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(Journal of Leukocyte Biology. 2001;70:121-129.)
© 2001 by Society for Leukocyte Biology

Potentiation of interferon-{gamma}-stimulated nitric oxide production by retinoic acid in RAW 264.7 cells

Liv M. I. Austenaa* and A. Catharine Ross*

* Department of Nutrition, The Pennsylvania State University, University Park, Pennsylvania, and
{dagger} Institute for Nutrition Research, University of Oslo, 0316 Oslo, Norway

Correspondence: A. Catharine Ross, Ph.D., Department of Nutrition, Pennsylvania State University, 126.S. Henderson Building, University Park, PA 16802. E-mail: acr6{at}psu.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nitric oxide (NO) production is essential for normal immunity. We have examined the capacity of retinoic acid (RA), a pleiotropic hormone necessary for normal immunity, to modulate NO production in RAW 264.7 cells. NO production induced by suboptimal concentrations of interferon-{gamma} (IFN-{gamma}) was significantly greater in cells cultured in low-retinoid medium and treated with all-trans-RA (10-10– 10-6 M, P <0.05), as well as with 9-cis-RA and several retinoids selective for the RA receptor subfamily of nuclear retinoid receptors. Similar results were obtained with lipopolysaccharide and monophosphoryl lipid A as stimuli. The RA-potentiated production of NO was positively correlated with inducible NO synthase (iNOS) protein (r =0.94, P <0.002), although the expression of iNOS mRNA was not altered. We hypothesize that modulation of the macrophage response to suboptimal immune stimuli by physiological concentrations of RA, as observed in these studies, may be important in establishing an optimal balance between T helper (Th) 1- and Th2-mediated immunity.

Key Words: lipopolysaccharide • monophosphoryl lipid A • immune regulation • T-helper-cell differentiation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The generation of nitric oxide (NO) plays a vital role in nonspecific host defense against intracellular and extracellular pathogens, viral infections, and tumor cells [1 , 2 ] and in the development of specific immunity. The importance of inducible NO production in the response to bacterial infection and endotoxic shock is demonstrated well by the increased disease susceptibility of mice lacking the gene for inducible nitric oxide synthase (iNOS) [3 , 4 ]. Among its wide-ranging effects, NO modulates macrophage activation and proliferation [5 ] and helps to shape the response of T cells by restricting proliferation and cytokine production from the T helper (Th) 1 subset [3 , 4 ]. Conversely, however, sustained production of NO is harmful to healthy tissues, and overproduction is implicated in several disease states including septic shock; localized inflammatory disorders such as asthma and arthritis; inflammatory bowel diseases; and autoimmune disorders including insulin-dependent diabetes mellitus and multiple sclerosis [reviewed in ref. 1 and 6]. Silencing of the iNOS gene by an antisense iNOS oligonucleotide in adult mice inhibited the induction of experimental autoimmune encephalomyelitis [7 ].

In the early 1900s, vitamin A deficiency was shown to be associated with reduced resistance to certain infections [8 , 9 ]. Vitamin A is now understood to be essential for normal immune system development, maintenance of mucosal barriers, and normal functioning of nonspecific and cell-mediated and humoral specific immunity. A reduction of T-cell precursors [10 ] and an abnormal shift toward the development of Th1-type immune responses at the expense of Th2-type immunity have been proposed as underlying causes of impaired immunity during vitamin A deficiency [11 , 12 ]. The increased severity of several infectious diseases observed in vitamin A-deficient animals and humans may be due, in part, to inappropriate development of Th1 versus Th2 immune responses.

Most physiological processes affected by vitamin A are mediated through the action of two families of nuclear retinoid receptors, retinoid acid (RA) receptor (RAR) and retinoid X receptor (RXR), which function as ligand-activated transcription factors for a large number of genes [reviewed in ref. 13 ]. All-trans RA binds with high affinity to members of the RAR subfamily, and 9-cis-RA binds to both RXR and RAR subfamily members [13 ]. Heterodimers composed of an RAR and an RXR bind to specific RA response elements (RAREs) in target genes; while the RXR may bind either as a homodimer to a different set of response elements, the retinoid X response elements (RXREs), or as a heterodimer with a number of other nuclear receptors, including those for vitamin D3, thyroid hormone, and peroxisome proliferators [14 ]. Numerous retinoids with selectivity for RAR or RXR have been synthesized because of their potential utility as drugs to promote cell differentiation and/or reduce proliferation [15 ].

