1 Unité Propre de Recherche et d'Enseignement Supérieur-Equipe d'Accueil 3447 Lésions-Réparation: Remodelage Cardiaque et Artériel, Faculté de Médecine, Université Henri Poincaré, Nancy I; 2 Département d'Hématologie Biologique, and 3 Département d'Anesthésie-Réanimation Chirurgicale, Centre Hospitalier Universitaire Brabois, 54500 Vandoeuvre-les-Nancy; 4 Institut de Génétique et de Biologie Moléculaire et Cellulaire, 67404 Illkirch; and 5 Département d'Anesthésie-Réanimation Chirurgicale, Centre Hospitalier Universitaire Hôpital Central, 54000 Nancy, France
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ABSTRACT |
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We have previously shown that
all-trans retinoic acid (atRA), the active metabolite of
vitamin A, enhances the activation of the inducible nitric oxide
synthase (NOS II) pathway, a component of innate immunity, in rats in
vivo. We investigated the relative contribution of retinoic acid
receptor- (RAR
) and retinoid X receptors (RXRs) to NOS II
activation triggered by LPS. Five-day supplementation with 10 mg/kg of
either atRA or the RAR
selective agonist Ro-40-6055, but not with 10 mg/kg of the pan-RXR agonist Ro-25-7386, enhanced the LPS-induced NOS
II mRNA, protein expression in liver, and plasma nitrite/nitrate
concentration. Both atRA and the RAR
agonist (but not the RXR
agonist) increased the number of peripheral T helper lymphocytes and
plasma interferon-
concentration. Synergism between retinoids and
LPS on NOS II activation within an organ coincided with synergism on
interferon regulatory factor-1 mRNA expression but not with the level
of expression of the RAR
protein. These results suggest that, in
vivo, atRA activates NOS II through RAR
and contributes to
characterizing the complex effect of retinoids on the host
inflammatory/immune response.
synthetic retinoids; inducible nitric oxide synthase; T helper lymphocyte; interferon type II; interferon regulatory factor-1
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INTRODUCTION |
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VITAMIN A DEFICIENCY has been associated with increased incidence of infection, which gained its reputation as an "anti-infective" vitamin. Nevertheless, the beneficial anti-infective effects of vitamin A supplementation are controversial (11, 36, 42). Vitamin A and its active metabolite all-trans retinoic acid (atRA) modulate host susceptibility to infection by interfering with both innate and adaptive immune as well as host inflammatory responses, but the detailed cellular and molecular mechanisms are poorly documented (33). In studies performed mainly in vitro, it was shown that vitamin A and related retinoids enhance proliferation and/or activation of neutrophils, monocytes/macrophages, dendritic cells, and T (both CD4 and CD8) and B lymphocytes (33). Vitamin A has also been shown to modulate the biological pathways involved in the host inflammatory response, such as the pathways of phospholipase A2 (19), cyclooxygenases (10), and, more recently, inducible nitric oxide synthase (NOS II), an important component of the innate immune response to pathogens (6).
Initial studies performed in vitro demonstrated that atRA attenuates
LPS or cytokine-induced activation of the NOS II pathway in several
cell types (8, 17, 27). Using cultured rat vascular smooth
muscle cells overexpressing a reporter gene under the control of the
murine NOS II gene promoter, Sirsjo et al. (34)
demonstrated that atRA decreased reporter gene expression via retinoic
acid receptor- (RAR
). However, we (9, 10) have
recently demonstrated that, in vivo, atRA supplementation before
challenge with LPS enhanced LPS-induced NOS II pathway expression and
activity. Several hypotheses could explain the differences between in
vitro and in vivo results. They include 1) organ- or
cell-specific expression of retinoid receptors, 2) organ- or
cell-specific metabolism of retinoids, and 3) the presence
in vivo of inflammatory/immune cell types not present in simpler in
vitro studies. This last hypothesis is based on our recent results
(9, 10) that showed that, in vivo, atRA-mediated
enhancement of LPS-induced NOS II activation in the liver was
associated with increased interferon-
(IFN-
) mRNA expression in
the liver and plasma concentration, an observation consistent with the
involvement of lymphocytes and/or natural killer (NK) cells.
