From the
Department of Molecular and Medical
Pharmacology, UCLA School of Medicine, Los Angeles, California 90095 and
¶Wolfson Institute for Biomedical Research,
University College London, London WC1E 6AE, United Kingdom
Received for publication, March 4, 2003 , and in revised form, May 6, 2003.
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ABSTRACT |
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INTRODUCTION |
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It is now becoming clear that NO plays a pivotal role in the regulation of
gene expression
(1418).
One key facet of this regulatory activity may be the control of iNOS induction
(1922).
Such a mechanism would constitute a self-regulating pathway by which NO
production from this NOS isoform could be fine tuned, which is essential
because iNOS is regulated transcriptionally rather than biochemically. We have
reported recently that NO has a patent biphasic effect on nuclear
factor-B (NF-
B) activity in murine macrophages and hence
possesses the ability both to up- and down-regulate the expression of a number
of pro-inflammatory proteins, including enzymes such as iNOS,
cyclooxygenase-2, and interleukin-6
(17). The dual effect of NO on
NF-
B has a pronounced effect on the activation profile of immune cells
and therefore has important implications for both the initiation and
suppression of an immune/inflammatory response. In particular, nanomolar
concentrations of NO, as might be produced by a constitutive isoform of NOS,
were shown to augment macrophage activation and expression of pro-inflammatory
proteins in response to LPS; in contrast, low micromolar concentrations of NO,
similar to those generated by the inducible isoform of NOS, inhibited gene
expression. These observations gave rise to the hypothesis
(17) that a constitutive NOS
isoform may play an equally important role in the macrophage by providing the
source of "low-output" NO, which facilitates the cellular response
to appropriate inflammatory stimuli via a positive effect on the activity of
NF-
B.
Endothelial nitric-oxide synthase (eNOS) has been detected at the mRNA and protein level in rodent and human macrophages. In non-induced J744.1 murine macrophages, Ca2+-dependent NOS activity has been detected in membrane extracts (23) and in human peripheral blood mononuclear cells. RT-PCR analysis has revealed the presence of eNOS (that is diminished after exposure to inflammatory stimuli) (24). Moreover, the human monocytic cell line U937 expresses both eNOS mRNA and protein; the enzyme activity is dependent on Ca2+ and calmodulin and results in cGMP accumulation (suggesting that these cells possess soluble guanylate cyclase (sGC)) (25). In addition, rat alveolar macrophages and RAW 264.7 cells express a constitutive NOS isoform and generate NO in the absence of inflammatory stimuli (26, 27). Despite these reports of constitutive NOS expression and activity, no function has been attributed to this protein in the macrophage; current dogma suggests that iNOS is the only physiologically relevant source of NO originating from the macrophage.
In the present study, we have used bone marrow-derived macrophages
(BMDMØs) from eNOS KO mice to ascertain the importance of this
constitutive NOS enzyme in the pathophysiological activity of these cells.
Herein, we demonstrate that macrophages lacking eNOS show considerably
diminished NF-B activity, iNOS expression, and NO production after
LPS-induced activation. Moreover, sGC can be found at both a mRNA and protein
level in murine macrophages, and the effect of eNOS-derived NO in regulating
pro-inflammatory gene expression is, at least in part, dependent on sGC
activation and cyclic GMP production; the effect of eNOS deficiency can be
mimicked by addition of the sGC inhibitor,
1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), and
reversed by the novel non-NO-dependent sGC activator, BAY 41-2272
(28). Thus, we have
established a key pathophysiological role for macrophage eNOS.
