocytes transfected with plasmid
pcDNAHO-1. B, Densitometric analysis of HO-1 expression levels.
Values are mean and SEM of 5 independent experiments; a value of 1
was assigned to the mock-transfected cells. ⴱ ⫽ P ⬍ 0.05, by paired
t-test. C, Culture supernatants from mock-transfected or pcDNAHO1–transfected monocytes were harvested, and levels of tumor necrosis
factor ␣ (TNF␣), interleukin-6 (IL-6), and IL-8 were determined by
enzyme-linked immunosorbent assay. Values are the mean and SEM
of 5 independent experiments; a value of 1 was assigned to the
mock-transfected cells. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01, by paired t-test.
HO-1 expression by CD14⫹ cells was then reduced using an HO-1–specific siRNA-expressing plasmid (Figures 5A and B). In contrast to the HO-1
transfectants, cytokine production was significantly elevated in psHO-1–transfected cells (Figure 5C). Moreover, much higher amounts of the cytokine and iNOS
were produced by psHO-1–transfected cells in the presence of exogenous TNF␣ (Figures 5A and C). These
associations indicate an inverse relationship between
HO-1 expression and inflammatory responses in monocytes. Since TNF␣ suppresses HO-1 expression, inflammation is augmented.
Elevated levels of HO-1 mRNA in PBMCs from
patients treated with infliximab. Based on the observation that TNF␣-dependent suppression of HO-1 protein
accelerates inflammation, the effect of the TNF␣ antagonist infliximab on HO-1 mRNA expression was monitored in RA patients. All patients had active disease,
based on DAS28 scores ⬎3.2 (Table 1). Consistent with
previous findings (11), baseline HO-1 mRNA levels in
471
PBMCs from RA patients and normal controls were
similar (data not shown). In the RA patients, C-reactive
protein (CRP) levels and erythrocyte sedimentation
rates (ESRs) fell significantly after infliximab treatment
(Table 1). In contrast, although the levels of HO-1
mRNA rose in PBMCs from 8 of the 12 patients, this
difference did not achieve statistical significance as compared with pretreatment levels (P ⫽ 0.12) (Figure 6A).
Infusion of infliximab has been reported to cause
a reduction in the number of monocytes (21,37). This
was confirmed in the current study, where the number of
monocytes, as determined by morphology, declined significantly after treatment (P ⬍ 0.01) (data not shown).
In parallel, levels of CD14 mRNA in PBMCs were also
decreased (Figure 6B). Since CD14⫹ monocytes are a
major source of HO-1 in PBMCs (Figure 1), levels of
HO-1 mRNA were reevaluated by adjusting for the
CD14 content. The results indicated that HO-1 mRNA
levels adjusted to reflect the frequency of monocytes,
which had increased after therapy (P ⬍ 0.05) (data not
shown).
The HO-1:CD14 mRNA ratio also significantly
increased after administration of infliximab (Figure 6C
and Table 1). Moreover, the change in the HO-1:CD14
mRNA ratio correlated inversely with the serum CRP
level, the ESR, and the DAS28 score (P ⬍ 0.05 by
Spearman’s rank correlation) (data not shown). The
level of TNF␣ mRNA fell significantly by day 14 (Figure
6D) and was inversely correlated with the HO-1:CD14
mRNA ratio (P ⬍ 0.05) (data not shown). Although the
reduction in the number of CD14⫹ cells may partly
explain this decline in TNF␣, no significant correlation
between CD14 and TNF␣ mRNA levels was observed.
Taken together, these data suggest that infliximab restores TNF␣-dependent deficiency of HO-1 in PBMCs,
which leads to an attenuation of inflammatory responses, including the synthesis of TNF␣.
