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,3
*
Unité Propre d’Enseignement Supérieur Associée 971068, Faculté de Médecine, 54505 Vandoeuvre;
Département d’Anesthésie-Réanimation Chirurgicale,
**
Laboratoire de Biologie Cellulaire, Centre Hospitalier Universitaire de Nancy, 54511 Vandoeuvre;
Institut National de la Santé et de la Recherche Médicale Unité X 308, 54000 Nancy; and
Laboratoire de Physiologie, Hôpital Maison Blanche, 51092 Reims, France
3To whom correspondence should be addressed. E-mail: d.longrois{at}chu-nancy.fr.
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • retinoids • prostaglandins • prostaglandin E synthase • nitric oxide • rats
|
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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–3)
. However, the effect
of vitamin A supplementation in patients prone to or presenting with
infectious diseases is controversial, with beneficial
(1
,4)
, deleterious (5
,6)
or neutral
(2
,7)
effects.
Modulation of the host immune and inflammatory response is supposed to
be one of the mechanisms responsible for the effects of vitamin A on
host-pathogen interactions (3)
.
All-trans-retinoic acid
(RA),4
the active metabolite of vitamin A, regulates the expression and/or
activity of several enzymes involved in the host defense against
pathogens, such as the inducible nitric oxide synthase (NOS2)
(8
,9)
, phospholipase A2
(PLA2) (10
,11)
and prostaglandin H
synthase (PGH-S) (10
,12
–16)
. These RA-mediated
effects on the host immune and inflammatory function have been
described mainly in vitro. However, compared with the in vivo effects,
opposite in vitro effects of RA have been reported by several groups
(17
–20)
including ours (8
,9)
. Taken
together, these observations are a strong incentive to document the
cellular and molecular mechanisms through which vitamin A and its
metabolites modulate the host immune and inflammatory response in vivo.
Prostanoids, including prostaglandins (PG), prostacyclins and
thromboxanes, are products of at least three enzymatic reactions as
follows: arachidonic acid (AA) is released from membrane
glycerophospholipids by several isoforms of PLA2
(21)
and give rise to the common intermediate
PGH2 by the PGH-S (22)
.
PGH2 is subsequently converted to various
prostanoids [PGE2, PGD2,
PGF2
, 6-keto-PGF1 and
thromboxane B2 (TxB2)] by
terminal synthases (23)
. Different isoforms of
PLA2, including a cytosolic
(cPLA2) and a secreted
(sPLA2) isoform, supply AA to PGH-S
(21)
. PGH-S catalyzes the committed step in the
synthesis of prostanoids and carries out two distinct activities, the
cyclooxygenase (COX) and peroxidase activities (22)
. Two
isoforms of the COX enzyme have been identified: 1) COX-1,
constitutively expressed in most tissues, is responsible for the
physiologic production of PG and thus maintains cellular homeostasis
(24)
; and 2) COX-2, induced in inflammatory
cells by cytokines, mitogens and bacterial products, is responsible for
the elevated production of PG during inflammation (25)
.
Terminal synthases catalyze the conversion of
PGH2 to biologically active prostanoids. Of
interest, two isoforms of prostaglandin E synthase (PGES) have been
characterized recently, i.e., a cytosolic isoform (cPGES) involved in
immediate PGE2 biosynthesis (26)
and
a membrane-associated isoform (mPGES) induced by proinflammatory
stimuli and involved in delayed PGE2 biosynthesis
(27)
.
Prostaglandins modulate both cellular and humoral immune
responses via inhibition of T-lymphocyte activation, natural killer
cell activity, B-cell activation and subsequent antibody production
(28)
. In addition, they have been shown to modulate
host-pathogen interactions (29
,30)
. More recently,
this modulatory role of PG has been confirmed in vivo using transgenic
mice whose COX-2 gene was knocked out (31)
.
We hypothesized that retinoids modulate the host inflammatory response,
at least in part, by modulation of the prostanoid biosynthetic pathway.
Therefore, this study was performed to document the effects of RA on
prostanoid synthesis in rats in vivo in the absence or presence of a
proinflammatory stimulus such as lipopolysaccharide (LPS). This
experimental design was chosen to mimic the clinical situation
characterized by vitamin A supplementation before challenge with a
pathogen as investigated in several clinical trials
(1
,2
,4
–7)
.
|
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Male Wistar Kyoto rats (250–350 g) were housed and treated in accordance with accepted practices for humane laboratory animal care. They consumed a standard diet (M20 Extralabo, Trouw Nutrition, Vigny, France)5 ad libitum.
