Retinoic acid and host-pathogen interactions: effects on inducible nitric oxide synthase in vivo

Yvan Devaux1, Sandrine Grosjean1,2, Carole Seguin1, Chantal David3, Brigitte Dousset4, Faiez Zannad1, Claude Meistelman2, Nicole De Talancé3, Paul-Michel Mertes1,3, and Dan Ungureanu-Longrois1,2

1 Laboratory of Experimental Medicine and Surgery, Faculté de Médecine, 54505 Vandoeuvre; 2 Department of Anesthesia and Intensive Care, 3 Laboratory of Cellular Biology, Centre Hospitalier Universitaire de Nancy, 54511 Vandoeuvre; and 4 Laboratory of Biochemistry, Centre Hospitalier Universitaire de Nancy, Hôpital Central, 54035 Nancy, France


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Vitamin A and its metabolite retinoic acid modulate the host response to pathogens through poorly characterized mechanisms. In vitro studies have suggested that retinoic acid decreases inducible NO synthase (NOS2, or iNOS) expression, a component of innate immunity, in several cell types stimulated with lipopolysaccharide (LPS) or cytokines. This study investigated the effect of retinoic acid on LPS-stimulated NOS2 expression in vivo. Wistar-Kyoto rats received all-trans retinoic acid (RA, 10 mg/kg) or vehicle intraperitoneally daily for 5 days followed by LPS (4 mg/kg) or saline intraperitoneally and were killed 6 h later. NOS2 activation was estimated by mRNA (RT-PCR) and protein (Western-blot) expression and plasma nitrate/nitrite accumulation. In sharp contrast to previous in vitro study reports, RA significantly enhanced NOS2 mRNA, protein expression, and plasma nitrate/nitrite concentration in LPS-injected rats but not in saline-injected rats. This was associated with increased expression of interleukin-2, interferon (IFN)-gamma and IFN regulatory factor-1 mRNAs in several organs and increased IFN-gamma plasma concentration. RA significantly increased mortality in LPS-injected rats. The NOS inhibitor aminoguanidine (50 mg/kg before LPS injection) significantly attenuated the RA-mediated increase in mortality. These results demonstrate for the first time that RA supplementation in vivo enhances activation of the LPS-triggered NOS2 pathway.

nitric oxide; retinoids; lipopolysaccharide; interferon type II; interferon regulatory factor-1


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE NUTRITIONAL STATUS of the host influences the incidence of infection after major surgery, trauma, or in the intensive care setting (11, 17, 24, 26). In addition, supplementation with several micronutriments has been reported to decrease the incidence of infection in intensive care unit patients through still poorly characterized mechanisms (16, 33).

Among micronutriments, vitamin A and its metabolites are known to decrease morbidity and mortality in several infectious diseases, which established its reputation as the "anti-infective vitamin" (3, 5, 37). Nevertheless, the beneficial effects of vitamin A as an anti-infective agent have not been found by all studies (7, 13, 41). Therefore, understanding the cellular and molecular mechanisms through which vitamin A acts as the anti-infective vitamin is essential.

It has been shown in vitro that the functions of macrophages, neutrophils, natural killer (NK) cells, and T- and B-lymphocytes are modulated by vitamin A and its metabolites (37). Furthermore, the production and/or secretion of several cytokines, such as interferon-gamma (IFN-gamma ), tumor necrosis factor-alpha (TNF-alpha ), interleukin (IL)-1, Il-2, Il-3, IL-4, and transforming growth factor-beta (TGF-beta ), are modulated by vitamin A and its metabolites (37). Vitamin A and its metabolites also regulate the expression and activity of several enzymes, such as phospholipase A2 (20) and, as was recently demonstrated, inducible nitric oxide synthase (NOS2, aka iNOS) (18, 32), that participate in the host inflammatory reaction to pathogens.

In an attempt to understand the cellular and molecular mechanisms through which vitamin A and its metabolites modulate the host pathogen interactions, we studied the effects of all-trans retinoic acid (RA) on the NOS2 biosynthetic pathway in LPS-injected rats. Despite their many conceptual limitations for the study of host-pathogen interactions, the experimental models of endotoxemia are well characterized in terms of NOS2 induction, cytokine production, and mortality (6, 35). NOS2 is an important component of the innate immune response to pathogens, and its activation affects host survival upon challenge with different pathogens (29). Uncontrolled activation of NOS2 has also been reported to contribute to the cardiovascular dysfunction of septic shock (47).

