1 Department of Basic Pharmaceutical Sciences, West Virginia University, Morgantown 26506; and 2 Pathology and Physiology Research Branch, National Institute for Occupational Safety and Health, Morgantown, West Virginia 26505
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ABSTRACT |
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Interleukin-10 (IL-10) is a pleiotropic cytokine
that controls inflammatory processes by suppressing the production of
proinflammatory cytokines that are known to be transcriptionally
regulated by nuclear factor-B (NF-
B). Although still
controversial, IL-10 has been shown to inhibit NF-
B activation
through a process that involves proteolytic degradation of inhibitory
subunit I
B-
. What is not known, however, is the mechanism by
which IL-10 exerts its effect on I
B-
degradation. The present
study investigates the possible role of reactive oxygen species (ROS)
and their inhibition by IL-10 in NF-
B activation and I
B-
degradation in macrophages. Treatment of the cells with
lipopolysaccharide (LPS) caused activation of NF-
B and rapid
proteolysis of I
B-
as determined by the electrophoretic mobility
shift assay, gene transfection, and Western blot. IL-10 pretreatment
inhibited both NF-
B activation and I
B-
degradation. Both of
these processes were also inhibited by ROS scavengers, catalase
(H2O2 scavenger), and sodium formate (·OH
scavenger) but were minimally affected by superoxide dismutase
(O
B-
degradation and generation of ·OH radicals in response to
LPS stimulation. These results demonstrate, for the first time, direct
evidence for the role of IL-10 in ROS-dependent NF-
B activation.
nuclear factor-B; tumor necrosis factor-
; oxygen free
radicals
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INTRODUCTION |
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ADVANCES IN
MOLECULAR and cellular biology have identified the cellular
mediators that regulate many disease processes. One such mediator,
interleukin (IL)-10, has been identified as an important regulator of
the immune and inflammatory systems (11, 21). IL-10 is an
anti-inflammatory cytokine that is regularly produced during
inflammatory processes in vivo, usually by the same cells that release
inflammatory mediators (e.g., monocytes, macrophages, T cells). It
inhibits the production of inflammatory cytokines such as IL-1, IL-6,
and tumor necrosis factor- (TNF-
) by macrophages stimulated with
lipopolysaccharide (LPS) and interferon (14).
In animal models of sepsis, IL-10 given before or soon after
gram-negative bacterial endotoxin reduced TNF- production,
hypothermia, and death (17). It was found that mice
treated with anti-IL-10 antibodies are more sensitive to LPS-induced
shock (18) and that IL-10-deficient mice developed severe
enterocolitis (19), indicating the essential
immunoregulatory role of IL-10. Because of its potent anti-inflammatory
effects, IL-10 has been implicated in a number of inflammatory
conditions such as sepsis, chronic arthritis, and inflammatory bowel disease.
Nuclear factor-B (NF-
B) is a transcription factor that regulates
gene transcription of many of the proinflammatory cytokines that are
suppressed by IL-10 (3). NF-
B is a ubiquitous
transcription factor that plays a central role in regulating gene
expression of a number of proinflammatory cytokines that are released
by activated macrophages (reviewed in Ref. 2). Thus
suppression of NF-
B activity is potentially a mechanism for
regulating inflammatory responses. NF-
B is most frequently composed
of two DNA-binding subunits, p50 and p65 (2), and is
present cytoplasmically in an inactive form in resting cells. The
nuclear translocation and DNA binding of NF-
B dimers is tightly
controlled by accessory proteins called I
B subunits
(3). NF-
B is activated by a diverse range of
stimulants, including LPS, ultraviolet light,
H2O2, and inflammatory cytokines such as IL-1
and TNF-
(1, 3). When cells are treated with NF-
B
inducers, rapid proteolysis of I
B-
proteins occurs, and NF-
B
dimers dissociate from the NF-
B-I
B complex (16, 33).