Preparation of reagents.
All chemicals and reagents were purchased from Sigma (Saint Quentin Fallavier, France) unless specified otherwise. Salmonella typhimurium LPS (Lot 96H4021) and the relatively specific NOS2 inhibitor aminoguanidine hemisulfate salt (AG) were dissolved in 9 g/L NaCl solution and administered intraperitoneally (i.p.) in 500 µL volumes. The COX-2-specific inhibitor SC-236 (4-[5-(4-chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide), kindly provided by Searle Research and Development (St. Louis, MO), was administered i.p. as a suspension of 5 g/L methylcellulose, 0.025% (v/v) Tween 20 in 500 µL sterile water. These drugs were prepared extemporaneously. All-trans retinoic acid was dissolved in 5% (v/v) dimethyl sulfoxide (DMSO) and first cold pression olive oil at a concentration of 10 g/L; aliquots were stored protected from light at -20°C.
Experimental protocols.
Four groups of rats were used as follows: Control, RA, LPS and RA + LPS. Rats of the RA and RA + LPS groups received daily injections of RA (10 mg/kg body, i.p.) for five consecutive days. Rats of the LPS and Control groups received olive oil plus 5% (vol/vol) DMSO under the same conditions. On d 5, LPS (4 mg/kg body; i.p.) was administered to the RA + LPS and LPS groups, whereas the RA and Control groups received the same volume (i.e., 500 µL) of 9 g/L NaCl solution. Unless stated otherwise, the dose of LPS was 4 mg/kg body.
For biochemistry experiments, rats were anesthetized 6 h after LPS injection with 100 mg sodium thiopental (Nesdonal Rhône Poulenc Rorer, Paris, France) i.p., and the thorax and abdomen were dissected. Blood samples were recovered by cardiac puncture, centrifuged at 600 x g for 10 min and plasma was stored at -70°C. Tissue samples from liver, lung, kidney, spleen and heart were excised, rapidly rinsed in ice-cold saline, frozen in liquid nitrogen and stored at -70°C until analysis.
For immunohistochemistry experiments, additional rats were treated similarly to those in the Control and RA + LPS groups. Six hours after LPS injection, tissues were fixed with paraformaldehyde (Serva electrophoresis GmbH, Heidelberg, Germany) through a cardiac cannula. Tissue samples were excised, further fixed for 4 h with paraformaldehyde, immersed in 15 g/L sucrose PBS (0.01 mol/L, pH 7.4), frozen and stored at -70°C.
The effects of AG and SC-236 on RA-mediated PG production
were studied as follows. Fifteen minutes before LPS administration,
rats from the RA + LPS group received either an injection of SC-236 (10
mg/kg body, i.p.; RA + LPS + SC-236 group) or an injection of AG (100
mg/kg body, i.p.; RA + LPS + AG group). These rats were killed 6 h
after LPS injection, and plasma and samples from the five organs
studied were recovered as described above. The doses of AG
(32)
and SC-236 (33)
were chosen from
published data on dose vs. efficacy vs. toxicity reports and previously
validated in our laboratory (9)
.
Additional protocols.
Experiments with higher doses of LPS were performed to demonstrate COX-2 induction. Rats were given an injection of LPS (15 mg/kg body; i.p.) and were killed 9 h later when COX-2 protein expression is supposed to reach a maximum. Plasma and samples from the five organs studied were recovered as described above.
Assessment of mRNA expression by semiquantitative reverse transcription-polymerase chain reaction (SQ RT-PCR).
Tissue mRNA expression for sPLA2, cPLA2, COX-1,
COX-2, and mPGES was estimated by SQ RT-PCR as previously described
(9)
. The following amplimers were chosen:
5'-CTGGAGTTTGGGCAAATGAT-3' (sense) and 5'-CCCTTTGCAAAACTTGTTGG-3'
(antisense) for sPLA2, 5'-ATTCTCCGGTGTGATGAAGG-3' (sense)
and 5'-GCTTCCAAACAGGTCAGGAG-3' (antisense) for cPLA2,
5'-GATGACGGGTCTGTCTTCGT-3' (sense) and 5'-TTCTTAGGGGGCTCCAGATT-3'
(antisense) for COX-1, 5'-TTCAAAAGAAGTTCTGGAAAAGGT-3' (sense) and
5'-GATCATGTCTACCTGAGTGTCTTT-3' (antisense) for COX-2,
5'-ATGACTTCCCTGGGTTTGGT-3' (sense) and 5'-GTCCCCCATTGTGGTATCTG-3'
(antisense) for mPGES, 5'-GGACTTCGAGCAGGAGATGG-3' (sense) and
5'-GCACCGTGTTGGCGTAGAGG-3' (antisense) for ß-actin. Densitometric
analysis was performed with a BioRad Gel Doc 1000 (BioRad Laboratories,
Iury-sur-Seine, France). Results are expressed as the ratio of
the optical density of the band of the PCR product of interest to that
of ß-actin.