The biological effects of atRA are mediated by two families of nuclear
receptors, retinoic acid receptors (RARs) and retinoid X receptors
(RXR, -
, and -
and their distinct isoforms) (7). All of these receptors belong to the steroid/thyroid receptor superfamily and work as ligand-dependent transcription factors. RARs
bind both atRA and 9-cis-RA with high affinity, whereas RXRs bind only 9-cis-RA (28). RARs heterodimerize
with RXRs, and RAR/RXR heterodimers bind to the RA response element
(RARE) present on the promoter of target genes to induce gene
expression. In addition, retinoid receptors can interact with other
transcription factors by direct protein-protein interaction and
modulate the expression of genes with promoters that lack RARE.
Synthetic retinoids have been developed and characterized in the
context of cancer chemoprevention (24). They possess
reduced toxicity compared with natural retinoids with preserved or
enhanced activity on their cognate receptors. Selective RAR or
RXR subtype ligands allow assessment of the relative contribution of
distinct receptors to the biological effects of retinoids
(38).
In an attempt to understand some of the causes of the discordant in
vitro vs. in vivo effects of retinoids on NOS II activation, the
present study was designed to 1) investigate which retinoid receptor subtypes are involved in the atRA-mediated enhancement of
LPS-triggered NOS II induction in vivo and test the hypothesis that
organ-specific expression of NOS II could be related to the level of
expression of retinoid receptors, and 2) investigate a
potential effect of atRA or specific retinoid receptor agonists on the
cellular types known to produce IFN- (i.e., lymphocytes and/or NK cells).
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MATERIALS AND METHODS |
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Animals
Male Wistar-Kyoto rats (250-350 g) were housed and treated in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (7th ed., Washington, DC: National Academy Press, 1996, aka National Research Council Guide).Chemicals and Reagents
All compounds used were purchased from Sigma (Saint Quentin Fallavier, France) unless specified otherwise. The RARExperimental Protocol
Rats were randomly divided into nine groups: atRA (n = 4) and atRA + LPS (n = 10) groups received daily intraperitoneal injections of atRA at 10 mg/kg body wt for 5 consecutive days; RARRNA Extraction and Semiquantitative RT-PCR
Semiquantitative RT-PCR was performed to estimate mRNA expression for NOS II, IFN-Extraction of total RNA. Total RNA was extracted from the different samples using Tri-Reagent (Euromedex, Souffelweyersheim, France). Total RNA concentration was measured in triplicate before and after dilution to ~1 µg/µl by spectrophotometric analysis at 260 nm. RNA purity was determined by the ratio A260/A280 (all samples between 1.6 and 2), and its integrity was confirmed by the existence of clear bands for 18S and 28S RNA after electrophoresis through a 0.8% agarose gel.
Reverse transcription.
To eliminate contaminating DNA, RNA samples were first digested
by DNAse I before reverse transcription. Thus 5 µg of total RNA were
mixed with 5 mM MgCl2, 1× PCR buffer II, 1 mM
dNTP, 2.5 µM oligo(dT)16, 1 U/µl RNasine (GeneAmp RNA
PCR kit; PE Applied Biosystems, Courtaboeuf, France), and 0.25 U/µl
DNase I (Pharmacia Biotech, Orsay, France) to a final volume of 20 µl
of diethyl pyrocarbonate-treated water and incubated for 30 min at
37°C followed by 5 min at 75°C. After 5 min on ice, the RNA was
mixed with 2.5 U/µl murine leukemia virus reverse transcriptase
(MuLV-RT; PE Applied Biosystems) and incubated for 45 min at 42°C
followed by 5 min at 90°C for denaturation of MuLV-RT. The cDNA
samples were stored at 20°C.
