Enhancement of the inducible NO synthase activation by retinoic acid is mimicked by RARalpha agonist in vivo

Carole Seguin-Devaux1, Yvan Devaux1, Véronique Latger-Cannard2, Sandrine Grosjean1,3, Cécile Rochette-Egly4, Faiez Zannad1, Claude Meistelman3, Paul-Michel Mertes1,5, and Dan Longrois1,3

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha (RARalpha ) and retinoid X receptors (RXRs) to NOS II activation triggered by LPS. Five-day supplementation with 10 mg/kg of either atRA or the RARalpha 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 RARalpha agonist (but not the RXR agonist) increased the number of peripheral T helper lymphocytes and plasma interferon-gamma 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 RARalpha protein. These results suggest that, in vivo, atRA activates NOS II through RARalpha 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha (RARalpha ). 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-gamma (IFN-gamma ) 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 (RXRalpha , -beta , and -gamma 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-gamma (i.e., lymphocytes and/or NK cells).


    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 RARalpha agonist Ro-4060-55 and the pan-RXR agonist Ro 2573-86 were provided by Dr. R. Klaus (Hoffmann-La Roche, Basel, Switzerland). Salmonella typhimurium LPS was dissolved in 0.9% NaCl at a concentration of 4 mg/ml; atRA, RARalpha , and RXR agonists were dissolved in 5% dimethyl sulfoxide (DMSO) and first cold-press olive oil at a concentration of 10 mg/ml; aliquots were stored protected from light at -20°C, and manipulations involving retinoids were conducted under reduced light conditions.

Experimental 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; RARalpha (n = 10) and RARalpha  + LPS (n = 10) groups received daily intraperitoneal injections of the RARalpha agonist (10 mg/kg) for 5 consecutive days; RXR (n = 8) and RXR + LPS (n = 8) groups received daily intraperitoneal injections of the RXR agonist (10 mg/kg) for 5 consecutive days; [RARalpha  + RXR] + LPS rats (n = 6) received daily intraperitoneally the combination of the [RARalpha  + RXR] agonists (5 mg/kg body wt each) for 5 consecutive days; and LPS (n = 9) and control (n = 4) groups received daily olive oil plus 5% DMSO for 5 days. On day 5, rats from the atRA + LPS, RARalpha  + LPS, RXR + LPS, and [RARalpha  + RXR] + LPS groups were injected intraperitoneally with LPS, and rats from the control, atRA, RARalpha , and RXR groups were injected with vehicle (0.9% NaCl solution). Six hours after LPS administration, rats were anesthetized intraperitoneally with 100 mg sodium thiopental (Nesdonal; Rhône Poulenc Rorer, Paris, France), and the thorax and abdomen were dissected. Blood samples were recovered by cardiac puncture, 4 ml were centrifuged at 600 g for 10 min, and plasma was stored at -70°C, and 2 ml were kept at room temperature and served for peripheral blood count and flow cytometry analysis. Tissue samples from liver, spleen, and lung were excised, rapidly rinsed in ice-cold saline, frozen in liquid nitrogen, and stored at -70°C until analysis. Animals were killed 6 h after the LPS injection because NOS II mRNA and protein expressions were maximal 4 and 6 h after LPS injection, respectively.

RNA Extraction and Semiquantitative RT-PCR

Semiquantitative RT-PCR was performed to estimate mRNA expression for NOS II, IFN-gamma , interferon regulatory factor (IRF)-1, and acyl-coenzyme A synthetase (ACS) in liver, spleen, and lung. These organs were chosen because of previously documented atRA-enhanced LPS-induced NOS II expression in liver and spleen but not in lung (9).

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 beta -actin and NOS II, IFN-gamma , 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 beta -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 beta -actin, and 30 cycles (94°C for 30 s, 60°C for 30 s, 72°C for 1 min) for IFN-gamma , IRF-1, ACS, and beta -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 beta -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 RARalpha [RPalpha (F)] (14), RARbeta [RPbeta (F)2] (30), and RARgamma [RPgamma (F)] (31).