The effect of immune and inflammatory stimulation on iNOS expression and NO production may differ significantly depending on the microenvironment and context of stimulation, including such factors as the type of cells, their state of maturation and differentiation, their prior exposure to activating or inhibitory stimuli, and the dose of NO-inducing stimuli to which they are exposed. As indicated by previous research, the ability of RA to modulate NO production may also differ within various contexts of infection or inflammation. In several studies conducted with cells that are already mature and activated, RA exhibited anti-inflammatory properties that appeared to be at least partly mediated by inhibition of iNOS gene expression. RA was shown to decrease proinflammatory-cytokine-, CD23-, or lipopolysaccharide (LPS)-induced NO production in vascular smooth muscle cells [16 , 17 ], peritoneal macrophages [18 ], keratinocytes [19 ], Kupffer cells [20 ], and mesangial cells [21 ]. However, in the context of RA-induced cell differentiation, RA may have the opposite effect, that is, enhancement of iNOS expression and NO synthesis. RA-induced enhancement of NO production has been observed in vivo [22 ] and in several cell types, including monocytic cells [23 ], breast cancer cells [24 ], epithelial cells [25 ], and neuroblastoma cells [26 ]. Additionally, RA treatment in vivo significantly enhanced production of iNOS mRNA, protein, plasma nitrate, and nitrate in LPS-injected but not saline-injected rats [22 ].

An understanding of how vitamin A deficiency and retinoid exposure affect cellular responses to immune stimuli is of both basic and practical importance. In the present experiments, RAW 264.7 cells were initially cultured in medium containing a low level of retinol to provide a model system of vitamin A deficiency. We then investigated the effects of RA on NO production and iNOS expression during immune activation by interferon (IFN)-{gamma} or LPS. The results indicate that exposure to RA sensitizes RAW 264.7 macrophages, enabling enhanced NO production in response to levels of immune stimuli that otherwise are suboptimal. We propose that RA-potentiated NO production may be important in regulating the differentiation of Th1 and Th2 cells and thereby in shaping the host’s immune response to infection.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
All-trans-RA and 9-cis-RA were purchased from Sigma Chemical Co. (St. Louis, MO). Retinoids of the UAB series were kindly provided by D. D. Muccio, University of Alabama at Birmingham, and retinoids of the Roche series were provided by L. H. Foley, Hoffmann-La Roche, Nutley, NJ. Retinoid stocks were dissolved at millimolar concentrations in 100% ethanol and stored at -80°C. Recombinant murine IFN-{gamma} was purchased from PharMingen (San Diego, CA), LPS (Pseudomonas aeruginosa Fisher Devlin immunotype 1) was from List Biological Laboratories (Campbell, CA), and monophosphoryl lipid A (MPL) was from Ribi (Hamilton, MT). N-Monomethyl-L-arginine (L-NMA) and its D-isomer (D-NMA) were from Sigma.

Cell culture and experimental protocol
The RAW 264.7 cells (ATTC TIB-71; American Type Culture Collection, Manassas, VA) were grown in high-glucose (4.5-g/L) Dulbecco’s modified Eagle’s medium (Gibco/BRL, Grand Island, NY) supplemented with 4 mM L-glutamine, penicillin (50 U/mL), streptomycin (0.05 mg/mL), and 10% heat-inactivated fetal bovine serum (FBS) (Gibco-BRL) and at 37°C in an environment with 5% CO2 and humidified air [27 ]. Cultures of RAW 264.7 cells used for experiments were replaced from frozen stock cultures no less than every 2 months. For the experiments, RAW 264.7 cells were seeded at a concentration of 2 x 106 cells/mL in Dulbecco’s modified Eagle’s medium with 1% heat-inactivated FBS together with a retinoid or vehicle as a control [0.01% ethanol (final concentration)], as indicated in the figure legends. After RA pretreatment, the medium was changed to medium containing the iNOS-inducing stimuli, as indicated in the figure legends.