PCR.
The duplex PCR was performed using primers for the housekeeping gene
-actin and NOS II, IFN-
, and IRF-1, as previously described (9). Additionally, duplex PCR of rat ACS in the liver was
performed using the amplimers 5'-TCACACACTGGGAGCAGAAG-3' (sense) and
5'-TTGGAAGAACCCAATTTTGC-3' (antisense), giving a PCR product of 502 bp.
PCR was performed in a DNA thermal cycler (Bio-Rad Laboratories,
Ivry-sur-Seine, France) with the use of 2 µl of cDNA sample in a
total reaction volume of 10 µl with 1 µM of each primer, 1× PCR
buffer II, 1 mM dNTP, 1.0 mM MgCl2, and 0.05 U/µl Ampli Taq DNA polymerase (Gene Amp RNA PCR kit; PE Applied
Biosystems). The PCR reactions for the gene of interest and
-actin
were performed in the same tube with both sets of primers. The PCR
conditions were as follows: denaturation at 94°C for 5 min,
amplification for 30 cycles (94°C for 30 s, 58°C for 30 s, 72°C for 1 min) for NOS II and
-actin, and 30 cycles (94°C
for 30 s, 60°C for 30 s, 72°C for 1 min) for IFN-
,
IRF-1, ACS, and
-actin. A final extension was performed at 72°C
for 10 min. A Gene Ruler 100-bp DNA Ladder Plus (Euromedex) was used to
determine the size of the PCR products. Preliminary experiments were
performed to document that the PCR was completed during the exponential
phase of amplification and that the amplification was linear. The
identity of the PCR product was confirmed by sequencing with the use of
the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit and
the ABI PRISM 310 (PE Applied Biosystems).
Densitometric analysis of PCR products.
The PCR products were separated on a 2% agarose gel containing 0.5 µg/ml ethidium bromide and viewed using UV light on a
transilluminator. Densitometry of the resulting bands was performed
with a Bio-Rad Gel Doc 1000 (Bio-Rad Laboratories). Results were
expressed as the ratio of the optical density of the band of the PCR
product of interest to that of -actin.
Western Blot Analysis of NOS II and RAR Proteins
The following specific rabbit polyclonal antibodies were used: anti-murine NOS II (Transduction Laboratories) and anti-human RARNuclear extract preparation.
Tissue samples were homogenized on ice in 2 vol of buffer
A (20 mM Tris · HCl, pH 8.0, 1 mM MgCl2, 20 mM
KCl, 1 mM DTT, 0.3 mM PMSF, 1 µM leupeptin, 100 U/ml aprotinin) by
use of a glass Dounce B homogenizer. The homogenates were then
centrifuged for 5 min at 1,500 g, and the crude nuclear
pellet was washed twice and resuspended in high-salt buffer
B (same composition as buffer A but with 0.6 M KCl and
25% glycerol). Extraction of nuclear proteins was performed on ice
during 45 min under gentle agitation. After centrifugation for 1 h
at 105,000 g, the supernatant was dialyzed for 2 h at
4°C with 50 mM Tris · HCl, pH 8.0, with the use of Cellu Sep
H1 membranes (Interchim, Montluçon, France). Protein
concentration was determined by the method of Lowry, and aliquots were
stored at 70°C.
Immunoblotting of NOS II and RAR proteins.