The aims of the present study were to 1) document the effects of RA on the NOS2 pathway in vivo in a model of nonlethal endotoxemia, 2) attempt to correlate the effects of RA on the NOS2 pathway with survival in this experimental model, and 3) suggest a possible mechanism through which RA may exert its effects on the NOS2 pathway in vivo.


    MATERIALS AND METHODS
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Animals

Male Wistar Kyoto rats (250-350 g) were housed and treated in accordance with accepted practices for humane laboratory animal care.

Preparation of Reagents

All chemicals and reagents were purchased from Sigma (Saint Quentin Fallavier, France) unless specified otherwise. Salmonella typhimurium lipopolysaccharide (LPS, Lot 96H4021) and aminoguanidine were dissolved in 0.9% NaCl at concentrations of 4 mg/ml and 50 mg/ml, respectively, and stored at 4°C. All-trans-RA was 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.

Experimental Protocols

Rats were divided into four groups. RA (n = 4) and RA+LPS (n = 10) groups received daily intraperitoneal injections of RA (10 mg/kg body wt) for 5 consecutive days, whereas LPS (n = 9) and Control (n = 4) groups received olive oil plus 5% DMSO in the same conditions. On day 5, LPS and RA+LPS groups were injected intraperitoneally with LPS (4 mg/kg body wt), and RA and Control groups were injected with the same volume (i.e., 500 µl) of 0.9% NaCl. Six hours after LPS administration, rats were anesthetized with 100 mg sodium thiopental intraperitoneally (Nesdonal, Rhône Poulenc Rorer, Paris, France), and the thorax and abdomen were dissected. Blood samples were recovered by cardiac puncture and centrifuged at 600 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.

Analysis of Rat mRNA Expression from Organ Samples by Semiquantitative RT-PCR

Semiquantitative (SQ) RT-PCR was performed to estimate mRNA expression for NOS2, the endothelial isoform of NOS (NOS3, aka eNOS), IFN regulatory factor-1 (IRF-1), osteopontin (OPN), natural killer cell markers (NKG2A and NKG2C) and several pro- and anti-inflammatory cytokines.

Extraction of total RNA. Total RNA was extracted from the different samples using Tri-Reagent (Euromedex, Souffelweyersheim, France), based on the acid guanidinium isothiocyanate-phenol-chloroform method. 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. In competitive RNA PCR studies, contaminating DNA can produce incorrect results because of its potential to act as a second competitor (22). Thus 5 µg of total RNA in 10 µl of diethylpyrocarbonate-treated water were mixed with 5 mM MgCl2, 1× PCR buffer 2, 1 mM dNTP, 2.5 µM oligo-d(T)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 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.

Duplex PCR. The duplex PCR was made using primers for the housekeeping gene beta -actin, NOS2, NOS3, IRF-1, OPN, and cytokines. In an attempt to identify the cellular source of some of the cytokines modulated by RA supplementation, the presence of markers for NK cells (NKG2A and NKG2C) and T-lymphocytes (IL-2) was investigated. The amplimer sequences, Genbank accession numbers, and lengths of the expected PCR products are presented in Table 1.

                              
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Table 1.   List of mRNAs of interest, expected size of amplified cDNAs, sequence of nucleotide primers, and Genbank accession numbers

PCR was performed in a DNA thermal cycler (Bio-Rad Laboratories, Ivry-sur-Seine, France) using 2 µl of cDNA sample in a total reaction volume of 10 µl with 1 µM of each primer, 1X PCR buffer 2, 1 mM dNTP, 1.5 or 1 mM MgCl2, 0.05 U/µl Ample Taq DNA polymerase (GeneAmp RNA PCR kit, PE Applied Biosystems). Because of the low-level expression of NOS3 compared with that of beta -actin, NOS3 PCR was performed in separate tubes, one with beta -actin primers and one with NOS3 primers. The PCR conditions were: 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 NOS2 and beta -actin, 35 cycles (94°C for 45 s, 60°C for 45 s, 72°C for 1.25 min) for NOS3, 35 cycles (94°C for 30 s, 64°C for 30 s, 72°C for 1 min) for NKG2A and NKG2C, 30 cycles (94°C for 30 s, 60°C for 30 s, 72°C for 1 min) for IRF-1 and cytokines, and 30 cycles (94°C for 30 s, 55°C for 30 s, 72°C for 1 min) for OPN. 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: r = 0.97, 0.98, and 0.96 for beta -actin, NOS2, and NOS3, respectively. PCR product identity was confirmed by restriction enzyme digestion using commercially available restriction enzymes (Boehringer Mannheim, Meylan, France) and sequencing with ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit and ABI PRISM310 (PE Applied Biosystems).