Determinations of nitrite in cell culture medium and cellular DNA
Levels of nitrite in cell culture medium were determined by a colorimetric assay involving the Griess reaction, as previously described [28 ]. To determine cell DNA content as a measure of cell number, a DNA-intercalating fluorochrome, Hoechst 33258 compound 2-[2-(4-hydroxyphenyl)-6-benzimidazolyl]-6-[1-methyl-4-piperazyl]-benzimidazole-3HCl (Sigma), was used [29 ]. Briefly, cells were grown and treated in 96-well plates; and at the desired time point the medium was aspirated, 100 µL of distilled deionized water was added to each well, and plates were incubated at 37°C for 30 min. After one freeze-thaw cycle, 100 µL of a solution containing Hoechst 33258 (at a concentration of 10 µg/mL in 0.05 M phosphate buffer, pH 7.4), 2 M NaCl, and 2 mM EDTA were added to each well. Herring sperm DNA was used as a DNA standard. Fluorescence was measured immediately using an excitation wavelength of 360 nm and an emission wavelength of 465 nm.

Western blot analysis
The RAW 264.7 cells were lysed in phosphate-buffered saline, pH 7.4, containing 1% sodium dodecyl sulfate and a Complete Mini Protease Inhibitor Cocktail tablet (Roche, Indianapolis, IN), after which the samples were passed through a 21-gauge needle and placed on ice for 45 min. The protein concentration was determined by a detergent-compatible protein assay (Bio-Rad, Hercules, CA). Between 20 and 40 µg of protein were dissolved in Laemmli sample buffer (Bio-Rad), boiled for 5 min, and then loaded on a sodium dodecyl sulfate-7.5% polyacrylamide gel (Bio-Rad) for size fractionation. The separated proteins were electroblotted onto a nitrocellulose membrane (Micron Separations, Westboro, MA). For Western blot analysis, the primary monoclonal antibody for iNOS (Transduction Laboratories, Lexington, KY) and the secondary peroxidase-linked anti-mouse immunoglobulin (Ig)G (Amersham Pharmacia Biotech, Piscataway, NJ) were diluted 1:2,000 and 1:5,000, respectively, in Tris-buffered saline, pH 7.4, with 0.1% Tween 20 (TBS-T) containing 5% dry milk, which was used as the blocking agent. The membrane was washed with TBS-T after blocking and after incubations with the primary antibody and the secondary antibody. Then the iNOS protein was detected by chemiluminescence using an enhanced chemiluminescence detection solution and Hyperfilm enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech) film according to the manufacturer’s directions. Densitometry was performed using NIH Image 1.56 software (National Institutes of Health, Bethesda, MD).

Isolation of total and poly(A)+ RNA and Northern blot analysis
The isolation of total RNA and the Northern blot analysis were performed essentially as previously described [30 ]. Briefly, total RNA was isolated using Trizol (Gibco-BRL), quantified spectrophotometrically, and examined for integrity by ethidium bromide staining [30 ]. Three micrograms of poly(A)+ RNA that had been isolated by affinity chromatography using oligo(dT) cellulose were size fractionated on a 1.2% agarose–0.66 M formaldehyde gel, transferred to a nylon membrane (Schleicher & Schuell, Keene, NH), and cross-linked with UV light [30 ]. The cDNAs for the RAR{alpha}, RARß, RAR{gamma}, RXR{alpha}, RXRß, and RXR{gamma} [31 ] used as probes were kindly provided by Pierre Chambon (Strasbourg, France). The probes were labeled with [{gamma}--32P]dCTP (3,000 Ci/mmol and 10 µCi/µL; Amersham) using the Prime-a-Gene Labeling System (Promega, Madison, WI). Prehybridization, hybridization, and autoradiography were performed as previously described [30 ].