Immunodetection of NOS II with the 5-bromo-4-chloro-3-indolyl
phosphate and nitroblue tetrazolium method was performed as previously
described (9). For RAR immunodetection, 80 µg of proteins of nuclear extracts from each tissue sample were fractionated by SDS-polyacrylamide gel electrophoresis (10-4%) and
electrotransferred to polyvinylidene difluoride membranes for 4 h
at 4°C as described (9). The equal amounts of tissue
proteins transferred were confirmed by staining the membrane with a
Ponceau red solution. The membranes were blocked overnight with 3%
blocking agent (Amersham Pharmacia Biotech) in TBST solution [25 mM
Tris, pH 7.5, 150 mM NaCl, 0.05% (vol/vol) Tween 20]. After two
washings with TBST, blots were incubated for 2 h at room
temperature under gentle agitation with 1:1,000 dilutions of each
antibody. After extensive washings with TBST, the membranes were
incubated for 30 min with peroxidase-conjugated protein A diluted
1:10,000 (Amersham Pharmacia Biotech). Membranes were washed in TBST
solution, and the immunocomplexes were revealed using an ECL Western
blotting analysis system (Amersham Pharmacia Biotech). Whole extracts
from COS-1 cells transfected with mouse RAR1, -
2, and -
1
expression vectors were used as positive controls for detection of RAR
proteins (14, 30, 31). High-range prestained
SDS-polyacrylamide gel electrophoresis standards (Bio-Rad) were used
for molecular mass determination. Densitometry of the resulting bands
was performed using a Bio-Rad GS-690 imaging densitometer.
Measurement of NO concentrations in
plasma.
The concentration of NO
was determined by ELISA, as previously
described (9). Tenfold plasma dilutions were performed,
all samples were tested in duplicate, and concentrations were expressed
in nanograms per milliliter.
Analysis of Blood Lymphocytes Subsets by Flow Cytometry
Peripheral blood count was assessed using a hematological counter (H2 Technicon, Bayer, France).Fluorescence labeling.
Peripheral blood cells were double stained with MAb directly
conjugated to either fluorescein isothiocyanate (FITC) or phycoerythrin (PE). This assay was performed in 100-µl samples of whole blood with
10 µl of MAb solution for 30 min at room temperature and in the dark.
Two-color staining was performed to determine the number of T helper
(Th) lymphocytes (CD5+/CD4+) and NK cells
(CD5/CD161+). All MAb were purchased from
Serotec (Oxford, UK): CD5 MAb (clone OX-19), CD4 MAb (clone W3/25), and
CD161 MAb (clone 10/78). The specificity of these MAbs has been
proved elsewhere (12, 22, 39). The MAb IgG1-FITC and
IgG1-PE (clone F8-11-13, Serotec) served as isotypic
controls. Red blood cells were then lysed with IOTest 3 lysing solution
(Beckman Coulter, Marseille, France) for 10 min at room temperature.
The samples were washed, centrifuged at 300 g for 5 min, and
suspended in CellWash (Becton-Dickinson, Meylan, France).
Flow cytometry analysis. Samples were analyzed on a FACScalibur flow cytometer (Becton-Dickinson) equipped with a 488-nm argon laser and the serial filter configuration. List mod data were acquired and analyzed with CellQuest software. The instrument's optical alignment, laser output, and photomultiplier settings were checked each day by means of calibration beads (Calibrite; Becton-Dickinson). The lymphocytes were identified by their low forward and side scatters and gated. Flow cytometry analysis was performed on 10,000 events. Results are expressed as absolute values (× 109/l).
Statistical Analysis
Statistical analysis was performed using the StatView IV software (Abacus Concepts, Berkeley, CA). Results are expressed as means ± SE. Comparisons among several groups were performed with nonparametric analysis of variance (Kruskall-Wallis test). Comparisons between two groups were performed with the Mann-Whitney test. A P value of <0.05 was considered statistically significant. ![]() |
RESULTS |
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After 3 days of supplementation, rats treated with the RAR
agonist had marked signs of hypervitaminosis A such as decreased food
intake, weight loss (10% body wt after 5 days of supplementation), hair loss, and diarrhea, whereas there were no signs for rats treated
with the pan-RXR agonist or atRA, thus confirming previous results
(9). Supplementation with the combination of RAR
and RXR agonists resulted in only moderate hair loss and unchanged body weight.
Effect of Supplementation with RAR and RXR Agonists on NOS II
Expression in Liver, Spleen and Lung
Effect on NOS II mRNA expression.