Densitometric analysis of PCR products. The PCR products were separated on a 2% agarose gel containing 0.5 µg/ml of ethidium bromide and were viewed by means of ultraviolet light on a transilluminator. Densitometry of the resulting bands was performed with a Bio-Rad Gel Doc 1000 (Bio-Rad). Results were expressed as a ratio of the optical density of the band of the PCR product of interest to that of beta -actin.

Western blot analysis of NOS2 and NOS3 protein expression. Tissue samples were homogenized with a Polytron PT 1200 (Kinematica, Littau, Switzerland) in 10 vol of lysis buffer (20 mM Tris · HCl, pH 7.4, 0.5 mM EDTA, 0.5 mM EGTA, 1 mM dithiothreitol, 1 µM leupeptin, 0.2 mM phenylmethylsulfonyl fluoride, 100 U/ml aprotinin). The homogenates were centrifuged at 3,000 g for 15 min. Protein concentration in the supernatant was measured by the method of Lowry. One hundred micrograms of proteins of each tissue sample homogenate were denatured by boiling for 10 min in sample buffer [0.5 M Tris · HCl, pH 6.8, 10% (wt/vol) SDS, 0.36% (vol/vol) glycerol, 0.06% (vol/vol) 2-beta -mercaptoethanol, 12% (wt/vol) bromophenol blue] and separated by electrophoresis on a 7.5-4% SDS-polyacrylamide gel (Mini Protean II, Bio-Rad). Electrophoresed proteins were transferred overnight at 4°C (Trans Blot Electrophoretic Cell, Bio-Rad) on polyvinylidene difluoride membranes (Sequi-Blot PVDF Membrane, Bio-Rad) in 20% methanol, 25 mM Tris, 192 mM glycine, pH 8.3. The membranes were blocked for 1 h with 3% bovine serum albumin (fraction V, Euromedex) in Tris-buffered saline [25 mM Tris, pH 7.5, 150 mM NaCl, 0.05% (vol/vol) Tween 20 (TBST solution)]. After washing for 10 min in TBST solution, the membranes were incubated for 2 h at room temperature with gentle agitation with 1:1,000 dilutions of either a rabbit anti-murine NOS2 polyclonal antibody or a rabbit anti-human NOS3 polyclonal antibody (both from Cayman Chemical, Ann Arbor, MI) for detection of NOS2 and NOS3, respectively. After being washed 5 times for 10 min, the membranes were incubated for 1 h with a 1:10,000 dilution of goat anti-rabbit IgG conjugated to alkaline phosphatase (Bio-Rad). Blots were washed in TBST solution, and the immunocomplexes were revealed with use of the nitro blue tetrazolium-5-bromo-4-chloro-3-indolyl phosphate method (36). A NOS2 mouse macrophage lysate obtained from RAW 264.7 cells stimulated with IFN-gamma and LPS and a human endothelial cell lysate derived from an aortic endothelial cell line (both from Transduction Laboratories, Lexington, KY) were used as positive controls for detection of NOS2 and NOS3 proteins, respectively. High-range prestained SDS-PAGE standards (Bio-Rad) were used for molecular mass determination. Densitometry of the resulting bands was performed using a Bio-Rad GS-690 imaging densitometer.

With regard to the SQ RT-PCR technique, the intensity of the bands was proportional to the quantity of protein submitted to the immunodetection: r = 0.98 for NOS2 and NOS3.