Estimation of protein expression by Western blot.
Western blot experiments were performed to estimate COX-1 and COX-2
protein expression in the five organs studied. Tissue samples were
prepared and 100 µg of proteins from each sample was
submitted to SDS-PAGE as previously described (9)
.
Proteins were transferred overnight at 4°C (Trans Blot
Electrophoretic Cell, BioRad) on polyvinylidene fluoride (PVDF)
membranes (Sequi-Blot PVDF Membrane, BioRad) in 6.2 mol/L methanol, 25
mmol/L Tris, 192 mmol/L glycine (pH 8.3). After blocking for 1 h
with 3 g/L blocking agent (Amersham Pharmacia Biotech Europe GmbH,
Freiburg, Germany) in Tris-buffered saline [25 mmol/L Tris, pH
7.5, 150 mmol/L NaCl, 0.05% (vol/vol) Tween 20 (TBST solution)],
membranes were incubated with 1:1000 dilutions of rabbit anti-human
COX-1 or COX-2 polyclonal antibodies (Santa Cruz Biotechnology, Santa
Cruz, CA and Oxford Biomedical Research, Oxford, MI) for detection of
COX-1 and COX-2 proteins. After washing, the membranes were incubated
for 30 min with a 1:20,000 dilution of goat anti-rabbit
immunoglobulin (Ig)G conjugated to horseradish peroxidase (Amersham).
Immunocomplexes were revealed using enhanced chemiluminescence (ECL
Western blotting, Amersham). Native ovine COX-1 and COX-2 proteins
purified from seminal vesicles and placenta, respectively (both from
Oxford Biomedical Research), were used as positive controls for COX
proteins detection. Densitometry of the resulting bands was performed
using a BioRad GS-690 Imaging Densitometer.
Immunohistochemical analysis of COX-2 protein expression.
Control and experimental tissue sections (20 µm thick) were collected on the same gelatin-coated slides and treated with the same immunohistochemical protocol. Sections were successively covered with different dilutions (1:40 to 1:200) of the polyclonal rabbit anti-human COX-2 serum (Santa Cruz) for 48 h at 6°C. After washing, the primary antibody was stained with a goat anti-rabbit IgG conjugated with fluorescein isothiocyanate (Jackson Immunoresearch Laboratories, West Grove, PA). Sections were counterstained with Evans blue before analysis with an epifluorescent microscope (Leica, France). The lack of immunostaining when sections were incubated with non-immune rabbit serum or PBS to replace the primary antibody recognizing the COX-2 protein verified the specificity.
Measurement of prostanoid concentrations in plasma and liver homogenates.
Prostaglandin E2 (PGE2), 6-keto-prostaglandin
F1 (6-keto-PGF1) and thromboxane
B2 (TXB2) are stable prostanoids derived from
PGH2, which is synthesized from AA through the COX pathway.
Their concentrations were determined by ELISA using PGE2,
6-keto-PGF1, and TXB2 ELISA kits (catalog #
404110, 404310 and 405110, respectively) following the manufacturer’s
instructions (Neogen, Lexington, KY). Dilutions (35-fold) of plasma
samples were used for the three assays. Liver samples were homogenized
with a Polytron PT 1200 in 5 volumes of extraction buffer (118.5 g/L
methanol and 1 g/L indomethacin in 0.1 mol/L PBS, pH 7.5). The
homogenates were centrifuged at 12,000 x g for 15
min. Homogenates were diluted 10-fold for PGE2 and
6-k-PGF1 assays and 50-fold for the TXB2
assay. The enzymatic reaction was stopped by the addition of 50
µL of 1 mol/L HCl and absorbance was read at 450 nm.