In all organs studied, primers specific for -actin yielded a single
band at 232 bp of equivalent intensity among all rats (Fig.
1A). In the absence of LPS,
NOS II mRNA expression could not be detected. After LPS injection,
there was reproducible induction of NOS II mRNA expression. In liver,
as previously reported (9), atRA enhanced the LPS-induced
NOS II mRNA expression (Fig. 1B). This effect was mimicked
by the RAR
agonist but not by the pan-RXR agonist (Fig.
1A). In spleen, atRA supplementation resulted in a
significant increase in NOS II mRNA expression in LPS-injected rats.
Supplementation with the RAR
agonist at 10 mg/kg or with [RAR
+ RXR] agonists at 5 mg/kg each resulted in a moderate
but not significant increase in NOS II mRNA expression compared with the LPS group. Rats from the RXR + LPS group had NOS II mRNA
expression similar to that of the LPS group. In lung, there were no
differences in NOS II mRNA expression among the groups challenged with
LPS (Fig. 1B).
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Effect on NOS II protein expression.
In all organs studied, in the absence of LPS there was no
detectable NOS II protein. In all LPS-treated rats, the NOS II-specific antibody revealed a double 130-kDa band (Fig.
2A). In the liver, densitometric analysis revealed a significantly higher expression of
NOS II protein in the atRA + LPS group compared with the LPS group
(Fig. 2B). This effect was mimicked by the RAR agonist but not by the RXR agonist. In spleen, supplementation with atRA enhanced significantly the LPS-induced NOS II protein expression compared with unsupplemented LPS-stimulated rats. Supplementation with
the RAR
or [RAR
+ RXR] agonists also enhanced the
LPS-induced NOS II protein expression, but this increase was not
statistically significant compared with the LPS group. In lung, there
were no differences in NOS II protein expression among the groups
treated with LPS (Fig. 2B).
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Effect on NO, and RXR groups had low but
detectable plasma concentrations of nitrate and nitrite, and no
differences were observed among the four groups (Fig.
3). Rats from the LPS group had
significantly higher plasma nitrate and nitrite concentrations than the
control group (P < 0.01). In rats supplemented with
atRA or the RAR
agonist at 10 mg/kg or with the combination of
RAR
and RXR agonists at 5 mg/kg each, LPS injection resulted in a
threefold increase in plasma nitrate and nitrite concentration compared
with unsupplemented rats. In contrast, rats of the RXR + LPS and
LPS groups had similar plasma nitrate and nitrite concentration.
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Effect of Supplementation with RAR and RXR Agonists on
Peripheral Blood Count and T Lymphocyte Subsets
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T lymphocyte (CD5+) and Th lymphocyte (CD5+/CD4+) numbers were deduced from the number of total lymphocytes obtained from peripheral blood count and the percentage of CD5-positive cells and CD5/CD4-double-positive cells, respectively. T lymphocytes were the main lymphocyte population in the peripheral blood of all rats, ranging from ~50 to 70% of total lymphocytes (data not shown). Only a minority of T lymphocytes (between 2 and 3%) carried out the interleukin-2 receptor activation marker CD25, and this was not modified in any group (data not shown).
Compared with the control group, the number of T and Th lymphocytes was
increased by the RAR agonist (magnitude of the effect comparable
with atRA-supplemented rats) but not by the RXR agonist (Fig.
4). All LPS-injected rats had a decreased
number of circulating T and Th lymphocytes compared with the non-LPS
groups. The decrease in circulating Th lymphocytes is consistent with
infiltration of these lymphocytes in the three organs, as attested by
the increased expression of IFN-
mRNA (Fig.
5).
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Effect of Supplementation with RAR and RXR Agonists on IFN-
Concentration in Plasma and IFN-
mRNA Expression in Liver, Spleen
and Lung
Effect on IFN- mRNA expression.