Measurement of NO2- and NO3- in plasma. The concentration of NO2- and NO3-, the stable end products of NO oxidation, was determined by the method of Green et al. (14). Plasma was centrifuged for 10 min at 12,000 g to remove coagulated proteins. One hundred microliters of plasma were added to 50 µl of bi-osmosed water and submitted to nitrate reduction by 0.1 U/ml nitrate reductase (EC 1.6.6.2, from Aspergillus species) in the presence of 5 µM FAD and 30 µM NADPH. Incubation with L-lactic dehydrogenase (EC 1.1.1.2.7, type II, from rabbit muscle) and 0.3 mM sodium pyruvate allowed NADPH to oxidize. Samples were mixed with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride, 2.5% H3PO4). After a 15-min incubation period at room temperature, the absorbance was read at 540 nm using a DU 640 B Beckman spectrophotometer (Beckman Instruments, Brea, CA). Nitrite concentration in each plasma sample was determined by extrapolation from a sodium nitrite standard curve (working range: 0.43-65 µM NO2-). All samples were tested in triplicate, and the background nitrite concentration of water was subtracted from the extrapolated nitrite concentration of samples.

Measurement of IFN-gamma and TNF-alpha concentrations in plasma. The concentrations of the cytokines IFN-gamma and TNF-alpha was determined by ELISA using commercially available kits following the manufacturer's instructions (R & D Systems, Minneapolis, MN). Before assay, plasma was centrifuged for 10 min at 3,500 g to remove coagulated proteins. Two- and tenfold plasma dilutions were performed for TNF-alpha and IFN-gamma assays, respectively. Background of substrate was subtracted from the values of samples. Cytokine concentrations were extrapolated from standard curves and were expressed in ng/ml.

Survival Studies

Forty two additional rats were treated as follows. Thirty-six rats were subjected to the same treatment as the RA+LPS group (10 mg/kg body wt RA and 4 mg/kg body wt LPS) and 6 rats were subjected to the same treatment as the LPS group (olive oil and 4 mg/kg body wt LPS). Eighteen rats of the RA+LPS group were injected intraperitoneally with the NOS inhibitor aminoguanidine (50 mg/kg) 15 min before LPS injection. Survival was measured hourly for the 24 h after LPS injection and was extended for at least 48 h.

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. Survival studies were analyzed with the Kaplan-Meier test. A P value < 0.05 was considered statistically significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Analysis of Rat NOS2 and NOS3 Expression and Activity

Effect of RA on NOS2 mRNA expression. In all organs studied, primers specific for NOS2 did not yield any PCR product for rats of Control and RA groups, whereas they yielded a single band of 578 bp for both the LPS and RA+LPS group. A representative liver mRNA expression profile is shown in Fig. 1A. Primers specific for beta -actin yielded a single band at 232 bp of equivalent intensity among all rats. Densitometric analysis revealed a twofold higher expression of NOS2 mRNA relative to that of beta -actin in RA+LPS group compared with LPS group for liver (Fig. 1B), kidney, and spleen (P < 0.05). No differences were observed for lung and heart (Table 2).


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Fig. 1.   Effect of retinoic acid (RA) supplementation on inducible NO synthase (NOS2) mRNA expression in the liver. A: representative RT-PCR profile of rat liver NOS2 mRNA expression. The NOS2 PCR product was detected as a 578-bp band and that of beta -actin as a 232-bp band. B: Densitometric analysis of NOS2 mRNA expression in liver. The optical density value of the NOS2 PCR product was divided by that of the beta -actin product. Results are expressed as means ± SE. *P < 0.05 vs. lipopolysaccharide (LPS) group; Dagger P < 0.01 vs. RA and control groups.


                              
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Table 2.   Effect of RA supplementation on NOS2 mRNA expression for the five organs studied

Effect of RA on NOS2 protein expression. In all organs studied, there was no detectable NOS2 protein in Control and RA groups. In contrast, the NOS2-specific antibody revealed a single band at 130 kDa for both LPS and RA+LPS groups. Figure 2A shows a representative Western blot of NOS2 protein expression in rat liver. Densitometric analysis revealed a twofold higher expression of NOS2 protein in the RA+LPS group compared with the LPS group for liver (Fig. 2B), kidney, and spleen (P < 0.05). No differences were observed for lung and heart (Table 3).