Nuclear 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 RARalpha 1, -beta 2, and -gamma 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<UP><SUB>2</SUB><SUP><UP>−</UP></SUP></UP>, NO<UP><SUB><UP>3</UP></SUB><SUP><UP>−</UP></SUP></UP>, and IFN-gamma concentrations in plasma. The concentration of NO<UP><SUB>2</SUB><SUP>−</SUP></UP> and NO<UP><SUB>3</SUB><SUP>−</SUP></UP>, the stable end products of NO oxidation, was determined in plasma by the method of Green et al. (16). All samples were tested in triplicate, and concentrations were expressed in micromoles. The concentration of IFN-gamma 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.


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After 3 days of supplementation, rats treated with the RARalpha 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 RARalpha and RXR agonists resulted in only moderate hair loss and unchanged body weight.

Effect of Supplementation with RARalpha 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 beta -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 RARalpha 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 RARalpha agonist at 10 mg/kg or with [RARalpha  + 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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Fig. 1.   Effect of supplementation with the retinoic acid receptor (RAR)alpha and retinoid X receptor (RXR) agonists on inducible nitric oxide synthase (NOS II) mRNA expression in liver, spleen, and lung. Rats were treated as described in MATERIALS AND METHODS. A: representative RT-PCR profile of rat liver NOS II mRNA expression for rats injected with LPS. The NOS II PCR product was detected as a 578-bp band and that of beta -actin as a 232-bp band. B: densitometric analysis of NOS II mRNA expression in liver, spleen, and lung. Results are expressed as means ± SE. *P < 0.05 vs. Control, all-trans (at)RA, RARalpha , and RXR groups; dagger P < 0.05 vs. LPS group; Dagger P < 0.05 vs. atRA + LPS group; §P < 0.05 vs. RARalpha  + LPS group, and #P < 0.05 vs. RXR + LPS group.

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 RARalpha 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 RARalpha or [RARalpha  + 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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Fig. 2.   Effect of supplementation with the RARalpha and RXR agonists on NOS II protein expression in liver, spleen, and lung. Rats were treated as described in MATERIALS AND METHODS. A: representative Western blot analysis of rat liver NOS II protein expression for rats injected with LPS. The NOS II protein was detected as a 130-kDa double band. B: densitometric analysis of NOS II protein expression in liver, spleen, and lung. Results are expressed as means ± SE. *P < 0.05 vs. Control, atRA, RARalpha , and RXR groups; dagger P < 0.05 vs. LPS group; Dagger P < 0.05 vs. atRA + LPS group; §P < 0.05 vs. RARalpha  + LPS group; and #P < 0.05 vs. RXR + LPS group.

Effect on NO<UP><SUB>2</SUB><SUP><UP>−</UP></SUP></UP> and NO<UP><SUB><UP>3</UP></SUB><SUP><UP>−</UP></SUP></UP> concentration in plasma. Rats from the control, atRA, RARalpha , 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 RARalpha agonist at 10 mg/kg or with the combination of RARalpha 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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Fig. 3.   Effect of supplementation with the RARalpha and RXR agonists on plasma nitrate and nitrite concentration (Griess reaction). Rats were treated as described in MATERIALS AND METHODS. Results are expressed as means ± SE. *P < 0.05 vs. Control, atRA, RARalpha , and RXR groups; dagger P < 0.05 vs. LPS group; Dagger P < 0.05 vs. atRA + LPS group; §P < 0.05 vs. RARalpha  + LPS group; and #P < 0.05 vs. RXR + LPS group.

Effect of Supplementation with RARalpha and RXR Agonists on Peripheral Blood Count and T Lymphocyte Subsets

In the peripheral blood of rats from all groups, monocyte and eosinophil populations corresponded to 1-4% of total leukocytes (data not shown). In the absence of LPS, supplementation with atRA or the RARalpha agonist, but not the RXR agonist, increased the number of circulating leukocytes, neutrophils, and lymphocytes in the peripheral blood compared with the control group (Table 1), but the increase in total lymphocyte number was not statistically significant with the RARalpha agonist. LPS (with or without retinoid supplementation) significantly decreased the number of peripheral leukocytes, neutrophils, and lymphocytes in all groups compared with the control group (Table 1).

                              
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Table 1.   Effect of supplementation with RARalpha and RXR agonists on peripheral blood count

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 RARalpha 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-gamma mRNA (Fig. 5).