NF-
B inducers cause rapid phosphorylation of I
B-
on two serine
residues (7, 30) after which it is ubiquitinated and
finally degraded by the proteosomes (9). Recently, the
I
B-
kinases (IKK) responsible for the phosphorylation have been
identified (27). The release of I
B-
exposes the nuclear localization sequence of NF-
B, and, as a result, NF-
B dimers rapidly appear in the nucleus where they bind DNA
(2). A number of genes contain NF-
B binding sites that
might control their inducible expression (20). Thus
certain genes regulated by NF-
B can be transcriptionally activated
within minutes after being exposed to the inducer.
The overall goal of this study was to investigate the effect of IL-10
on NF-B activation in macrophages and to elucidate the molecular
mechanism involved in this process. Macrophages play an important role
in host defense against noxious substances and are involved in a
variety of disease processes, including autoimmune diseases,
infections, and inflammatory disorders (22). Although the
role of IL-10 as an anti-inflammatory molecule has been well
established (21), the mechanism by which it exerts its
action is still not completely understood, and the experimental results
are controversial. Whereas most studies have shown that IL-10 inhibits
NF-
B activation (24, 31), there are others that report
quite the opposite (12, 29). Similarly, the data on the
effect of IL-10 on I
B-
degradation are contradictory (10,
25). These differences can be reasonably explained by the fact
that IL-10 has a great diversity of action, and the effect of IL-10 on
each cell is dependent on many factors, not all of which are understood.
Because macrophages are known to generate reactive oxygen species (ROS)
in response to stimuli (23) and because ROS have been
implicated as the common messenger involved in NF-B activation by
diverse stimuli (5, 6), we hypothesized that IL-10 may exert its effect on NF-
B activation by changing the redox status of
stimulated cells through an ROS-dependent pathway. The specific questions being addressed in this study are: 1) Do ROS play
a role in the IL-10-mediated NF-
B signaling pathway? 2)
What are the effects of IL-10 on NF-
B activation and I
B-
degradation in macrophages? 3) What are the key reactive
species involved in the process? This is the first study that directly
demonstrates the inhibitory effect of IL-10 on ROS production.
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MATERIALS AND METHODS |
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Cell culture. The macrophage cell line RAW 264.7 was obtained from the American Type Culture Collection (Manassas, VA). The cells were grown in DMEM (GIBCO BRL, Life Technologies) supplemented with 10% FBS, 2 mM L-glutamine, and 100 U/ml penicillin-streptomycin. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2. Before use, cells were trypsinized briefly or scraped mechanically and centrifuged. They were plated at ~1 × 106 cells/ml in 12-well tissue culture plates 1 day before transfection studies.
Liposomal transfection.
Approximately 1 × 106 cells were plated on a 12-well
plate and allowed to grow for 24 h before the transfection. The
plasmid DNA NF-B luciferase (1 µg/ml) was diluted in 200 µl of
DMEM, and the DNA condensing agent protamine sulfate (1 µg/ml; Sigma, St. Louis, MO) was added to the DNA. The liposome (LipofectAMINE, 12 µg/ml; GIBCO BRL, Life Technologies) was diluted in 200 µl of DMEM.
The diluted DNA and liposome samples were combined and incubated at
room temperature for 15-20 min. Cells with transfection reagents
were incubated for 4 h. Transfection medium was then replaced with
growth medium containing 10% FBS. Thirty-six hours after transfection,
the cells were stimulated with LPS in either the presence or absence of
other test agents. Twelve hours after stimulation, luciferase activity
was measured as described below. To account for the potential cytotoxic
effect caused by the transfecting agent, LPS, or other test agents
during the experiments, total cell protein was determined and used to
normalize the measured luciferase activity. All transfections were
conducted under sterile conditions, and duplicate plates were tested
for each condition.