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Fig. 2.   Effect of RA supplementation on NOS2 protein expression in the liver. A: representative Western blot analysis of rat liver NOS2 protein expression. The NOS2 protein was detected as a 130-kDa band. B: densitometric analysis of NOS2 protein expression in liver. Results are expressed as means ± SE. § P < 0.005 vs. LPS group; Dagger  P < 0.01 vs. RA and control groups.


                              
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Table 3.   Effect of RA supplementation on NOS2 protein expression for the five organs studied

Effect of RA on NOS3 mRNA expression. Expression of NOS3 mRNA was studied only in the liver. NOS3 mRNA expression was (mean ± SE of relative NOS3 to beta -actin mRNA abundance) 0.83 ± 0.08, 0.95 ± 0.03, 0.88 ± 0.05, and 0.83 ± 0.05 for control, RA, LPS, and RA+LPS groups, respectively (no statistical difference).

Effect of RA on NOS3 protein expression. Western blot of NOS3 protein expression was performed only in liver. NOS3 protein expression was (mean ± SE of densitometric analysis arbitrary units) 2.71 ± 0.11, 2.97 ± 0.15, 2.48 ± 0.16, and 2.71 ± 0.17 for control, RA, LPS, and RA+LPS groups, respectively (no statistical difference).

Measurement of NO2- and NO3- concentration in plasma. As shown in Fig. 3, rats in the RA group had low but detectable plasma concentrations of nitrate/nitrite, not statistically different from those of the control group (3.38 ± 0.25 vs. 4.32 ± 0.42 µM, respectively). Rats in the LPS group had significantly higher plasma nitrate/nitrite concentrations (P < 0.01). RA supplementation (RA+LPS rats) resulted in a further threefold increase in plasma nitrate/nitrite concentration (P < 0.005).


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Fig. 3.   Effect of RA supplementation on plasma nitrate/nitrite concentration (Griess reaction). RA significantly increased the concentration of plasma nitrate/nitrite compared with LPS rats (17.12 ± 2.7 vs. 6.45 ± 0.9 µM, §) and with RA rats (17.12 ± 2.7 vs. 3.38 ± 0.25 µM, Dagger ). Results are expressed as means ± SE. §P < 0.005 vs. LPS group; Dagger  P < 0.01 vs. RA and control groups.

Analysis of Pro- and Anti-Inflammatory Cytokines Known to Modulate NOS2 Gene Expression

The comparison of the cytokine profiles is an attempt to understand the mechanisms that lead to NOS2 overexpression in the RA+LPS group compared with the LPS group.

Effect of RA on cytokines mRNA expression. Messenger RNA expression of several proinflammatory (IL-1beta , IL-6, TNF-alpha , IFN-gamma ) and anti-inflammatory (IL-4, IL-10, TGF-beta 2) cytokines, known to modulate NOS2 activation, was studied in the liver. In addition, IFN-gamma mRNA expression was studied in kidney, lung, heart, and spleen.

RA per se, compared with control rats, increased mRNA expression in the liver for IL-1beta , IL-2, and TNF-alpha , decreased mRNA expression for IL-6, and did not change mRNA expression for IL-4, IL-1beta , and TGF-beta 2 (Table 5). There was a significantly increased mRNA expression for IFN-gamma and IL-10 in the RA+LPS group compared with the LPS group (Table 4 and Table 5). The difference between the two groups for the IFN-gamma mRNA expression was organ specific with significant differences in liver and spleen but not in kidney, lung, and heart (Table 4). In contrast, mRNA expression for IL-1beta , IL-4, IL-6, TNF-alpha , and TGF-beta 2 was similar in the RA+LPS and LPS groups (Table 5).

                              
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Table 4.   Expression profile of IFN-gamma and IRF-1 mRNA for the five organs studied


                              
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Table 5.   Expression profile of several cytokines, OPN, NKG2A, and NKG2C mRNAs in the liver

Effects of RA on TNF-alpha and IFN-gamma plasma concentrations. Quantitative measurement of TNF-alpha and IFN-gamma production was performed by ELISA in plasma samples.

TNF-alpha and IFN-gamma plasma concentrations were below detection limits of the tests (5 and 10 pg/ml, respectively) in both control and RA groups. As expected, LPS injection resulted in significantly increased plasma concentrations of both TNF-alpha and IFN-gamma . Supplementation with RA (RA+LPS group) resulted in a further increase of plasma cytokine concentration (Table 6).