For reverse transcription (RT)-PCR analysis of RARß and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), used as a control, the Perkin-Elmer Gene Amp PCR system 24000 (PE Biosystems, Foster City, CA) was used. Reverse transcription was performed using 1 µg of RNA in a 25-µL reaction. The cDNA was diluted 1:4 before 5 µL of this solution was used for one PCR in a total volume of 25 µL. The final concentration of each primer was 0.3 µM for analysis of RARß and 0.2 µM for analysis of GAPDH. For detection of RARß, 24 PCR cycles were run, and for GAPDH, 20 cycles were run. Ten microliters of PCR product were separated on a 2% agarose gel in Tris-acetic acid-ethylenediaminetetraacetate buffer, pH 8.0, containing 250 µg of ethidium bromide per mL and photographed. The primers for GAPDH (Clontech Laboratories, Palo Alto, CA) yielded a 982-bp PCR product. The primers for RARß were designed using Primer3 software (http://www.genome.wi.mit.edu/genome_software/other/primer3.html). The forward primer for RARß was 5'-TCC-ACA-CCT-AGA-GGA-TAA-GC-3', the reverse primer was 5'-TGG-TAC-TCT-GTG-TCT-CGA-TG-3', and the PCR product was 227 bp.

Quantitative real-time PCR analysis was used to determine iNOS mRNA expression. Duplicate or triplicate aliquots of RNA were analyzed on 96-well plates using the Perkin-Elmer/Applied Biosystems Division (PE/ABD) 7700 Sequence Detector. The iNOS forward primer was 5'-CAG-CTG-GGC-TGT-ACA-AAC-CTT-3', the probe was 5'-CGG-GCA-GCC-TGT-GAG-ACC-TTT-GA-3', and the reverse primer was 5'-TGA-ATG-TGA-TGT-TTG-CTT-CGG-3'. The 5' label was 6-carboxyfluorescein (FAM) and the 3' -label was 6-carboxytetramethylrhodamine. The primers and probe for iNOS were designed using the PE/ABD Primer Express software based on the mouse cDNA sequence reported by Kone et al. [32 ]. Each value for iNOS mRNA was normalized relative to the 18S RNA analyzed in the same well.

Statistical analysis
One-factor analysis of variance (ANOVA) was performed using the software SuperANOVA (Abacus Concepts, Berkeley, CA). Differences were considered statistically significant according to Fisher’s protected least-significant-difference test (P <0.05).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
RA enhances IFN-{gamma}-, LPS-, or MPL-stimulated NO production
RAW 264.7 cells treated with all-trans-RA exhibited significant potentiation of NO production after stimulation with concentrations of IFN-{gamma} that, alone, induced only submaximal NO production (Fig. 1A ). In concentrations of IFN-{gamma} that induced maximal NO production, RA had no additional effect. When the results from experiments using suboptimal doses of IFN-{gamma} were calculated as the nanomoles of nitrite in the cell culture medium per microgram of cell DNA, iNOS specific activity was significantly greater in RA-treated cells than in vehicle-treated cells (data not shown). RA also potentiated the response of RAW 264.7 cells to stimulation with LPS and MPL (Fig. 1B and 1C , respectively).



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  		Figure 1. Potentiation of NO production by RA in RAW 264.7 cells stimulated with IFN-{gamma}, LPS, or MPL. Cells were pretreated for 24 h with 10-7 M all-trans-RA or vehicle (0.1% ethanol, EtOH). Then the medium was changed to medium containing IFN-{gamma} (A), LPS (B), or MPL (C) at the indicated concentrations, together with 10-7 M all-trans-RA or vehicle. After 24 h, the nitrite assay for NO production was performed immediately on cell culture medium. The data shown are the means ± SD, n = 3–4. *, P < 0.05 versus vehicle control.