Prostanoid concentrations were extrapolated from standard curves and
were expressed in µg/L for plasma and in
pg/µg of protein for liver homogenates. Protein
concentration in the supernatant was measured by the method of Lowry et
al. (34)
.
Measurement of sPLA2 and total PLA2 activity in liver homogenates.
Secreted and total PLA2 activities were measured with
commercially available kits (catalog # 765001 and 765021, respectively)
following the manufacturer’s instructions (Cayman Chemical, Ann Arbor,
MI). Liver samples were homogenized with a Polytron PT 1200 in 5
volumes of extraction buffer [25 mmol/L Tris-HCl, pH 7.4, 300
mmol/L NaCl, 20 mmol/L CaCl2, 1 µmol/L
leupeptin, 1 x 105 U/L aprotinin, 0.2
mmol/L phenylmethylsulfonyl fluoride, 1% Triton X-100 (v/v) and 0.5%
Nonidet P-40 (v/v)]. The homogenates were centrifuged at 12,000
x g for 15 min and protein concentration in the
supernatant was measured by the method of Lowry et al.
(34)
. Absorbance was read at 405 nm, and 10.0
(mmol/L-1) · cm-1 was used as the
adjusted (for the path length of the solution in the well) molar
extinction coefficient. PLA2 activity was expressed in
nmol/(min · µg protein).
Measurement of nitrate and nitrite in plasma.
Nitrate and nitrite are the stable end products of NO oxidation. Their
plasma concentration was used as an indicator of NO production and was
determined as previously described (9)
. All samples were
tested in triplicate and the background nitrite concentration of water
was subtracted from the extrapolated nitrite concentration of samples.
Statistical analysis.
Statistical analysis was performed using the StatView IV software (Abacus Concepts, Berkeley, CA). Results are expressed as means ± SEM. Comparisons among several groups were performed with nonparametric ANOVA (Kruskall-Wallis test). Comparisons between two groups were performed with the Mann Whitney test. Differences with a P-value < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Control rats had low but detectable plasma and liver
concentrations of 6-keto-PGF1,
PGE2 and TXB2. In plasma
and liver, RA alone significantly increased the concentrations of
6-keto-PGF1 and PGE2
compared with Controls (Table 1
). Retinoic acid increased TXB2 concentration in
the liver but not in the plasma. Compared with Control rats, LPS
treatment significantly enhanced plasma and liver concentrations of
6-keto-PGF1, PGE2 and
TXB2. The effects of RA or LPS were of similar
magnitude. Supplementation with RA in LPS-injected rats (RA + LPS
group) synergistically increased plasma and liver concentrations of
6-keto-PGF1, PGE2 and
TXB2 compared with rats given RA or LPS alone.
|
Compared with rats from the Control group, rats from the RA + LPS group exhibited a moderate but significant increase in sPLA2 mRNA expression in several organs, notably in the liver (0.91 ± 0.04 and 1.46 ± 0.06 relative sPLA2/ß-actin mRNA abundance for Control and RA + LPS groups, respectively). However, there were no increases in sPLA2 and total PLA2 activities in LPS- and/or RA-injected rats compared with control rats (results not shown). Taken together, these results are consistent with a modest effect of RA in LPS-challenged rats on PLA2 mRNA expression and unchanged PLA2 activity.
Effect of RA supplementation on COX-1 and COX-2 mRNA expression
Expression of COX-1 mRNA.
In all organs tested, RA alone had no effect on COX-1 mRNA expression
compared with the Control group (Table 2
). Injection of LPS alone (LPS group) significantly decreased COX-1 mRNA
expression compared with the RA and Control groups in the liver and
spleen. In the spleen, a further decrease in COX-1 mRNA expression was
observed in RA + LPS rats compared with LPS or RA rats. No differences
were seen for the kidney, lung and heart among the four experimental
groups.
|
. Administration of RA or LPS alone induced COX-2 mRNA
expression in the liver, spleen and kidney. Moreover, a further
increase of COX-2 mRNA expression was observed in rats from the RA +
LPS group in the liver and kidney. COX-2 mRNA was not detectable in the
lung and heart of any of the groups (Table 2)
. Expression of COX-2 mRNA
was higher in rats given 15 mg/kg LPS and killed 9 h later
(Fig. 1A
). In addition, these rats had detectable COX-2 mRNA expressions in the
lung and heart, two organs in which 4 mg/kg LPS had no effect.
|
Western blot analysis of COX-1 and COX-2 protein expression.