In liver and spleen, in the absence of LPS, rats from the control,
atRA, RAR
, and RXR groups had low but detectable mRNA expression of
IFN-
. LPS injection significantly increased IFN-
mRNA expression
(Fig. 5A). There was a nearly twofold increase in IFN-
mRNA relative expression in the RAR
+ LPS and [RAR
+ RXR] + LPS groups, just as in the atRA + LPS group, compared with
the LPS group, whereas the RXR + LPS group was not statistically different from the LPS group (Fig. 5B). In lung, the level
of expression of IFN-
was low in the nine groups studied, and no difference was observed among all groups.
Effect on IFN- concentration in plasma.
Plasma IFN-
concentration was below the detection limit of the test
in the control, atRA, RAR
, and RXR groups. LPS injection resulted in
significantly increased plasma concentrations of IFN-
(Fig.
6). Supplementation with atRA resulted in
a fourfold increase in IFN-
plasma concentrations. Supplementation
with the RAR
agonist (RAR
+ LPS group) significantly
increased plasma IFN-
concentration compared with the LPS group, but
this concentration was significantly lower compared with the atRA + LPS group (P < 0.05). Rats of the RXR + LPS and
[RAR
+ RXR] + LPS groups exhibited IFN-
plasma
concentrations similar to those in the LPS group.
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Effect of Supplementation with RAR and RXR Agonists on IRF-1
mRNA Expression in Liver, Spleen, and Lung
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Effect of Supplementation with RAR and RXR Agonists on
Expression Level of RAR Proteins in Liver, Spleen, and Lung
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DISCUSSION |
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The major findings of this study are that 1) the
RAR agonist, but not the pan-RXR agonist, mimics the effects of atRA
on the LPS-induced NOS II pathway activation; 2) the
increased IFN-
mRNA expression in peripheral organs is probably
related to the atRA and RAR
agonist-mediated increase of circulating
CD4+ T lymphocyte number and subsequent organ infiltration
upon stimulation with LPS; 3) the ability of atRA or the
RAR
agonist to increase NOS II mRNA and protein expression in a
given organ after stimulation with LPS coincided with increased
IRF-1 mRNA expression; and 4) the level of NOS II mRNA and
protein expression within a given organ is not related to the level of
expression of the RAR
protein.
The rat models of vitamin A supplementation and "low-dose" LPS
stimulation have been previously characterized and discussed (5,
9). The doses of RAR and RXR agonists were arbitrarily chosen
at 10 mg/kg to be compared with the biological effects of atRA at 10 mg/kg in LPS-stimulated rats in vivo. Rats injected with the RAR
agonist exhibited marked signs of hypervitaminosis, whereas rats
treated with the RXR agonist and atRA had no signs of toxicity. This
finding is in agreement with reports concerning the toxicity of
receptor-selective synthetic retinoids as cancer-chemopreventive agents
(24).
RAR Agonist, but Not Pan-RXR Agonist, Mimics Effects of atRA on
LPS-Induced NOS II Pathway Activation
Because RARs require heterodimerization with RXRs for efficient
DNA binding and gene regulation (29), our results suggest that the enhancement of LPS-induced NOS II pathway activation by atRA
is mediated in vivo through an RAR-RXR heterodimer. Our findings
confirm the results of Sirsjo et al. (34), showing that,
in vitro, atRA modulates the interleukin-1
-induced NOS II mRNA
expression through RAR
, and the results of Austenaa and Ross
(1). Nevertheless, the in vitro and in vivo effects are qualitatively different. These differences could be related to the
delay of exposure to retinoids (1). Another possible
explanation could be the presence in vivo of cell types able to amplify
the inflammatory/immune response, a hypothesis investigated in the present study.