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MATERIALS AND METHODS |
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BMDMØ Isolation and CharacterizationBone marrow-derived macrophages were isolated and cultured as described previously (29). L929 fibroblasts (CCL-1; American Type Culture Collection) were cultured in Dulbecco's modified Eagle's medium (high glucose; 4.5 g/liter) with L-glutamine and sodium pyruvate and supplemented with 10% heat-inactivated New Zealand fetal bovine serum (FBS) (Invitrogen), 100 units/ml penicillin, 100 µg/ml streptomycin, 50 µM 2-mercaptoethanol, and 1% 100x minimal Eagle's medium essential amino acids without L-glutamine (all from Invitrogen) at 37 °C in a humidified incubator containing 10% CO2 in air. Cells were passaged by trypsinization and then seeded to confluence. After 57 days of culture, the supernatant (growth medium) was collected, centrifuged (593 x g, 10 min), then filtered through a 0.2 µm membrane, aliquoted, and used as a source of macrophage colony-stimulating factor for the conditioning medium.
Femurs were removed from male wild-type (WT) and eNOS knockout (KO) mice, 68 weeks of age, and placed in collection medium (Dulbecco's modified Eagle's medium with HEPES and without NaHCO3 supplemented with 10% heat-inactivated New Zealand fetal calf serum (low endotoxin), and 100 units/ml penicillin, 100 µg/ml streptomycin, and 50 µM 2-mercaptoethanol). Bone marrow cells were then flushed from the femurs into the collection medium and centrifuged (461 x g, 10 min) before resuspension in conditioning medium (Dulbecco's modified Eagle's medium; high glucose; 4.5 g/liter with L-glutamine and sodium pyruvate and supplemented with 10% heat-inactivated New Zealand FBS (low endotoxin), 100 units/ml penicillin, 100 µg/ml streptomycin, 10% L929 supernatant, 5% heat-inactivated horse serum, 1% 100x minimum Eagle's medium essential amino acids without L-glutamine, and 50 µM 2-mercaptoethanol). Cells were counted using trypan blue exclusion, and between 6 and 10 x 106 cells were seeded in conditioning medium in a Teflon bag (formed from heat-sealing Teflon fluorocarbon film (Dupont) such that the cells grew on the hydrophobic side). The bags were incubated at 37 °C in a humidified incubator containing 10% CO2 in air. After 10 days, cells were pelleted by centrifugation (259 x g, 10 min) and resuspended in RPMI 1640 medium (with 25 mM HEPES) supplemented with 10% heat-inactivated New Zealand FBS (low endotoxin), 2 mM glutamine, 100 units/ml streptomycin, and 100 µg/ml penicillin (complete medium).
After harvesting, the cell populations obtained were characterized by fluorescence-activated cell sorter analysis of the cell surface marker F4/80, which is specific for murine macrophages, and the lymphocyte-specific CD3. Four aliquots of 2 x 105 cells from WT or KO animals were incubated with fluorescein isothiocyanate-conjugated antibodies (all supplied by Serotec, Oxford, UK) raised against F4/80 and CD3 and isotype control antibodies (rat IgG2b negative control and rat IgG2a negative control), which had been diluted to a concentration of 300500 ng/ml in 200 µl of PBS containing 1% heat-inactivated New Zealand FBS (both Invitrogen). After 2 h of incubation at room temperature, cells were washed twice by centrifugation (200 x g, 2 min) with PBS/1% FBS. Cells were then resuspended in 200 µl of fixation solution (1% paraformaldehyde in PBS) and incubated at room temperature for 15 min. The cells were centrifuged as before and resuspended in 200 µl of PBS/1% FBS. The flow cytometry was performed using a FACScalibur machine (Becton Dickinson, Oxford, UK). The statistical analysis was performed using Cell Quest software.
