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
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ABSTRACT |
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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)- and IFN regulatory factor-1 mRNAs in several organs and
increased IFN-
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
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INTRODUCTION |
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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- (IFN-
), tumor necrosis factor-
(TNF-
),
interleukin (IL)-1, Il-2, Il-3, IL-4, and transforming growth
factor-
(TGF-
), 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.
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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 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 atExperimental 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 atAnalysis 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
-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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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 -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--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-
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.
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- and TNF-
concentrations in plasma.
The concentrations of the cytokines IFN-
and TNF-
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-
and IFN-
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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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
-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
-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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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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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 -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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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-1, IL-6,
TNF-
, IFN-
) and anti-inflammatory (IL-4, IL-10, TGF-
2) cytokines, known to modulate NOS2 activation, was studied in the liver.
In addition, IFN-
mRNA expression was studied in kidney, lung,
heart, and spleen.
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Effects of RA on TNF- and IFN-
plasma concentrations.
Quantitative measurement of TNF-
and IFN-
production was
performed by ELISA in plasma samples.
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Investigation of NOS2 Pathway Activation
IFN- 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-.
INF-
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.
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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DISCUSSION |
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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- 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 · kgRetinoic 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-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-1RA 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- to NOS2 induction. IFN-
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-1
phosphorylation in liver homogenates in vivo. We cannot
therefore, for the moment, confer any role to phosphorylated (active)
STAT-1
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-/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.
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ACKNOWLEDGEMENTS |
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The secretarial assistance of Mmes. Claude Baillot and Rebecca Clement are gratefully acknowledged.
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FOOTNOTES |
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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.
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