                              
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Table 6.   Effect of RA supplementation on TNF-alpha and IFN-gamma plasma concentrations

Investigation of NOS2 Pathway Activation

IFN-gamma increases NOS2 mRNA expression by increased transcription of the IRF-1 gene. IRF-1 mRNA expression was significantly higher in the RA+LPS group compared with the LPS group in liver, kidney, and spleen but not in lung and heart (see Table 4 and Refs. 1, 25).

Cellular source of IFN-gamma . INF-gamma is produced only by activated T lymphocytes and natural killer cells (2, 44). We therefore investigated the effects of RA supplementation on mRNA expression of IL-2 and OPN [markers of T lymphocytes activation (2, 10)], NKG2A, and NKG2C [markers of NK cells (4)]. In addition, IL-12, a macrophage-derived cytokine known to activate T lymphocytes and NK cells (44), was also studied.

Interleukin-2 mRNA expression in the liver was significantly higher in RA+LPS rats compared with LPS rats (Table 5). Osteopontin mRNA expression was not different among the four groups of rats in the liver (Table 5) and was not detectable in the spleen (results were not shown).

Messenger RNA encoding NKG2A and NKG2C were identically expressed in liver of all rats. Interleukin-12 mRNA expression in the spleen was detectable but was similar among the four groups of rats (results not shown). On the other hand, IL-12 was undetectable in the liver of animals from the different groups (Table 5).

Analyzed together, these results are consistent with an effect of RA on T lymphocyte activation as assessed by the increased IL-2 mRNA expression in RA+LPS rats compared with LPS rats.

Survival Studies

Mortality after the observation period (48 h) was zero when rats were injected with RA or LPS alone (unpublished observations). The six rats treated with LPS survived and were killed 2 days later. The 18 rats treated with RA+LPS died between the 5th and the 12th h after LPS injection, corresponding to the period of highest NOS2 induction. Among the 18 rats injected with aminoguanidine, only two rats died; the others were killed 2 days after LPS injection (Fig. 4). Thus the NOS inhibitor aminoguanidine significantly (P < 0.0001) increased survival from 0% (all RA+LPS rats died) to 89% (2 out of 18 aminoguanidine-treated rats died).


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Fig. 4.   Survival studies. LPS rats (---) were treated with 4 mg/kg LPS, RA+LPS rats () with 10 mg/kg RA (5 injections) and 4 mg/kg LPS, and RA+LPS+aminoguanidine rats (+) with 10 mg/kg RA (5 injections), 50 mg/kg aminoguanidine, and 4 mg/kg LPS. Each point represents 1 dead rat.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The main findings of the present study are as follows. In sharp contrast to previous in vitro studies, all-trans RA administered as replicate doses reproducibly and significantly enhances the LPS-triggered activation of the NOS2 biosynthetic pathway in vivo. The increased NOS2 activation upon RA supplementation was associated with highly increased mortality in this experimental model of endotoxemia. The mechanisms leading to the RA-mediated increase of LPS-induced NOS2 activation could be related, at least in part, to T-cell activation with resulting increased IFN-gamma gene transcription and secretion and/or IRF-1 mRNA expression.

Methodological Discussion

The rat model of nonlethal endotoxemia has been previously characterized by members of our group (6, 35). After injection of 4 mg/kg of LPS intraperitoneally, NOS2 mRNA and protein expression was detectable as early as 2 h and peaked at 6-8 h (unpublished results). For the survival studies, NOS activity was blocked with the relatively specific NOS2 inhibitor, aminoguanidine (15) at doses reported to be efficient and devoid of toxic effects (43). The regimen of RA supplementation (replicate doses) has previously been reported by van Pelt and de Rooij (48) and shown to have biological effects, whereas single doses were devoid of measurable biological effect. This was confirmed by our preliminary experiments, where single doses (5-10 mg/kg) of RA had no effect on NOS2 biosynthetic pathway in LPS-injected rats (data not shown). A toxic effect of RA is unlikely, because replicate doses of RA had no effect on rat behavior or mortality or on visual inspection of the peritoneal cavity. In addition, there were no signs of hypervitaminosis A, such as weight loss, hair loss, or skin scaling, after 5 days of 10 mg · kg-1 · day-1 RA treatment. This is in agreement with Look et al. (27) who showed toxic effects after 7 days of supplementation with 100 mg · kg-1 · day-1 RA in mice.