Western Blot AnalysisCells were seeded into 12-well culture
plates (1 x 106 cells/well) in 1 ml of complete medium and
incubated overnight at 37 °C in a humidified incubator containing 5%
CO2 in air. After activation with LPS (100 ng/ml) for appropriate
times, the medium was removed and stored at 20 °C for nitrite
measurement (see below). Cells were then washed with 0.5 ml of PBS and
homogenized for 20 min on ice in whole-cell homogenization buffer (50
mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 2
mM EDTA, 8 mM EGTA, and 10 µg/ml protease inhibitor
mixture (benzamidine, antipain, leupeptin, and aprotinin)) and transferred to
1.5-ml tubes. The homogenate was then centrifuged (13,793 x g,
5 min, 4 °C), and the supernatant was retained. Protein concentrations
were determined by Bio-Rad protein assay (Bio-Rad). Equal volumes of protein
were subjected to 7.5% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis under reducing conditions. The proteins were transferred to
nitrocellulose membranes (Amersham Biosciences) with a semidry blotter
(Amersham Biosciences) at 120 mA for 6090 min. The membranes were then
incubated with shaking in 5% milk in wash buffer (PBS/0.1% Tween 20) for 1 h
at room temperature. The membrane was washed twice (15 min/wash) in wash
buffer before incubation overnight, at 4 °C with gentle shaking, with
primary antibody (iNOS, Transduction Laboratories;
sGC, Cayman
Chemicals) diluted 1:2000 (
iNOS) or 1:1000 (
sGC) in 1% milk
(iNOS) or 5% milk (
sGC) in wash buffer. The membrane was washed six
times (5 min/wash) and then incubated, with gentle shaking for 2 h at room
temperature, with horseradish peroxidase-conjugated
-rabbit IgG
(Vector) diluted 1:5000 in 1% milk in wash buffer. The membrane was washed as
done previously, and proteins were visualized using enhanced chemiluminescence
(Amersham Biosciences). Bands were quantified by densitometry (NIH Image).
Electrophoretic Mobility Shift AssayCells (1 x 107) were seeded in complete medium in 10-cm tissue culture dishes and then incubated and activated as described above. After activation with LPS (100 ng/ml) for appropriate times, the cells were scraped into the medium and collected by centrifugation (150 x g, 5 min, 4 °C). Pellets were resuspended in 800 µl of cold lysis buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EGTA, 0.1 mM EDTA, 0.5 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride) and transferred to a 1.5-ml tube, after which 50 µl of 10% Nonidet P-40 was added to each. Tubes were centrifuged (150 x g, 5 min, 4 °C), and the resulting nuclear pellets were washed with an additional 800 µl of lysis buffer by centrifugation as before. 60 µl of cold nuclei extraction buffer was added to each tube, which was then incubated on ice for 20 min, followed by centrifugation (14,000 x g, 10 min, 4 °C). The resulting supernatants were then retained as nuclear extracts. Protein concentrations were measured using the Bio-Rad Protein Assay II (Bio-Rad), and samples were diluted to equal concentrations.
Double-stranded oligonucleotide (NF-B (
B sequence)
5'-GGGGATTTCCC-3'; Santa Cruz Biotechnology, Inc. (Santa Cruz,
CA)) was end-labeled using [
-32P]ATP, 7000 Ci/mmol (ICN,
Irvine, CA), and T4 polynucleotide kinase (Promega, Madison, WI). The
following components were incubated in a 1.5-ml tube for 30 min at 37 °C:
1 µl of manufacturer's 10x buffer; 1 µl of 0.1 M
dithiothreitol, 1 µl of [
-32P]ATP, 1 µl of T4
polynucleotide kinase, 4 µl of H2O, and 2 µl of
oligonucleotide (3.5 pmol). Afterward, 40 µl of TE buffer, pH 7.6 (10
mM Tris, 1 mM EDTA) was added to stop the reaction.