DISCUSSION
The present study demonstrates that TNF␣
down-regulates HO-1 expression by human peripheral
blood monocytes and that reduced HO-1 expression
facilitates the development of inflammatory responses,
including the synthesis of TNF ␣ . Thus, a selfperpetuating cycle linking TNF␣ production and low
HO-1 expression may exist. Pharmacologic blockade of
this interaction represents a promising strategy for the
treatment of inflammatory disorders. Indeed, the clinical efficacy of infliximab, a TNF antagonist, was associ-
472
KIRINO ET AL
Figure 5. Effects of transfecting monocytes with the short interfering RNA (siRNA)
expression vector psHO-1. A, Levels of heme oxygenase 1 (HO-1) and inducible nitric oxide
synthase (iNOS) were determined by immunoblotting of CD14⫹ cells that had been
transfected with the siRNA expression vector psHO-1 or with the scrambled siRNA
expression vector psCont, with or without tumor necrosis factor ␣ (TNF␣; 10 ng/ml). B,
Densitometric analysis of HO-1 expression levels. Values are the mean and SEM of 5
independent experiments; a value of 1 was assigned to the psCont-transfected cells. ⴱ ⫽ P ⬍
0.05; ⴱⴱ ⫽ P ⬍ 0.01, by paired t-test. C, Culture supernatants from psHO-1–transfected or
psCont-transfected monocytes were harvested, and levels of TNF␣, interleukin-6 (IL-6), and
IL-8 were determined by enzyme-linked immunosorbent assay. Production of IL-6 and IL-8
was also evaluated in the presence of exogenous TNF␣ (10 ng/ml). Values are the mean and
SEM of 5 independent experiments; a value of 1 was assigned to the psCont-transfected
cells. ⴱ ⫽ P ⬍ 0.05; § ⫽ P ⬍ 0.0001, by paired t-test.
ated with increased HO-1 mRNA expression and decreased TNF␣ synthesis in RA patients.
Although HO-1 is categorized as an inducible
enzyme in response to various stimuli (1), the present
study indicates that substantial amounts of this protein
are present in freshly isolated peripheral blood monocytes from healthy donors. Since HO-1–deficient cells
and individuals are susceptible to inflammatory stimuli
and oxidative stresses (9,10), the constitutive expression
of HO-1 protein may contribute to the maintenance of
homeostasis.
The present study focuses on the interaction
between TNF␣ and HO-1 expression in circulating
monocytes, since TNF␣ plays a pivotal role in the
pathogenesis of chronic inflammatory diseases such as
RA (15). The HO-1 promoter contains both AP-1 and
NF-␬B binding sites, which can be activated by TNF,
suggesting that this cytokine may act as an HO-1 inducer. Indeed, previous studies indicate that HO-1
expression is up-regulated in human endothelial and
monocytic cell lines, such as U937 and THP-1, by TNF␣
(17–20), a finding confirmed in preliminary studies in
our laboratory (data not shown). Nevertheless, the
present study revealed that unlike tumor-derived mono-
TNF␣ DOWN-REGULATION OF HO-1 IN HUMAN PERIPHERAL MONOCYTES
Figure 6. Changes in heme oxygenase 1 (HO-1) mRNA levels following infliximab treatment of rheumatoid arthritis (RA) patients. The
mRNA was extracted from peripheral blood mononuclear cells obtained from 12 RA patients before and 2 weeks after their first
infliximab treatment. Levels of mRNA for HO-1, CD14, and GAPDH
were determined by real-time polymerase chain reaction. A, HO-1 and
B, CD14 mRNA levels in the individual patients were standardized
against GAPDH. The ratio of HO-1 mRNA to that of GAPDH was
expressed in arbitrary units (AU). C, Ratio of HO-1 to CD14 mRNA
in individual patients. D, Relative change in tumor necrosis factor ␣
(TNF␣) mRNA expression. A value of 1 was assigned to the pretreatment levels. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01, by Wilcoxon’s signed rank
test.
cytic cell lines (19), TNF␣ had suppressive effects on
HO-1 expression in human peripheral monocytes, a
finding consistent with that reported for chondrocytes
(16). It is important to determine what causes the
discrepancies among the cells.
Despite the suppressive effect of TNF␣ on HO-1
mRNA expression, the reporter gene assay showed no
effect of this cytokine on HO-1 gene transcription by
monocytes. Rather, TNF␣ promoted the degradation of
HO-1 mRNA. A similar acceleration of mRNA degra-
473
dation by TNF has been reported for ␤5 integrin in
osteoclasts, although the molecular detail of the relevant
mechanisms remains unclear (34). Since multiple cytoplasmic enzymes and factors are involved in mRNA
turnover (38), further study will be needed to identify
the mechanism by which TNF␣ accelerates the degradation of HO-1 mRNA.