Retinoic Acid Enhances the LPS-Triggered Activation of NOS2 Pathway In Vivo

Our results show that, in sharp contrast to previous in vitro studies that demonstrated that RA attenuates NOS2 expression and activity in rat peritoneal macrophages (32) or vascular smooth muscle cells (18), in vivo administration of RA is associated with significant and reproducible enhancement of LPS-triggered NOS2 expression and activity. RA alone in single or replicate doses did not induce NOS2 pathway activation, as evidenced by the absence of measurable NOS2 mRNA or protein in RA rats and by similar nitrite/nitrate concentrations in plasma of RA rats and control rats. In addition, the vehicle used to solubilize RA did not increase the LPS-mediated NOS2 expression, because NOS2 pathway activation was identical in rats that received LPS or LPS with the vehicle of RA (data not shown). These results are consistent with a specific effect of RA on the NOS2 biosynthetic pathway.

NOS2 overexpression was observed in liver, kidney, and spleen but not in the lung and heart. This is consistent with the organ-dependent distribution of retinoids (46, 49). Taken together, these observations are strong arguments that persistently increased concentrations of RA in several organs such as the liver, spleen, or kidney could explain the organ-specific differences in NOS2 overexpression observed in the present study.

RA specifically activated the NOS2 pathway, because the expression of a functionally related gene (NOS3) was not modified by RA.

Two important questions concerning our results are related to 1) the discordance between the in vitro and the in vivo effect of RA on the NOS2 pathway and 2) the molecular and cellular mechanisms responsible for the effect of RA observed on the NOS2 pathway in vivo.

Discordance Between In Vitro and In Vivo Effects of RA on the NOS2 Pathway

Several reports have already documented discordant in vivo [increased antitumor activity of rat alveolar macrophages (19)] vs. in vitro [attenuation of IFN-gamma and LPS-induced cytostatic activity of murine peritoneal macrophages (31)] effects of RA on macrophage activation (42, 45). This discordance could be related to 1) the high concentrations (micromolar) of RA used in vitro; physiological concentrations of RA are ~10 nM in plasma and 100 nM in tissue (34); 2) the complex and organ-specific metabolism of RA (34); and 3) the different timing of vitamin A/RA supplementation and the pathogen/inflammatory challenge in vitro vs. in vivo (23, 40).

Biological Significance of RA-Mediated Increase of LPS-Triggered NOS2 Pathway Activation

Our results demonstrate that 1) the RA-mediated increase of NOS2 gene expression and activity is associated with increased mortality in this rodent endotoxemia model, and 2) the inhibition of NOS2 enzymatic activity by aminoguanidine significantly improved survival. How do these findings reconcile with the previously reported beneficial effect of vitamin A and its metabolites on host survival in infection (3, 5)? This has probably to do with the beneficial vs. detrimental effect of NOS2 induction on host survival dependent on the type of pathogen studied. Indeed, in models of endotoxemia, NOS2 activation is clearly deleterious (28), whereas NOS2 induction is beneficial for host survival in many other circumstances of exposure to pathogens (29). Nevertheless, the timing of vitamin A supplementation, the vitamin A status before supplementation, or the pathogens involved could have different effects on the host/pathogen interactions and result in beneficial (21, 38), neutral (13, 41), or detrimental (7) effects on the host. Further studies must attempt to document the detailed cellular and molecular mechanisms that mediate the effects of vitamin A on specific host/pathogen interactions.

Molecular and Cellular Mechanisms Responsible for the Effect of RA on the NOS2 Pathway In Vivo

RA alone did not by itself induce NOS2 expression but enhanced the effect of LPS. A computer search of the published 1,845-bp rat NOS2 gene promoter (12) revealed a sequence consistent with a putative RA response element (RARE). Further studies will be necessary to document whether the RARE in the NOS2 promoter is active. In addition, recent reports have convincingly demonstrated that retinoids can modulate gene transcription in the absence of RA response elements (8).