Oligonucleotides were separated from unreacted nucleotides using Sephadex G-25
spin columns (Amersham Biosciences). The slurry in the column was removed from
its buffer by an initial 1-min spin at 1000 x g. Then, the
total volume of the kinase reaction (50 µl) was applied to the resin and
centrifuged for an additional 1 min at 1000 x g. The eluate
then contained radiolabeled oligonucleotide. Counts per minute were
subsequently determined using a scintillation counter, and the eluate was
diluted to
100,000 per µl. 12 µg of nuclear extract was
incubated in a 1.5-ml tube containing 10 µg of acetylated bovine serum
albumin and 1 µg of poly(dI-dC) in electrophoretic mobility shift assay
buffer (25 mM HEPES, 100 mM KCl, 5% Ficoll, 2% glycerol,
0.025% Nonidet P-40, 0.05 mM EDTA, 0.05 mM EGTA, 2
mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl
fluoride) for 30 min at room temperature. 100,000 cpm (1 µl) radiolabeled
consensus oligonucleotide (
0.1 pmol) was subsequently added to each
sample and incubated at room temperature for an additional 30 min. Specificity
of probe binding was determined by adding 100-fold excess of unlabeled probe
(data not shown). Protein-DNA complexes were subsequently resolved in a 5%
native Tris/taurine-buffered gel, 100 V for
45 min. Gels were dried and
exposed to x-ray film overnight at 80 °C. Bands were analyzed by
densitometry (NIH Image).
RNA Extraction and RT-PCRRNA was extracted from cells (5 x 106) using the RNeasy mini kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. In addition, the RNase-free DNase set (Qiagen) was used during RNA extraction to destroy any DNA present.
RT-PCR was performed using the Qiagen OneStep RT-PCR kit (Qiagen) using the
manufacturer's Q Solution protocol and reagents, with 1 µl of RNA and 0.6
µM forward and reverse primers. Reverse transcription was
carried out at 50 °C for 30 min, followed by 15 min at 95 °C to
inactivate the reverse transcriptase and activate the DNA polymerase. Thermal
cycling conditions for PCR were as follows: 35 cycles of denaturation at 94
°C for 1 min, annealing at 60 °C for 1 min, and polymerization at 72
°C for 1 min, followed by a final extension at 72 °C for 10 min.
RT-PCR products were resolved by agarose gel electrophoresis (2% gel) and
stained with ethidium bromide. The primer sequences (Qiagen Operon, Alameda,
CA) used were: sGC1 sense,
5'-GGTCACCATGTGTGGACAGG-3'; sGC
1 antisense,
5'-CCAGCTCTCCACACTGCTGG-3'; sGC
1 sense,
5'-GCATGCATCTGGAGAAGGG-3; and sGC
1 antisense,
5'-CCGAGGCATCCGCTGTCC-3'.
Nitrite MeasurementNitrite accumulation was determined by mixing equal volumes of cell culture medium and Griess reagent (0.5% sulfanilamide, 0.05% naphthylethylenediamine dihydrochloride, and 2.5% H3PO4) with absorbance (A540A620) read on a Molecular Devices 96-well microplate reader (Menlo Park, CA); standard curves were constructed with known concentrations of NaNO2.
Cyclic GMP MeasurementCells were seeded into 12-well culture plates (1 x 106 cells/well) in 1 ml of complete medium and incubated overnight at 37 °C in a humidified incubator containing 5% CO2 in air. Medium was aspirated and replaced with complete medium containing 0.25 mM isobutylmethylxanthine, and plates were incubated for 10 min. Cells were stimulated with LPS (100 ng/ml) or BAY 41-2272 (10 µM) and incubated for 10, 20, and 30 min. Three wells were used for each condition. The medium was then aspirated, and intracellular cGMP was measured using the Biotrak cGMP enzyme immunoassay system (Amersham Biosciences) according to the manufacturer's instructions.
Data AnalysisAll statistical analyses were performed using GraphPad Prism (GraphPad Software Inc., San Diego, CA). Densitometric analyses were performed using NIH Image. All data are plotted graphically as mean values with vertical bars representing standard error of the mean (S.E.). A Student's t test was used to assess differences between experimental conditions. A probability (p) value of <0.05 was taken as an appropriate level of significance.