Positive and negative controls were used to verify the specificity of
the antibodies used to detect COX-1 and COX-2 proteins. The
COX-2-specific antibody revealed a single band at 
74 kDa for the
COX-2 but not for the COX-1 positive control. COX-2 protein expression
was not detectable in any of the organs of rats injected with 4 mg/kg
LPS and killed 6 h later, whereas it was reproducibly increased in
the five studied organs of rats injected with 15 mg/kg LPS and killed
9 h later (Fig. 1
B). The COX-1–specific antibody
revealed a single band at 70 kDa for the COX-1 but not for the COX-2
positive control. As expected, COX-1 protein was constitutively
expressed in the liver of all rats. RA alone, but not LPS,
significantly increased COX-1 protein expression in the liver compared
with the Control group. Liver COX-1 protein expression in RA + LPS rats
was not significantly greater than in rats that received RA alone
(Fig. 2
). Taken together, these results are consistent with very low
level/absent expression of the COX-2 protein (6 h after injection of 4
mg/kg LPS) and with moderately increased COX-1 protein expression after
RA treatment.
|
) and RA + LPS (Fig. 3
C) groups stained with the
COX-2-specific antibody but not when the sections were incubated with
normal rabbit serum or PBS (Fig. 3
A). Immunofluorescent
staining decreased with the dilution of the primary antibody, thus
confirming the staining specificity. The high magnification (X50) is
consistent with specific immunostaining either in endothelial cells of
the liver sinusoids or in infiltrating monocytes/macrophages (Fig. 3
C'). Staining was more intense in liver
sections from RA + LPS compared with Control rats. Nevertheless,
staining intensity was low and patchy in all sections, thus confirming
the Western blot experiment and consistent with very low level COX-2
protein expression.
|
Figure 4A
shows a representative expression profile of mPGES mRNA in the liver of
the four groups of rats. Rats from the Control group did not express
detectable mPGES mRNA in the liver, whereas RA supplementation (RA
group) induced the expression of mPGES mRNA. LPS challenge resulted in
a higher increase of mPGES mRNA expression. More importantly, LPS
challenge in the RA-supplemented rats induced a further increase of
liver mPGES mRNA expression. The level of mRNA encoding mPGES in the
liver of RA + LPS rats was 10-fold that of RA rats and near twofold
that of LPS rats (Fig. 4
B).
|
Administration of SC-236, a putative selective inhibitor of COX-2
activity, to RA + LPS rats resulted in decreased plasma and liver
PGE2 concentrations. This decrease reached values
not different from the Control group in the liver but not in the plasma
(Fig. 5A
). Administration of SC-236 did not modify NOS2, NOS3, COX-1 or COX-2
mRNA expressions (data not shown). We also investigated a potential
effect of SC-236, mediated by the decreased PGE2
concentration, on the NOS2 pathway, as assessed by measurement of
plasma nitrate and nitrite concentration. The SC-236–mediated decrease
in PGE2 concentration had no effect on NOS
activity in this experimental model (Fig. 5
B).
|
As expected, AG administered before LPS injection completely
inhibited the increase of plasma nitrate and nitrite concentrations
observed in RA + LPS rats (Fig. 6A
), confirming that this increase was effectively a consequence of the
induction of NOS2 expression and activity (9)
.
Interestingly, the inhibition of NO production by AG resulted in a
concomitant inhibition of PGE2 release.
PGE2 concentration decreased to control values in
the liver but not in the plasma (Fig. 6B)
. Treatment with AG had no
effect on NOS2, NOS3, COX-1 or COX-2 mRNA expressions (data not shown).
|
|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Methodological discussion
Van Pelt and de Rooij (35)
reported that
single-dose RA supplementation was ineffective, whereas replicate
doses had measurable biological effects. Look et al. (36)
showed toxic effects in mice after 7 d of treatment with 100 mg
RA/(kg · d). The 90% lower dose used in this study explains the
lack of toxic effects. Plasma nitrate and nitrite concentrations were
used as indicators of NO production. The diet contained 78 mg/kg
NaNO3 and was nitrite free; dietary intake could
therefore influence the plasma nitrite and nitrate concentration.
Nevertheless, rats from all groups were fed the same diet; thus all
rats had the same intake of NaNO3.