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Fig. 4.   Effect of supplementation with the RARalpha and RXR agonists on T and Th lymphocyte number. Rats were treated as described in MATERIALS AND METHODS. T lymphocyte number was deduced from the peripheral blood count and the percentage of CD5-positive cells obtained by flow cytometry. Th lymphocytes (CD5+/CD4+) in whole blood were double stained with monoclonal antibodies against CD5 and CD4. Flow cytometry analysis was performed on 10,000 lymphocytes. Results are expressed as absolute values (× 109/l). Results are expressed as means ± SE. *P < 0.05 vs. Control group; dagger P < 0.05 vs. atRA, RARalpha , and RXR groups.



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Fig. 5.   Effect of supplementation with the RARalpha and RXR agonists on interferon (IFN)-gamma mRNA expression in liver, spleen, and lung. Rats were treated as described in MATERIALS AND METHODS. A: representative RT-PCR profile of rat spleen IFN-gamma mRNA expression for rats injected with LPS. The IFN-gamma PCR product was detected as a 489-bp band and that of beta -actin as a 232-bp band. B: densitometric analysis of IFN-gamma mRNA expression in liver, spleen, and lung. Results are expressed as means ± SE of relative IFN-gamma /beta actin mRNA abundance. *P < 0.05 vs. Control, atRA, RARalpha , and RXR groups; dagger P < 0.05 vs. LPS group; Dagger P < 0.05 vs. atRA + LPS group; §P < 0.05 vs. RARalpha  + LPS group; and #P < 0.05 vs. RXR + LPS group.

Effect of Supplementation with RARalpha and RXR Agonists on IFN-gamma Concentration in Plasma and IFN-gamma mRNA Expression in Liver, Spleen and Lung

Effect on IFN-gamma mRNA expression. In liver and spleen, in the absence of LPS, rats from the control, atRA, RARalpha , and RXR groups had low but detectable mRNA expression of IFN-gamma . LPS injection significantly increased IFN-gamma mRNA expression (Fig. 5A). There was a nearly twofold increase in IFN-gamma mRNA relative expression in the RARalpha  + LPS and [RARalpha  + 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-gamma was low in the nine groups studied, and no difference was observed among all groups.

Effect on IFN-gamma concentration in plasma. Plasma IFN-gamma concentration was below the detection limit of the test in the control, atRA, RARalpha , and RXR groups. LPS injection resulted in significantly increased plasma concentrations of IFN-gamma (Fig. 6). Supplementation with atRA resulted in a fourfold increase in IFN-gamma plasma concentrations. Supplementation with the RARalpha agonist (RARalpha  + LPS group) significantly increased plasma IFN-gamma 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 [RARalpha  + RXR] + LPS groups exhibited IFN-gamma plasma concentrations similar to those in the LPS group.


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Fig. 6.   Effect of supplementation with the RARalpha and RXR agonists on plasma IFN-gamma concentration in LPS-stimulated rats. Rats were treated as described in MATERIALS AND METHODS. Plasma IFN-gamma concentration was below the detection limit of the test in Control, atRA, RARalpha , and RXRs rats. Results are expressed as means ± SE. *P < 0.05 vs. Control, atRA, RARalpha , and RXR groups; dagger P < 0.05 vs. LPS group; Dagger P < 0.05 vs. atRA + LPS group; §P < 0.05 vs. RARalpha  + LPS group; and #P < 0.05 vs. RXR + LPS group.

Effect of Supplementation with RARalpha and RXR Agonists on IRF-1 mRNA Expression in Liver, Spleen, and Lung

In liver, IRF-1 mRNA expression was significantly higher in the atRA, RARalpha , RXR, and LPS groups compared with the control group (Fig. 7A). After LPS injection, in rats supplemented with the RARalpha or the RARalpha  + RXR agonists or atRA, there was a significantly higher increase of IRF-1 mRNA expression compared with LPS rats. In rats from the RXR + LPS group, IRF-1 mRNA expression was not statistically different from the LPS group. In spleen, LPS injection resulted in a significant increase in IRF-1 mRNA in rats from the LPS group compared with rats from the control, RARalpha , and RXR groups. Rats of the atRA + LPS, RARalpha  + LPS, and [RARalpha  + RXR] + LPS groups exhibited a further significant but moderate increase in IRF-1 mRNA expression compared with the LPS group (Fig. 7B). In lung, a significant increase in IRF-1 mRNA was observed after LPS treatment, but no differences in IRF-1 mRNA relative expression were observed among all the groups receiving LPS.