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RESULTS |
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Western blot analysis was carried out in whole-cell extracts from the eNOS KO macrophages to confirm that the targeted protein was not being expressed (Fig. 2). A comparison with extract from human umbilical vein endothelial cells as positive control indicated that eNOS was not expressed in KO BMDMØs.
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Effect of eNOS Deletion on iNOS Protein Expression and
ActivityActivation of WT BMDMØs with LPS (100 ng/ml)
produced a time-dependent increase in the expression of iNOS protein.
Expression peaked between 12 and 24 h and had started to wane by 48 h (Figs.
3). Concomitantly, the stable
NO metabolite, nitrite, accumulated in the culture medium
(Fig. 4). Such an activation
profile closely resembles that which we have published previously for RAW
264.76 macrophages (17),
providing further evidence that the BMDMØs used in this study possess
an authentic macrophage phenotype. However, in BMDMØs from eNOS KO
mice, both the expression and activity of iNOS protein were significantly
altered. Although the time course of expression remained unchanged, the levels
of protein expression and activity were reduced in eNOS KO cells at all time
points (Figs. 3 and
4). Indeed, in eNOS KO
BMDMØs, iNOS protein levels and nitrite production reached 59 ±
7% (n 3; p < 0.05) and 53 ± 8% (n
3; p < 0.05) of control (WT) values, respectively.
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Effect of eNOS Deletion on NF-B Activity in
BMDMØsTo investigate whether the effect of eNOS-derived NO
on iNOS expression and activity was occurring through a modulation of
NF-
B, the activity of this transcription factor was assessed by
electrophoretic mobility shift assay. NF-
B activity was absent in
resting cells (Fig. 5).
Stimulation of eNOS WT and KO BMDMØs with LPS (100 ng/ml) resulted in
increased NF-
B activity, which peaked at 90 min
(Fig. 5). However, NF-
B
activity was significantly decreased in eNOS KO cells as compared with WT, an
effect particularly apparent after 60 and 90 min of activation, where activity
was approximately half that in WT cells
(Fig. 5).
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Identification of sGC in BMDMØsTo determine whether
the sGC enzyme was expressed in BMDMØs, RT-PCR and Western blot
analyses were conducted on cytosolic extracts. mRNA for both the
1 and
1 subunits of murine sGC was clearly
identified in BMDMØs from WT and eNOS KO mice. Moreover, in WT mice,
1 and
1 subunit protein was identified by
immunoblotting using a commercially available antibody that recognizes both
subunits (Fig. 6).
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Effect of the sGC Inhibitor, ODQ, on iNOS Expression in BMDMØsBecause the amounts of NO produced by eNOS are akin to concentrations associated with activation of sGC (i.e. low [nM]) and this enzyme had been identified in the BMDMØs, investigations were conducted to ascertain whether sGC activation played an obligatory role in the regulation of iNOS expression. BMDMØs extracted from WT animals were pre-incubated with the sGC inhibitor ODQ (31) (10 µM) or the sGC activator BAY 41-2272 (0.130 µM). The cells were then activated with 100 ng/ml LPS, and iNOS expression was monitored after 9 and 24 h (chosen to represent early and later time points of activation, respectively).
BAY 41-2272 elicited a concentration-dependent potentiation of
LPS-stimulated iNOS expression after both 9 and 24 h of activation, with a
maximum increase of 165 ± 15% and 154 ± 14% at 9 and 24 h,
respectively (n 4; p < 0.05 for both;
Fig. 7). The potentiating
effect of the cGMP-generating agent was reversed by ODQ (10 µM;
Fig. 8), confirming the
importance of sGC in the regulation of macrophage activation. Moreover, the
presence of ODQ alone led to a reduction in LPS-stimulated iNOS expression at
both 9 and 24 h after activation (Fig.
8), providing further evidence that sGC activation is necessary
for the augmenting effect of (endogenous) eNOS-derived NO on iNOS
expression.