Stable transfection. Cells were transfected with 1 µg of both CMV-mIL-10 (a kind gift from Dr. H. Tahara, University of Pittsburgh) and pCDNA3 with the protocol above for transient transfection. Two days after transfection, the medium was replaced with fresh complete medium containing 20 µl of 75 µg/ml G418 solution. Every 2 days, the medium was changed, and fresh G418 was added; this was cultured for 2 wk. Survived colonies were picked and tested for their ability to produce IL-10 as determined by ELISA. To ensure a homogeneous population of cells, positive colonies were subcloned and rescreened. The control cell line (RAW control) was prepared the same way except that a control CMV plasmid lacking IL-10 was used.
Measurement of luciferase activity. Luciferase synthesized during the in vitro translation was quantitated by the assay of enzyme-dependent light production using a luciferase assay kit (Promega, Madison, WI). Cells were washed two times with PBS and incubated at room temperature for 10 min in the presence of 250 µl of lysis buffer (Promega) and then centrifuged at 12,000 g. Ten microliters of each sample were placed in a 5-ml polystyrene test tube, and the tubes were then loaded in an automated luminometer (Bio-Rad, Hercules, CA). At the time of measurement, 100 µl of luciferase substrate were injected automatically in each sample, and total luminescence was measured over a 20-s time interval. Output is quantitated as relative light units. Protein content in the supernatant was determined by the bicinchoninic acid (BCA) protein assay reagent (Pierce, Rockford, IL). Luminescence detected was standardized per microgram protein present in the supernatant.
Determination of TNF- protein.
In experiments where inhibition of TNF-
produced by LPS stimulation
had to be studied, cells were stimulated with the indicated amount of
LPS for 6 h at 37°C. Cell-free supernatants were then collected
and assayed for cytokines by ELISA kits specific for murine TNF-
,
and protein levels were measured as specified by the manufacturer
(Endogen). The sensitivity of the assays ranged from 15 to 31 pg/ml.
The coefficient of variation for all cytokine assays was <10%.
Oligonucleotides.
Oligonucleotides (ON) were purchased from GIBCO BRL, Life Technologies.
The synthesized single-strand ON were deprotected and desalted, and
complementary strands were denatured at 80°C for 5 min and annealed
at room temperature. The double-strand ON were labeled with
[32P]dCTP (Amersham, Arlington Heights, IL) by using
Klenow fragment (Bethesda Research Laboratories, Gaithersburg, MD), and
the labeled ON were used as probes in the electrophoretic mobility
shift assay (EMSA). The ON used in this study contained the NF-B
sequence of the IL-6 gene promoter (74-TGGGATTTTCCCATGAGTCT-54).
Nuclear extraction.
Nuclear extracts were prepared as follows: 5 × 107
cells were treated with 500 µl of lysis buffer [50 mM KCl, 0.5%
Nonidet P-40, 25 mM HEPES, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml leupeptin, 20 µg/ml aprotinin, and 100 µM dithiothreitol (DTT)] and kept on ice for 4 min. Nuclei were pelleted by
centrifugation at 20,000 rpm for 1 min, and supernatants were
discarded. The nuclei were washed one time with the same buffer
without Nonidet P-40, and the nuclei were extracted in 300 µl of
extraction buffer (500 mM KCl, 10% glycerol, 25 mM HEPES, 1 mM PMSF,
10 µg/ml leupeptin, 20 µg/ml aprotinin, and 100 µM DTT). After
centrifugation at 20,000 rpm for 5 min, the supernatants were
harvested. The protein concentration of the resulting nuclear protein
extract was determined by BCA protein assay reagent (Pierce), and the
samples were diluted to 1 µg/µl with extraction buffer. The nuclear
proteins were stored at 70°C.
EMSA.