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Fig. 7.   Effect of supplementation with the RARalpha and RXR agonists on interferon regulatory factor (IRF)-1 mRNA expression in liver, spleen, and lung. Rats were treated as described in MATERIALS AND METHODS. A: representative RT-PCR profile of rat liver IRF-1 mRNA expression. The IRF-1 PCR product was detected as a 523-bp band and that of beta -actin as a 232-bp band. B: densitometric analysis of IRF-1 mRNA expression in liver, spleen, and lung. Results are expressed as means ± SE of relative IRF-1/beta actin mRNA abundance. *P < 0.05 vs. Control group; dagger P < 0.05 vs. atRA, RARalpha , and RXR groups; Dagger P < 0.05 vs. LPS group; §P < 0.05 vs. atRA + LPS group; #P < 0.05 vs. RARalpha  + LPS group; and $P < 0.05 vs. RXR + LPS group.

Effect of Supplementation with RARalpha and RXR Agonists on Expression Level of RAR Proteins in Liver, Spleen, and Lung

Immunoblotting of whole cell extracts made from COS-1 cells transfected with mouse RARalpha 1, -beta 2, and -gamma 1 expression vectors, respectively, with RARalpha -, -beta -, and -gamma -specific antibodies revealed a single band at 51 kDa. By use of this technique, RARalpha and RARbeta proteins could be detected in liver extracts and at a low level in lung extracts (Fig. 8A). However, these two receptors were undetectable in spleen (Fig. 8B). RARgamma was undetectable in the three organs. Figure 8C shows the effect of the different treatments on the expression of RARalpha and RARbeta in liver. Compared with the control group, only the atRA- and LPS-treated groups depicted a significant decrease in RARalpha protein expression, and RARbeta was not affected. No biologically consistent pattern (related to different retinoids and/or LPS administration) of changes in RARalpha or RARbeta protein expression was observed among the other groups.


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Fig. 8.   Effect of supplementation with the RARalpha and RXR agonists on RARalpha and RARbeta proteins expression in liver, spleen, and lung. Rats were treated as described in MATERIALS AND METHODS. A: representative Western blot analysis of RARalpha and -beta protein expression in liver, spleen, and lung for 1 rat of Control group. B: densitometric analysis of RARalpha and -beta protein expression in liver, spleen, and lung for the Control group. C: densitometric analysis of RARalpha and -beta protein expression in liver. Results are expressed as means ± SE. *P < 0.05 vs. all groups except LPS group; dagger P < 0.05 vs. RARalpha group.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The major findings of this study are that 1) the RARalpha agonist, but not the pan-RXR agonist, mimics the effects of atRA on the LPS-induced NOS II pathway activation; 2) the increased IFN-gamma mRNA expression in peripheral organs is probably related to the atRA and RARalpha agonist-mediated increase of circulating CD4+ T lymphocyte number and subsequent organ infiltration upon stimulation with LPS; 3) the ability of atRA or the RARalpha 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 RARalpha protein.

The rat models of vitamin A supplementation and "low-dose" LPS stimulation have been previously characterized and discussed (5, 9). The doses of RARalpha 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 RARalpha 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).

RARalpha Agonist, but Not Pan-RXR Agonist, Mimics Effects of atRA on LPS-Induced NOS II Pathway Activation

The results presented here confirm our previous observations that atRA enhanced the LPS-induced NOS II activation. Here, we bring convincing evidence that the effects of atRA are mimicked by the RARalpha agonist. Indeed, the RARalpha agonist, but not the pan-RXR agonist, could mimic (although not entirely) the effects of atRA. RXRs are able to heterodimerize not only with RARs but also with other members of the steroid/thyroid hormone receptor superfamily, including peroxisome proliferator-activated receptor (PPAR), thyroid hormone receptor, and vitamin D3 receptor (28). The pan-RXR agonist used in this study (Ro-25-7386) activates RXR/PPAR heterodimers. Indeed, this compound was able to increase ACS mRNA in liver of rats from the RXR and RXR + LPS groups compared with the control and LPS groups, respectively (data not shown), as it was previously reported for other RXR ligands (25). However, despite the fact that the compound Ro-25-7386 is biologically active in this model, no change in NOS II expression and activity was observed compared with the LPS group. These results suggest no role for RXR as homodimers or as heterodimers with nuclear receptors other than RAR on NOS II pathway activation. These observations are consistent with in vivo and in vitro reports demonstrating that the RXR agonist alone cannot mimic the biological effects of the RAR agonist (38).