Retinoic acid acts synergistically with LPS to increase plasma and liver prostanoid concentrations
RA or LPS, alone or in combination, increased plasma and liver
concentrations of 6-keto-PGF1,
PGE2 and TXB2. In
particular, PGE2 concentration increased 400-fold
in plasma of rats from the RA + LPS group compared with the Control
group (Table 3). Modulation of the PG biosynthetic pathway by RA has
been studied only in vitro, and both activation (14
,15)
and inhibition (12
,37)
have been observed. These
contradictory effects may depend on cell type, co-inducers and RA
concentrations used. Our results are the first to show a synergistic
activation of the PG biosynthetic pathway by RA and LPS in vivo.
Investigation of mechanisms responsible for the increased prostanoid concentrations after RA supplementation in vivo
Our results demonstrate a modest enhancement of mRNA expression of sPLA2 in the liver of rats from RA, LPS or RA + LPS groups, which was not associated with a detectable increase in enzyme activity. This suggests that the highly increased PG concentration in the RA + LPS group is not due to increased sPLA2 expression or activity.
Effect of RA on COX Pathway
Low-level induction of COX-2 mRNA and very low level/absent protein
expression in vivo.
RA induced modest COX-2 mRNA expression in the liver, spleen and
kidney, but not in the lung and heart, consistent with the complex and
organ-specific metabolism of RA (38
,39)
. The
previously reported effects of RA on COX-2 expression in vitro are
controversial, with both inductive (10
,13)
and suppressive
(12)
effects being described.
The effects of RA on COX-2 expression were similar to those of 4 mg/kg
LPS, which induced modest COX-2 mRNA expression in the liver, spleen
and kidney, but not in the lung and heart. The absence of major COX-2
induction after 4 mg/kg LPS challenge contrasts with previous reports
showing COX-2 induction in many distinct cell types [for review, see
(25)
] and is probably related to the relatively low dose
of LPS. Indeed, when additional rats were stimulated with 15 mg/kg LPS
for 9 h, COX-2 mRNA and protein expressions were much higher (Fig. 1)
. Therefore, the low mRNA expression observed by RT-PCR and the
barely detectable COX-2 protein expression in rats stimulated with 4
mg/kg LPS for 6 h are consistent with low-level COX-2 pathway
activation in this experimental model. Later time points have been
investigated in rats stimulated with 4 mg/kg LPS and confirm the low
level/absent expression of COX-2 protein (Devaux et al., unpublished
observations).
Effect of RA in the absence or presence of LPS on COX-1 mRNA and
protein expression in vivo.
Because COX-2 protein expression was not induced by RA and/or LPS
treatment, we sought to determine whether an increase in COX-1 mRNA and
protein expression could account for the high concentration of PG
observed in rats from the RA, LPS and especially the RA + LPS groups.
Interestingly, COX-1 protein expression was significantly but
moderately increased by RA compared with Control rats in liver. Our
results (Table 3 and Fig. 2
) are consistent with a modest or absent
effect of RA administered alone on COX-1 mRNA expression and a moderate
increase in COX-1 protein expression in vivo. They are in contrast to
the cell type–dependent modulation of COX-1 expression by RA
previously reported in vitro, i.e., decreased expression in rat
tracheal epithelial cells (10)
and increased expression in
macrophages (14)
. Nevertheless, the 400-fold increase in
plasma and 15-fold increase in liver PGE2
concentrations cannot be explained solely by the moderate increase of
COX-1 protein expression.
Effect of RA on mPGES pathway
Because PGE2 is the most commonly released
prostanoid and is a mediator of inflammatory reactions as well as
host-pathogen interactions (28
–31)
, we focused on the
mechanisms responsible for its synthesis. The terminal synthase
metabolizing the common prostanoid intermediate
PGH2 to PGE2 was recently
identified as being a member of the "membrane
associated proteins involved in
eicosanoid and glutathione
metabolism" (the MAPEG superfamily) and named PGES
(40)
. Cytosolic (cPGES) (26)
and
membrane-associated (mPGES) (27)
forms of PGES have
been identified in rats. Our results show that mPGES mRNA expression
was undetectable in the liver from Control rats, as already reported
(27)
. RA or LPS alone induced mPGES mRNA expression and
their combination was synergistic (Fig. 4)
. This is the first report of
an induction of mPGES pathway by LPS in the rat liver and, more
importantly, of a potentiation of this induction by RA. Taken together,
these results are consistent with an effect of RA on mPGES expression
rather than PLA2 or COX expression.