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An additional set of experiments, essentially identical studies to those described above, were conducted in eNOS KO BMDMØs. In this case, ODQ alone had no effect on LPS-induced iNOS expression, as would be expected in cells lacking eNOS-derived NO. However, iNOS expression was significantly enhanced in the presence of BAY 41-2272, and this increase could be blocked by the concomitant addition of ODQ (Fig. 8). Indeed, the increase in iNOS expression in the presence of BAY 41-2272 in eNOS KO BMDMØs was significantly greater than that observed in WT cells, suggesting that activation of sGC could compensate for the loss of eNOS-derived NO.
Cyclic GMP Accumulation in BMDMØs in Response to LPS
The observation that modulation of sGC activity resulted in a marked
alteration in iNOS expression suggested a role for cGMP in the regulation of
LPS-induced macrophage activation. Therefore, experiments were conducted to
define a role for cGMP in LPS-induced iNOS expression by exposing WT
BMDMØs to LPS (100 ng/ml) and BAY 41-2272 (10 µM) and
assessing intracellular cGMP concentrations. In unactivated WT cells, basal
cGMP content was 10.7 ± 2.1 fmol/well (i.e. 3 x
106 cells). After addition of LPS (100 ng/ml), cGMP concentrations
increased in a time-dependent manner for 30 min such that at this time point,
levels were 150% of control (Fig.
9). A similar increase in cGMP concentrations was also observed
(in the absence of LPS) in the presence of BAY 41-2272 (10 µM;
Fig. 9). However, LPS was
unable to elicit an increase in cGMP concentrations in eNOS KO cells, whereas
BAY 41-2272 produced a similar increase in cGMP levels compared with WT
BMDMØs (data not shown).
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Cell ViabilityCell viability, as measured by trypan blue exclusion, was not altered significantly by any of the experimental conditions (data not shown).
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DISCUSSION |
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Because NF-B is known to be essential for iNOS expression in murine
and human macrophages, electrophoretic mobility shift assays were used to
investigate whether the effects of eNOS-derived NO observed in the present
study were exerted via an action on NF-
B. In BMDMØs derived from
eNOS KO mice, NF-
B activity after exposure to LPS was significantly
reduced compared with that in WT cells. Thus, the augmenting actions of
"low-output" NO generated by eNOS appear to be mediated, at least
in part, via an action on NF-
B. These observations are in accord with a
previous study in transgenic mice overexpressing eNOS that exhibit altered
hemodynamics and resistance to LPS when compared with WT animals
(32) and suggest that
eNOS-derived NO may be important in vivo in expediting host defense.
In addition, enhancement of NF-
B activity and pro-inflammatory protein
expression by NO may underlie control of osteoclast function, which appears to
be regulated by a combination of constitutive and inducible NOS activity
in vitro and in vivo
(33,
34).
To investigate possible mechanisms by which NO produced by eNOS could
modulate iNOS expression, the effect of modulators of sGC were investigated.
Recent evidence suggests that sGC within intact cells is activated by NO with
an EC50 20 nM
(35), which is commensurate
with the concentrations of NO produced by constitutive NOS isozymes and which
we have shown previously to potentiate LPS-induced NF-
B activity and
iNOS expression (17).
Moreover, there is a precedent for regulation of gene expression by cGMP. In
human glomerular mesangial cells, 8-bromo-cGMP (a non-hydrolysable analogue)
potentiates interleukin-1
and tumor necrosis factor-
activation
of iNOS protein expression and nitrite production
(14). Also, in primary glial
cell cultures, cGMP analogues and the PDE 5 inhibitor, zaprinast, increase
LPS-stimulated NO release and iNOS expression, whereas the G-kinase inhibitor
KT5823 has an inhibitory effect
(36). In the present study,
sGC mRNA and protein were identified in murine macrophages, supporting a novel
role for this enzyme in inflammation. What is more, exposure of BMDMØs
to LPS (and the sGC activator BAY 41-2272) resulted in accumulation of cGMP
within the cell, thereby linking exposure of the macrophage to an inflammatory
stimulus with sGC activation/cGMP production. The cGMP dependence of the
effect of NO on iNOS expression was assessed further using the sGC activator
BAY 41-2272 and the sGC inhibitor ODQ. The results from these studies
substantiated our initial findings that eNOS-derived NO is important in
regulating macrophage activation, because the presence of ODQ reduced iNOS
expression in response to LPS, whereas BAY 41-2272 augmented iNOS expression.