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 RARalpha -RXR heterodimer. Our findings confirm the results of Sirsjo et al. (34), showing that, in vitro, atRA modulates the interleukin-1beta -induced NOS II mRNA expression through RARalpha , 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-gamma mRNA Expression in Peripheral Organs Is Probably Related to atRA and RARalpha Agonist-Mediated Increase of Circulating CD4+ T Lymphocyte Number and Subsequent Organ Infiltration Upon Stimulation with LPS

We have previously shown that atRA-mediated enhancement of LPS-induced NOS II expression was associated with increased IFN-gamma plasma concentration and mRNA expression in several organs (consistent with lymphocyte infiltration) as well as IRF-1 mRNA expression (9). In the present study, we show that the effects of atRA on IFN-gamma mRNA expression are mimicked by the RARalpha agonist. Nevertheless, the combination of RARalpha and pan-RXR agonists could not entirely mimic the effects of atRA on plasma IFN-gamma concentration. This is consistent with experiments with cultured cells where none of the various combinations of RAR-RXR-selective agonists could mimic all the effects induced by atRA (32).

Because only T lymphocytes and NK cells produce IFN-gamma , 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 RARalpha 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 RARalpha 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-gamma mRNA (Fig. 5).

Ability of atRA or RARalpha Agonist to Increase NOS II mRNA and Protein Expression in a Given Organ After LPS Stimulation Coincided with Increased IRF-1 mRNA Expression

An important question raised by our previous results (9) concerned the mechanisms that underlie the organ-specific synergistic effect of atRA and LPS to enhance NOS II activation. Expression of NOS II mRNA after LPS stimulation was enhanced by supplementation with atRA or the RARalpha or [RARalpha  + RXR] agonists, but not with the pan-RXR agonist, only in organs where there was an increase in IRF-1 mRNA expression after LPS administration (Fig. 7). Although this could be purely coincidental, it could explain the organ-specific effect of retinoid supplementation, given the documented direct effect of retinoids on IRF-1 mRNA expression (26). IRF-1 is a transcription factor known to be necessary for NOS II gene expression even when other transcription factors are present (20). Among other situations, IRF-1 is induced upon stimulation with IFN-gamma , a cytokine shown to enhance the activation of NOS II induced by other proinflammatory stimuli and to induce organ dysfunction (2, 3, 40, 41).

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-gamma , and 2) this effect is mimicked by the RARalpha agonist.

Level of NOS II mRNA and Protein Expression Within a Given Organ Is Not Related to Expression Level of RARalpha Protein

The RAR and RXR subtypes are expressed in a cell type-specific pattern during embryogenesis in the adult organism and in malignant tissue, suggesting that the sensitivity to retinoids of a given tissue is defined, in part, through the expression pattern of RAR/RXR subtypes (15). RAR transcripts are present in many adult and embryonic tissues. RARalpha has been found to be the major transcript in all tissues, whereas RARbeta is restricted to fewer tissues and RARgamma to the skin (21). We investigated the expression of RARalpha , -beta , and -gamma proteins, which are the functional molecules for regulation of gene transcription rather than the mRNAs. Western blot analysis revealed that both RARalpha and RARbeta proteins were constitutively expressed in liver and not in spleen. RARalpha and RARbeta proteins were detectable but expressed at a low level in lung.

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 RARalpha or RARbeta protein expression, which is in contrast with in vitro studies (18, 21); 2) in the presence or absence of retinoid supplementation, RARalpha 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 RARalpha agonist. This observation confirms the involvement of RARalpha 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-gamma -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.


    ACKNOWLEDGEMENTS

The technical assistance of Chantal David and Viviane Camaeti is gratefully acknowledged.


    FOOTNOTES

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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Am J Physiol Endocrinol Metab 283(3):E525-E535
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