 
The enhancement of NO production by RA was dependent on incubating cells with a low concentration of FBS (1%) in the culture medium from the time of RA pretreatment and throughout the experiment (data not shown). The retinoid concentrations of human and rodent plasma are in the ranges of 2–3 µM for retinol and 5–15 nM for RA [33 ]. The concentration of retinol in FBS is ~0.7 µM (A. C. Ross, unpublished data), which would equal 7 nM in culture medium containing 1% FBS. Therefore, the cell cultures not receiving RA were deficient in retinoids, similar to a state of vitamin A deficiency.

The potencies of all-trans-RA and 9-cis-RA were compared in dose-response experiments. IFN-{gamma}-stimulated NO production was enhanced when all-trans-RA was added in the range from 10-9 to 10-6 M (P<0.05) (Fig. 2A ). Potentiation of NO production was greatest in the physiological range of 10-7 to 10-8 M, whereas at 10-5 M, all-trans-RA abolished NO production (data not shown). Enhanced NO production was also observed in cells treated with 9-cis-RA, but 9-cis-RA was less potent than all-trans-RA (P <0.05) (Fig. 2A) . RA alone did not elicit NO production (data not shown).



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  		Figure 2. Dose-response comparison of all-trans-RA and 9-cis-RA and the effect of RA pretreatment and kinetics of NO production in RAW 264.7 cells. Cells were pretreated for 24 h (A and C) or 6–24 h (B) with all-trans- or 9-cis-RA at concentrations from 10-12 to 10-6 M (A), 10-7 M all-trans-RA (B and C), or vehicle (0.1% ethanol, EtOH). Then the medium was changed to medium containing IFN-{gamma} (A, 5 U/mL; B and C, 3 U/mL) and all-trans- or 9-cis-RA or vehicle at the same concentrations as for the pretreatment. Immediately after 24 h (A, B) or at 0, 6, 12, 18, and 24 h (C), the nitrite assay was performed on cell culture medium. The data shown are the means ± SD, n = 3–4. *, P < 0.05 versus vehicle control.

 
The enhancement of NO production by all-trans-RA required retinoid pretreatment for at least 12 h prior to stimulation with IFN-{gamma} (Fig. 2B) , LPS, or MPL (data not shown). Pretreatment with RA for 12, 18, and 24 h potentiated NO production by 2.0-, 3.2-, and 3.7-fold, respectively (P <0.02).

The kinetics of NO production (nitrite accumulation) in RAW 264.7 cell culture medium was examined in cells pretreated with RA or vehicle only and stimulated with a suboptimal dose of IFN-{gamma} (3 U/mL). Nitrite was readily detectable in the cell culture medium 12–18 h after stimulation with IFN-{gamma} (Fig. 2C) . Nitrite was not detected any earlier in RA-treated cells than in vehicle-treated cells, but once detected the rate of nitrate accumulation was higher in RA-treated cells.

To ensure that the source of nitrite in the cell culture medium is L-arginine, L-NMA was added as an inhibitor of the NOS enzymes [6 ] during maximal cell stimulation with IFN-{gamma} (20 U/mL) and LPS (40 µg/mL). With equimolar concentrations (0.4 mM) of L-arginine and L-NMA, the accumulation of nitrite in the medium was about half of that observed without L-NMA (Fig. 3 ). At an 8:1 molar ratio of L-NMA to L-arginine, nitrite formation was reduced by ~90%. The D-NMA isomer, added as a negative control, did not affect either IFN-{gamma}- or LPS-stimulated NO production.



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  		Figure 3. Inhibition of NO production by L-NMA. RAW 264.7 cells were stimulated with IFN-{gamma} (20 U/mL) and LPS (40 µg/mL) alone or in the presence of L-NMA or D-NMA at the indicated concentrations. Immediately after 24 h, the nitrite assay was performed on cell culture medium. The data shown are the means ± SD, n = 4. *, P < 0.001.

 
Lack of effect of RA on cell growth and IFN-{gamma}-induced growth inhibition
To determine whether enhanced NO production is related to an increase in the number of RA-treated cells, experiments were conducted with RAW 264.7 cells treated with IFN-{gamma} and RA separately and together. An analysis of the results of seven independent experiments showed that IFN-{gamma} treatment alone caused a significant (P <0.05) growth inhibition that was observed after 48 h of culture and that IFN-{gamma} with or without RA inhibited growth after 72 h (Fig. 4 ). RA treatment alone did not inhibit cell growth.