Effect of SC-236, a putative COX-2 inhibitor, on PGE2 plasma and liver concentrations
Additional experiments with SC-236, a drug characterized as a
COX-2–specific inhibitor, revealed that the increase in
PGE2 concentration in rats from the RA + LPS group was
completely abolished by SC-236 in the liver and partially abolished in
the plasma (Fig. 5)
. This observation is surprising because COX-2
protein was barely or not induced under these experimental conditions.
Nevertheless, inhibition of mPGES activity by another COX-2–specific
inhibitor, NS-398, has recently been observed and presumed to be
related to similar structural properties in the active sites of COX-2
and mPGES (41)
. We can thus assume that SC-236 inhibited
mPGES rather than COX-2 activity in our experiments.
Crosstalk between the NOS2 pathway and PGE2 biosynthesis
Because we have recently reported that LPS challenge in
RA-supplemented rats induced significant NOS2 expression and
activity (9)
, we sought to determine whether NOS2-derived
NO could be responsible for the high PGE2
concentration. We show here that inhibition of NOS2 activity by
aminoguanidine in RA + LPS rats decreased plasma nitrate and nitrite
concentration, as expected, but also decreased plasma (partially) and
liver (completely) PGE2 concentrations. These
decreases were not linked to an effect of AG on NOS2, COX-1 and COX-2
mRNA expressions (data not shown). In experiments with COX-1– and
COX-2–deficient mice, Clancy et al. (42)
demonstrated
that NO exerts divergent effects on the COX isoforms, activating COX-1
while inactivating COX-2. Our results are consistent with these
observations and suggest that under these experimental conditions,
NOS2-derived NO activates COX-1. NO has been shown to activate COX-1
via S-nitrosation of the cysteine residues in the catalytic
domain (43)
. In addition, peroxynitrite, the coupling
product of superoxide anion and NO, is a substrate for the peroxidase
and an activator of the cyclooxygenase activities (44)
.
In conclusion, the present study demonstrates that RA acts synergistically with LPS to highly increase prostanoid concentration in vivo by several mechanisms involving a moderate increase of COX-1 protein expression, induction of mPGES and NOS2-derived activation of the COX-1 pathway. These results may contribute to the characterization of the complex effects of vitamin A and its metabolites on the host inflammatory reaction.
|
FOOTNOTES |
|---|
2 These authors contributed equally to this
work.
4 Abbreviations used: AA, arachidonic acid; AG,
aminoguanidine; COX-1, cyclooxygenase type 1 (constitutive isoform);
COX-2, cyclooxygenase type 2 (inducible isoform); DMSO, dimethyl
sulfoxide; Ig, immunoglobulin; i.p., intraperitoneal; LPS,
lipopolysaccharide; NOS2, inducible nitric oxide synthase; NOS3,
endothelial nitric oxide synthase; PG, prostaglandins;
PGE2, prostaglandin E2; PGES, prostaglandin E
synthase; cPGES, cytosolic PGES; mPGES, membrane-associated PGES;
6-keto-PGF1, 6-keto-prostaglandin F1;
PGH-S, prostaglandin H synthase; PLA2,
phospholipase-A2; sPLA2, secreted
PLA2; cPLA2, cytosolic PLA2; RA,
all-trans-retinoic acid; PVDF, polyvinylidene fluoride;
SQ RT-PCR, semiquantitative reverse transcription-polymerase
chain reaction; TXB2, thromboxane B2.
5 Composition of the diet was as follows (g/kg):
crude protein, 187; fat, 33; crude fiber, 37; ash, 56; moisture, 124;
dry matter, 876. Mineral mixture (g/kg): calcium, 10.6; phosphorus,
6.8; sodium, 2.5; chloride, 5.1; potassium, 9.4; magnesium, 1.4. Trace
elements (g/kg): iron, 0.13; copper, 0.01; manganese, 0.06; zinc, 0.07.
Vitamin mixture: vitamin A, 14.1 IU/kg; vitamin E, 59
mg/kg; vitamin B-5, 22.4 mg/kg; vitamin PP, 103.3 mg/kg. Total
metabolizable energy [kcal/kg (MJ/kg)]: 3010 (12.59). Nitrate as
NaNO3, 78 mg/kg. Nitrate as NaNO2 and viable
organisms were not detected.
Manuscript received March 13, 2001. Initial review completed April 17, 2001. Revision accepted June 29, 2001.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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