Consequently, the potentiating effects of NO on NF-
B activity and
subsequent protein expression described in this study are likely to occur, at
least in part, via activation of sGC and the production of cGMP. However, the
effects of eNOS gene deletion on macrophage activation were greater than those
elicited by sGC modulatory compounds,suggesting that eNOS-derived NO exerts
both cGMP-dependent and -independent effects to augment macrophage
activation.
The mechanisms underlying the potentiating effects of NO/cGMP on macrophage
activation, as demonstrated in this study, remain unclear. In primary glial
cell cultures, cGMP augments LPS activation of iNOS expression through
activation of G-kinase. Furthermore, the NO-donor
S-nitroso-N-acetyl-D,L-pencillamine
has been shown to activate NF-B and induce tumor necrosis
factor-
mRNA and protein expression in feline cardiac myocytes, and
this is blocked by pretreatment with ODQ or a G-kinase inhibitor
(37); both observations
intimate the involvement of G-kinase. Interestingly, G-kinase has been shown
to phosphorylate an I
B
-glutathione S-transferase fusion
protein on Ser32 in vitro
(37). Because Ser32
is one of the residues that is phosphorylated before proteasomal degradation
of I
B
(38), this
represents a mechanism by which the sGC-cGMP-G-kinase pathway could initiate
the activation of NF-
B. In accord with the potential role of sGC-cGMP
in macrophage activation, LPS stimulation of rat cerebellar astrocytes leads
to an increase in cGMP levels, which causes a gradual decrease in the
expression of the sGC
1 subunit
(39). Therefore, it seems that
although LPS can stimulate cGMP production, this occurs at earlier time points
of activation, whereas at later stages the sGC enzyme is turned off by the
down-regulation of protein expression. This represents a way in which the
potentiating effects of NO on pro-inflammatory protein expression, by the
activation of sGC and production of cGMP, are blocked at later stages when
pro-inflammatory proteins should be down-regulated. Moreover, high-output NO
inhibits both the expression and activity of the constitutive NOS isozymes
(40,
41), turning off the signal
conveyed by eNOS-derived NO. In concert, these observations intimate that NO
exerts distinct effects on transcription factor activity and pro-inflammatory
protein expression by the use of both constitutive and inducible NOS isoforms
and also by cGMP-dependent and -independent mechanisms.
Potential mechanisms by which NO may augment cellular activation in a
cGMP-independent manner may also be multi-faceted. For example, NO may act
directly on IB kinase
to increase phosphorylation and
degradation of I
B
(42). Alternatively, NO may
act upstream of I
B kinase on a molecule such as
p21ras, which is involved in the LPS-induced activation of
NF-
B (43) and known to
be activated by S-nitrosation of Cys118
(44,
45). Mitogen-activated protein
kinases, which lie downstream of p21ras, also appear to be
susceptible to modulation by NO and are known to be involved in induction of
iNOS expression in LPS-stimulated RAW 264.7 macrophages
(46).