The DNA-protein binding reaction was conducted in a 24-µl reaction
mixture including 3 µg of nuclear protein extract, 1 µg of
poly(dI · dC) (Sigma), 3 µg of BSA, and 12 µl of 2xY
buffer, which contained 24% glycerol, 24 mM HEPES, 8 mM
Tris · HCl, 2 mM EDTA, and 2 mM 1,4-dithiothreitol. The
mixture was incubated on ice for 10 min. A total of 105
counts/min of 32P-labeled ON probe was added, and the
mixture was incubated for an additional 20 min at room temperature and
then loaded on a 5% acrylamide gel (GIBCO BRL) that had been prerun at
110 volts for 30 min with 0.5× Tris-borate-EDTA buffer. The loaded gel
was run at 200 V for 90 min and then was dried and exposed to X-OMAT film overnight (Eastman Kodak, Rochester, NY) for autoradiography. The
film was developed after overnight exposure at 70°C.
Western blots.
For intracellular IB-
degradation studies, whole cell proteins
were extracted. Whole cell extracts were subjected to 12% SDS-PAGE.
Resolved proteins were transferred to a nitrocellulose membrane and
incubated with affinity-purified rabbit polyclonal anti-I
B-
serum
raised against a peptide corresponding to amino acids 297-317
(mapping within the carboxy-terminal domain of the human I
B-
molecule). After three 10-min washes with PBS-Tween 20, the membranes
were incubated with peroxidase-conjugated anti-rabbit immunoglobulin,
and the antigen-antibody complexes were detected using enhanced
chemiluminescence Western blotting detection reagents according to the
manufacturer's instructions.
Free radical measurements. The electron spin resistance (ESR) spin-trapping technique was used to detect short-lived free radical intermediates (32). Spin trapping is necessary because of the reactive nature of the free radicals to be studied. This technique involves an addition-type reaction of a short-lived radical with a diamagnetic compound (spin trap) to form a relatively long-lived free radical product, the so-called spin adduct, which can be studied by conventional ESR. The intensity of the spin adduct signal corresponds to the amount of short-lived radicals trapped, and the hyperfine splittings of the spin adduct are generally characteristic of the original, short-lived, trapped radical.
All measurements were conducted with a Varian E9 ESR spectrometer and a flat-cell assembly. Hyperfine splittings were measured (to 0.1 G) directly from magnetic field separations using potassium tetraperoxochromate and 1,1-diphenyl-2-picrylhydrazyl as standards. Reactants were mixed in test tubes in a total volume of 0.5 ml. The reaction mixture was then transferred to a flat cell for ESR measurement. All measurements were carried out using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO; Aldrich) as a spin trap.Oxygen consumption measurements. Oxygen consumption measurements were carried out with a Gilson oxygraph equipped with a Clark electrode (Gilson Medical Electronics, Middleton, WI). These measurements were made from mixtures containing 1.0 × 106 cells/ml and various treatments in a total volume of 1.5 ml. The oxygraph was calibrated with media equilibrated with oxygen of known concentrations.
Statistical analysis. Each study group consisted of four experiments. Statistical analysis between study groups was performed with paired two-tailed Student's t-test. The level of significance was P < 0.05.
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RESULTS |
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Inhibition of LPS-induced TNF- production by IL-10.
It has been suggested that NF-
B mediates the transcriptional
activation of TNF-
(2). We tested whether IL-10 could
inhibit NF-
B-dependent TNF-
expression. RAW 264.7 macrophages
were stimulated with LPS (10 ng/ml) for 6 h, and cell culture
supernatants were then assayed by ELISA for TNF-
released.
Stimulation of cells with LPS resulted in the release of high
concentrations of TNF-
. Pretreatment of cells with recombinant mouse
IL-10 (1-50 ng/ml) 2 h before stimulation with LPS (10 ng/ml)
resulted in a dose-dependent inhibition (50-72%) in TNF-
production (Fig. 1A). We also
tested the inhibitory effect of IL-10 on TNF-
production in a stably transfected cell line (RAW IL-10) that constitutively expresses IL-10
protein. Stimulation of RAW IL-10 with LPS showed much lower levels of
TNF-
compared with the regular (RAW 264.7) or control cell line (RAW
control; Fig. 1B).