Hypothetical Mechanism for RA Increase of LPS-Mediated NOS2 Induction

The initial step was to determine whether RA supplementation resulted in changes in the cytokine mRNA expression profile on LPS stimulation. For this, macrophage (IL-1beta , IL-6, IL-10, and TGF-beta 2) or lymphocyte and NK cell (IL-4, IFN-gamma , TNF-alpha , and OPN)-derived cytokines known to modulate NOS2 expression were studied. Moreover, TNF-alpha and IFN-gamma concentrations were quantitatively measured in plasma.

RA per se selectively modified the mRNA expression of several of the cytokines studied (Table 5). This selectivity is consistent with specific effects of RA alone on cytokine expression. Interestingly, RA alone increased mRNA expression of IL-2. The RA-mediated enhancement of NOS2 pathway activation in LPS-injected rats is consistent with lymphocyte (but not NK cell) activation (increased IL-2 mRNA expression in several organs) through IL-12- and OPN-independent pathways (10). Taken together, these results are consistent with a specific effect of RA on lymphocyte activation (37).

The subsequent step was an attempt to identify the signal transduction events leading from IFN-gamma to NOS2 induction. IFN-gamma activates latent cytoplasmic proteins termed signal transducers and activators of transcription (STATs) through phosphorylation of tyrosine residues (9). STAT-1 protein expression was detected (Western blot) in the liver but was not different in RA+LPS compared with LPS groups (results not shown). Despite many efforts, we could not reproducibly study STAT-1alpha phosphorylation in liver homogenates in vivo. We cannot therefore, for the moment, confer any role to phosphorylated (active) STAT-1alpha protein in NOS2 gene transcription in response to RA in this in vivo model. Activated STAT proteins have been reported to induce the transcription factor IFN regulatory factor 1 (IRF-1), which is strictly necessary for NOS2 induction (1, 25). Our results revealed similar levels of IRF-1 mRNA in rats of LPS and RA groups but a significantly higher level in the RA+LPS group. These results are consistent with constitutive IRF-1 expression as demonstrated by Sims et al. (39) and enhanced IRF-1 mRNA expression upon RA supplementation and LPS stimulation. In addition, RA was shown to directly activate IRF-1 gene expression in cultured NB4 cells derived from bone marrow of patients suffering from acute promyelocytic leukemia (30).

Our results suggest that the increased expression of IRF-1, either as a result of IFN-gamma /STAT-1 signal transduction (e.g., in the liver and spleen) or as a direct effect of RA on IRF-1 mRNA expression (e.g., in the kidney), contributes to the RA-mediated enhancement of LPS-triggered NOS2 activation.

Limitations of the Present Study

There are several limitations of the present study. The first is the fact that extrapolation of the results observed with RA to vitamin A and/or its metabolites such as retinol should be done cautiously because of metabolite-specific pharmacological effects and/or toxicity. The second is that, as in any in vivo study, our experiments cannot demonstrate all cellular and molecular mechanisms that account for the biological effects of retinoic acid as modulator of the host pathogen interactions. Nevertheless, several hypotheses, which need further exploration both in vivo and in vitro, have been raised by our experiments.

In conclusion, we have demonstrated that retinoic acid supplementation in rats injected with lipopolysaccharide is associated with increased NOS2 pathway activation and decreased host survival. The in vivo effects of retinoic acid on NOS2 expression are in sharp contrast to previously reported in vitro effects. These results may contribute to the understanding of the mechanisms through which retinoic acid modulates the immune function in vivo.


    ACKNOWLEDGEMENTS

The secretarial assistance of Mmes. Claude Baillot and Rebecca Clement are gratefully acknowledged.


    FOOTNOTES

Y. Devaux and C. Seguin were financed by Association de Recherche et d'Information Scientifique en Cardiologie and Unité Propre Enseignement Supérieur Associée 971068. S. Grosjean received a grant from the Société Française d'Anesthésie Réanimation and the Programme Lavoisier (Ministère des Affaires Etrangères).

Address for reprint requests and other correspondence: D. Ungureanu-Longrois, Dept. of Anesthesia and Intensive Care, C.H.U. Brabois, Rue du Morvan, 54511 Vandoeuvre Cedex, 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.

Received 21 March 2000; accepted in final form 13 June 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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