Increased IFN- mRNA Expression in Peripheral Organs Is Probably
Related to atRA and RAR
Agonist-Mediated Increase of Circulating
CD4+ T Lymphocyte Number and Subsequent
Organ Infiltration Upon Stimulation with LPS
Because only T lymphocytes and NK cells produce IFN-, we
investigated the effects of atRA and the different agonists on these two cell types. Supplementation with retinoids had no effect on NK cell
number (data not shown). However, supplementation with atRA or the
RAR
agonist, but not with the pan-RXR agonist, in the absence of LPS
increased the number of circulating T and Th lymphocytes
(CD5+/CD4+) compared with the control group.
These results are in partial agreement with previous reports (13,
18, 43). The results of this study cannot elucidate the
mechanism(s) responsible for the increased number of peripheral T and
Th lymphocytes in atRA or RAR
agonist-supplemented rats; this could
be the result of increased proliferation in the absence of an antigenic
stimulus (23) or, more likely, related to inhibition of
lymphocyte apoptosis, as suggested by Szondy et al.
(37). After the LPS challenge, there was a decrease in
circulating T and Th lymphocyte counts in both supplemented and
nonsupplemented rats, consistent with lymphocyte infiltration in the
different organs as attested by increased expression of IFN-
mRNA
(Fig. 5).
Ability of atRA or RAR Agonist to Increase NOS II mRNA and
Protein Expression in a Given Organ After LPS Stimulation Coincided
with Increased IRF-1 mRNA Expression
Although purely speculative for the moment, these results are
consistent with the hypothesis that 1) retinoids could
enhance LPS-induced NOS II activation by increasing IRF-1 expression
directly (26) and/or upon activation in the presence of
IFN-, and 2) this effect is mimicked by the RAR
agonist.
Level of NOS II mRNA and Protein Expression Within a Given Organ Is
Not Related to Expression Level of RAR Protein
We explored the hypothesis that supplementation with retinoids before
challenge with LPS could change the level of expression of RAR proteins
in an organ-specific pattern and that this could explain the
organ-specific enhancement of NOS II expression after LPS. Our results
do not support this hypothesis, because 1) supplementation with retinoids modestly decreased or did not affect RAR or RAR
protein expression, which is in contrast with in vitro studies (18, 21); 2) in the presence or absence of
retinoid supplementation, RAR
protein expression was low in both
spleen and lung, but in supplemented rats, LPS injection enhanced NOS
II expression in spleen but not in lung. Taken together, these results
suggest that retinoid-mediated enhancement of NOS II upon challenge
with LPS is not related to the level of expression of the different retinoid receptors.
In summary, these results demonstrate for the first time in vivo that
the atRA-mediated enhancement of NOS II pathway activation is mimicked
by an RAR agonist. This observation confirms the involvement of
RAR
in the modulation of the NOS II pathway (34) but
provides additional evidence that the in vivo and in vitro effects are
opposite. A plausible hypothesis to explain this difference is that, in
vivo, retinoids activate IFN-
-producing cells, most probably Th
lymphocytes, and amplify the Th1 phenotype induced by LPS in target
organs. Our results also demonstrate that, in vivo, supplementation
with retinoids has organ-specific effects through complex mechanisms
still to be defined.
Taken together, our results contribute to the characterization of the complex immunomodulatory effects of vitamin A supplementation and, by showing organ-specific effects, could help in understanding why clinical trials of vitamin A supplementation can have beneficial (42), neutral (4, 11), or deleterious effects (35, 36) on the infected host.
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ACKNOWLEDGEMENTS |
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The technical assistance of Chantal David and Viviane Camaeti is gratefully acknowledged.
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FOOTNOTES |
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C. Seguin-Devaux and Y. Devaux were supported by a grant from the Association de Recherche et d'Information Scientifique en Cardiologie.
Address for reprint requests and other correspondence: D. Longrois, Département d'Anesthésie-Réanimation Chirurgicale, CHU Brabois, Rue du Morvan, 54500 Vandoeuvre-les-Nancy, France (E-mail: d.longrois{at}chu-nancy.fr).
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.
May 21, 2002;10.1152/ajpendo.00008.2002
Received 9 January 2002; accepted in final form 8 May 2002.
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