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Effect of IL-10 on NF-B binding activity and I
B-
degradation.
EMSA studies were performed on stably transfected cells to study the
effect of IL-10 on NF-
B activation (Fig.
2). Although LPS stimulation enhances
NF-
B binding activity of RAW 264.7 and RAW control cells, this
LPS-induced NF-
B activity was inhibited in the RAW IL-10 cell line.
This suggests that IL-10 is responsible for NF-
B inhibition.
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Role of ROS in LPS-stimulated macrophages.
The role of free radical reactions in LPS-induced NF-B activation
was examined using specific ROS inhibitors, including superoxide dismutase (SOD; O
B activity, whereas SOD pretreatment had no effect on
LPS-induced NF-
B activity. It should be noted that all three
scavengers were tested at different concentrations; however, only
optimal concentrations of each scavenger are presented here.
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Effect of IL-10 on hydroxyl radical generation.
Our results suggest that ·OH radical may be the key species involved
in NF-B activation. To confirm that ·OH radicals were actually
formed during LPS activation, ESR studies using the spin trap DMPO were
carried out. ESR spin trapping is a method that is specific and
sensitive and is considered to be the best technique for detection and
identification of free radical generation (32). Cells were
treated with LPS in the presence or absence of IL-10 and ROS
scavengers. Figure 5 shows that, in the
presence of LPS, an ESR spectrum consisting of a 1:2:2:1 quartet
pattern, which is characteristic of the DMPO-·OH adduct
(8), was observed. LPS-induced adduct formation increased
with time and reached a peak in 1 h. Pretreatment of cells with
IL-10 inhibited LPS-induced free radical generation (Fig. 5).
Pretreatment with a control protein, albumin, did not have any
inhibitory effect on LPS-induced ·OH generation. Addition of the
·OH scavenger sodium formate to the system decreased the intensity of
the DMPO-·OH signal, thus confirming the formation of ·OH induced
by LPS. Interestingly, catalase, the function of which is to scavenge
H2O2, also inhibited ·OH formation. These
results are consistent with previous studies by our group that
demonstrated that H2O2 can react with
endogenous metal ions to form ·OH via the Fenton reaction
(23). It is interesting to note that, when IL-10 was added
to a chemical system (Fenton reaction), it did not inhibit ·OH
generation. These results suggest that IL-10 is not a free radical
scavenger. Instead, it interacts with the cellular machinery in such a
way that free radical production is inhibited. Oxygen consumption
studies also showed that IL-10 was able to inhibit molecular oxygen
consumption in LPS-stimulated RAW 264.7 cells (Fig.
6).
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DISCUSSION |
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IL-10 is an anti-inflammatory cytokine that plays an important regulatory role in inflammatory diseases (11, 21). Studies with IL-10 knockout mice have shown that a key function of IL-10 is the control of inflammatory responses (19). In vitro and in vivo studies suggest that IL-10 could be a potentially useful therapeutic agent for the treatment of acute and chronic, systemic and localized inflammatory reactions.
Although numerous studies have been performed to identify the signaling
pathways targeted by IL-10, the mechanism is still not completely
understood. We hypothesized that IL-10 mediates part of its inhibitory
effects via its effect on ROS. Our studies show that IL-10 pretreatment
inhibits TNF- production of stimulated macrophages because of its
ability to decrease NF-
B-DNA binding and NF-
B-induced
transcription. Further analysis showed that IL-10 inhibits LPS-induced
I
B-
degradation.