Although a role for eNOS in macrophage activation has been established in
this study, the mechanism(s) underlying eNOS stimulation remains to be
elucidated. It has been reported previously that RAW 264.7 macrophages produce
NO under basal conditions and that this can be increased in a
Ca2+-dependent manner
(27). Furthermore, LPS can
stimulate eNOS activity via increasing intracellular free
Ca2+ levels. L-type
Ca2+ channel antagonists increase cytoplasmic levels of
IB
and reduce nuclear levels of NF-
B p65 subunit and
subsequent iNOS expression in LPS-treated rat Kupffer cells
(47). This suggests that an
increase in Ca2+ levels might be involved in LPS-induced
NF-
B activation and expression of iNOS. Furthermore, thapsigargin and
the Ca2+ ionophore A23187
[GenBank]
increase nitrite production in
murine macrophages exposed to LPS
(48). The results of the
current study are in accord with these observations because an increase in
Ca2+ at the time of LPS stimulation may activate eNOS,
which produces low levels of NO to potentiate NF-
B expression. Although
the addition of compounds that modulate intracellular
Ca2+ levels can affect iNOS expression and activity, it
has also been reported that LPS itself does not trigger changes in
intracellular Ca2+ levels, suggesting that alternative
mechanisms for eNOS activation may be important
(49,
50). Endothelial NOS can also
be activated by phosphorylation via the phosphatidylinositol 3-kinase
(PI-3K)/Akt pathway in response to shear stress in endothelial cells
(51,
52), and this pathway may also
exist in macrophages. LPS has been shown to increase the activity of PI-3K in
RAW 264.7 murine macrophages
(53), and vascular endothelial
growth factor can stimulate intercellular adhesion molecule 1 expression via a
PI-3K/Akt/NO-dependent process in brain microvascular endothelial cells
(54). Because intercellular
adhesion molecule 1 is up-regulated by NF-
B, this represents evidence
that NO from phosphorylation-activated eNOS might activate this transcription
factor. Moreover, the PI-3K inhibitor LY294002 inhibits LPS-stimulated iNOS
mRNA expression and NO production by blocking the degradation of
I
B
(55), and
tumor necrosis factor-
has been shown to activate the PI-3K/Akt
pathway, leading to phosphorylation of eNOS in human microvascular endothelial
cells (56). In vivo
treatment of rats with LPS leads to an increase in brain eNOS mRNA levels with
a corresponding rise in Ca2+-dependent NOS activity
(57).
In summary, this is the first report of a role for macrophage eNOS in
regulating cellular activation and pro-inflammatory gene expression. NO
produced by this isozyme appears pivotal in the cellular response to an
inflammatory stimulus, and this role is mediated, in part, via modulation of
NF-B. Moreover, activation of sGC and production of cGMP appear to be
important in this novel regulatory role for eNOS, assigning a new
(patho)physiological function to sGC in immune cells. A complete understanding
of the mechanism underlying this pro-inflammatory effect of eNOS-derived NO
may have important implications for our understanding of diseases such as
sepsis and provides the rationale for the design of novel therapeutics.
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FOOTNOTES |
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Recipient of a Wellcome Trust International Prize Traveling Research
Fellowship.
|| Recipient of a Wellcome Trust Senior Fellowship in Basic Biomedical Sciences. To whom correspondence should be addressed: Wolfson Institute for Biomedical Research, University College London, Cruciform Bldg., Gower St., London WC1E 6AE, United Kingdom. Tel.: 44-(0)20-7679-6611; Fax: 44-(0)20-7813-2846; E-mail: a.hobbs{at}ucl.ac.uk.
1 The abbreviations used are: NO, nitric oxide; iNOS, inducible nitric oxide
synthase; eNOS, endothelial NOS; LPS, lipopolysaccharide; NF-B, nuclear
factor-
B; RT, reverse transcription; BMDMØ, bone marrow-derived
macrophage; sGC, soluble guanylate cyclase; ODQ,
1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one; cGMP, cyclic
guanosine-3',5'-monophosphate; FBS, fetal bovine serum; WT, wild
type; KO, knockout; ATP, adenosine triphosphate; PI-3K, phosphatidylinositol
3-kinase.
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