Accumulating evidence shows that HO-1 protein
helps to protect the host from pathologic inflammatory
reactions (1). Results of the present study indicate that
forced expression of HO-1 reduces the production of
proinflammatory cytokines by monocytes. Elimination
of the HO-1 protein increased inflammatory cytokine
production. These findings are consistent with the conclusion that HO-1 has a beneficial effect on inflammatory disorders.
HO-1 is present in the joint lesions of patients
with RA (12,39) and in the joints of animals with
adjuvant-induced or collagen-induced arthritis (40,41).
Our previous study established that HO-1 reduced the
production of proinflammatory cytokines by RA synovial cell lines (12). HO-1 is also implicated in the bone
destruction of RA joints, since HO-1 attenuates
inflammation-induced osteoclastogenesis and bone loss
in vivo and in vitro (39). HO-1–expressing cells were
primarily located in the lining and sublining layer, while
few were detected at the cartilage–pannus junction,
where TNF␣–producing cells are abundant (39,42).
Thus, the localization of HO-1–deficient cells is intimately related to the destruction of bone and cartilage in
RA joint lesions (39). These data suggest that HO-1
plays a role in the bone destruction as well as inflammation in RA patients.
Previous studies suggest that the antirheumatic
effects of gold agents are mediated, at least in part, by
the induction of HO-1 (12,27). Yet, it is clear that
corticosteroids and other DMARDs, including sulfasalazine and D-penicillamine, do not directly modulate the
expression of HO-1 in synovial cells from RA patients
(12). However, as shown in Figure 2, the effects of
auranofin may be modulated by the level of TNF␣ in the
joint.
Infliximab is an established therapy for RA
(43,44). While its pharmacologic targets are membranebound or soluble TNF␣ molecules, infliximab has unexpectedly wide immunomodulatory activity (45). For
example, the clinical efficacy of this agent persists far
longer than its half-life. We previously demonstrated
that the number of TNF␣-secreting PBMCs decreased
in patients with Behçet’s disease receiving infliximab
(46). This observation suggested that the TNF agonist
474
KIRINO ET AL
not only neutralized soluble TNF␣, but also modulated
macrophage function by binding to membraneassociated TNF␣ (45). The present study shows that
TNF␣ synthesis decreases when HO-1 expression increases in patients receiving infliximab. However, the
monoclonal antibody did not directly effect HO-1 expression in monocytes in the absence of TNF␣. It is
likely that infliximab blocked the TNF␣-dependent suppression of HO-1 expression in monocytes, resulting in
reduced TNF␣ synthesis.
In summary, the present study demonstrates that
TNF-dependent suppression of HO-1 expression in
monocytes accelerates inflammatory responses and promotes further TNF synthesis. TNF antagonists serve to
disrupt this vicious circle, contributing to the remission
of inflammation. Additional strategies designed to interfere with the interaction between TNF and HO-1 may
also be of benefit in the treatment of inflammatory
disorders.
ACKNOWLEDGMENTS
The authors are greatly indebted to Dr. Dennis M.
Klinman (Center for Biologics Evaluations and Research,
Food and Drug Administration, Bethesda, MD) for his review
and invaluable suggestions in preparing the manuscript. The
authors also thank Mr. Tom Kiper (Yokosuka, Japan) for his
review of the manuscript.
AUTHOR CONTRIBUTIONS
Dr. Ishigatsubo had full access to all of the data in the study
and takes responsibility for the integrity of the data and the accuracy
of the data analysis.
Study design. Dr. Ishigatsubo.
Acquisition of data. Drs. Kirino, Takeno, and Murakami.
Analysis and interpretation of data. Drs. Kirino, Takeno, Murakami,
M. Kobayashi, H. Kobayashi, Miura, Ideguchi, Ohno, Ueda, and
Ishigatsubo.
Manuscript preparation. Drs. Kirino, Takeno, and Ishigatsubo.
Statistical analysis. Drs. Kirino, Takeno, and Ishigatsubo.
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