Macrophages provide an important line of host defense against noxious
substances. One of the essential mechanisms by which this is
accomplished is the production of reactive oxygen metabolites such as
O
NF-B inducers produce ROS (2), and the observation that
antioxidants and radical scavengers inhibit the activation of NF-
B
strongly supports the idea that oxygen radicals are involved in the
release of I
B-
from the NF-
B-I
B complex. Recent studies have indicated that ROS can activate a number of cytokine genes, including the TNF-
gene. It has also been speculated that the protease activities involved with I
B-
are under the control of
the cell's redox status (2). This speculation is
strengthened by the observation that the antioxidant pyrrolidone
dithiocarbamate prevents inducible decay of I
B-
in response to
phorbol 12-myristate 13-acetate (4, 16, 28).
Oxygen free radicals are known to facilitate the progress of
inflammatory reactions. Because NF-B has been identified as an
oxidative stress-responsive transcription factor that is activated by a
diverse range of inducers, it has been postulated that ROS may be the
common messenger involved in the signal transduction pathway leading to
NF-
B activation (2). Our results show that ROS are
involved in NF-
B activation and I
B-
degradation. Specifically, ·OH seems to be the key player in ROS-mediated I
B-
degradation. ·OH radicals in macrophages are generated via the metal-catalyzed Fenton reaction [Mn+ + H2O2
M(n+1)+ + OH
+ ·OH] using H2O2 as
the source (23). These results are consistent with those
observed by Schreck et al. (26). They observed that H2O2 on its own does not activate the purified
NF-
B-I
B complex; therefore, it appears that a metabolite of
H2O2 caused the release of I
B-
. This
metabolite is probably the ·OH radical.
Because IL-10 inhibits IB-
degradation, we tested the effect of
IL-10 on ·OH radical generation. IL-10 inhibits LPS-induced ·OH
radical generation, as demonstrated by ESR. The fact that IL-10 does
not inhibit ·OH radicals in a chemical system suggests that IL-10
does not act as a free radical scavenger. Instead, it probably induces
signals that interface with the cellular signals that generate ROS in
response to LPS stimulation. The results of oxygen consumption studies
further suggest that IL-10 inhibits cellular production of ROS by
reducing molecular oxygen consumption induced by LPS. A recent study by
Schottelius et al. (25) has shown that IL-10 inhibits
TNF-
-induced activation of IKK, which is responsible for
phosphorylation and subsequent degradation of I
B-
. Bonizzi et al.
(5) have suggested that ROS generation may be required for
cell-specific kinase activity. It is interesting to note that, when
IL-10 was added to a chemical system (Fenton reaction), it did not
inhibit ·OH generation. These results suggest that IL-10 is not a
free radical scavenger. Instead, it interacts with the cellular
machinery in such a way that free radical production is inhibited.
Based on our results and those reported earlier, we propose the
following mechanism of action for IL-10. IL-10 inhibits ROS production,
which in turn inhibits IKK activity and hence I
B-
degradation.
This results in a decrease in NF-
B activity and hence decreases the
expression of inflammatory cytokines such as TNF-
. This whole
cascade of events is responsible for the anti-inflammatory properties
of IL-10 in macrophages. Although the proposed mechanism is in line
with the evidence shown here, the linkage between LPS, free radicals,
and IL-10 is still not completely understood. Although we have shown
that IL-10 inhibits LPS-induced free radical generation, the exact
pathway is still not clear, and further studies are being done.
In conclusion, IL-10 inactivates macrophage production of reactive oxygen radicals, thereby decreasing inflammation. The results in this study provide a new light into the action of IL-10. Finally, the mechanism of IL-10 in this cell type may not be reflective of its action in other cell types. As rightly discussed by Bowie and O'Neill (6), signal transduction pathways in different cell types need to be addressed individually.
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ACKNOWLEDGEMENTS |
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This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-62959 and by the National Institute for Occupational Safety and Health.
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FOOTNOTES |
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Address for reprint requests and other correspondence: Y. Rojanasakul, West Virginia Univ. School of Pharmacy, Dept. of Basic Pharmaceutical Sciences, P. O. Box 9530, Morgantown, WV 26506 (E-mail: Yrojanasakul{at}hsc.wvu.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 27 July 2000; accepted in final form 26 December 2000.
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