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  		Figure 4. Cell growth is inhibited by IFN-{gamma} but not by RA. RAW 264.7 cells were pretreated for 24 h with RA or vehicle (0.1% ethanol, EtOH), after which the medium was changed to medium containing IFN-{gamma} and all-trans-RA or vehicle, all-trans-RA alone, or vehicle alone. After 24 h, the medium was changed again to medium containing IFN-{gamma} and all-trans-RA or vehicle, all-trans-RA alone, or vehicle alone, and cells were incubated until 48 h. Cells were harvested at this time or after another 24-h IFN-{gamma} stimulation and incubation (noted as 72 h). Cell DNA content was assayed. The data shown are the means ± SD of data from 7 independent experiments. * denotes P < 0.05 compared to ethanol-treated cells at each time point.

 
RAR and RXR subtypes in RAW 264.7 cells, enhancement of IFN-{gamma}-stimulated NO production, and induction of RARß by RA
The retinoid receptor isoforms that are expressed by RAW 264.7 cells have not to our knowledge been reported previously. Northern blot analysis of poly(A)+ RNA (Fig. 5 ) detected RAR{alpha}, RARß, RAR{gamma}, RXR{alpha}, and RXRß; but RXR{gamma} was undetectable. To determine whether the potentiation of NO production by RA is most likely mediated through the RAR or RXR pathways, several receptor-selective retinoids were compared for their ability to enhance IFN-{gamma}-stimulated NO production as well as for their ability to induce expression of RARß, whose gene contains an RARE (ßRARE) [34 ]. Table 1 shows the reported receptor selectivity for the retinoids tested [34a–34f], the enhancement by each retinoid of IFN-{gamma}-stimulated NO production, and their abilities to induce RARß mRNA. As determined by RT-PCR analysis, all of the RAR-selective retinoids [Ro40-6055 (Am580), Ro19-0645, and all-trans-UAB8] induced RARß expression, as did all-trans-RA and 9-cis-RA; these retinoids also stimulated significant enhancement of NO production. The RXR-selective retinoids (Ro25-6603, Ro25-7386, and 9-cis-UAB30) either had no significant effect compared with control cells or were less effective than all-trans-RA or Ro40-6055, an RAR-{alpha}-selective retinoid (Table 1) . In combination with an RAR-selective retinoid, Ro25-6603 and Ro25-7386 neither enhanced nor inhibited activity. With the exception of 9-cis-UAB30—reported to be RXR selective [35 ]—which did increase expression of RARß mRNA but did not significantly increase NO production, there was a consistent relationship between the ability of a retinoid to induce RARß and its ability to potentiate NO production stimulated by IFN-{gamma}.



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  		Figure 5. Retinoid receptor expression in RAW 264.7 cells. Three micrograms of poly(A)+ RNA was used for Northern blotting for RAR{alpha}, RARß, and RAR{gamma} and for RXR{alpha}, RXRß, and RXR{gamma} as described in Materials and Methods.

 

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  		Table 1. Ability of Various Receptor-Selective Retinoids to Potentiate IFN-{gamma}-Induced Nitric Oxide Production and Induce Expression of Retinoic Acid Receptor RARß

 
RA enhances iNOS protein levels and NO production without a concomitant change in iNOS mRNA
To investigate whether NO production (nitrite accumulation) in cell culture medium was correlated with an increase in iNOS mRNA and/or protein levels, we performed a quantitative real-time PCR to assess iNOS mRNA levels and Western blotting to assess iNOS protein expression. iNOS mRNA increased above background levels after 4 h of stimulation with IFN-{gamma} (Fig. 6 ). Maximal levels were reached after 6 h, and expression returned to background levels after 18–24 h (data not shown). However, in three independent experiments, there was no difference in the iNOS mRNA level attributable to treatment with RA, but stimulation with IFN-{gamma} with and without RA resulted in equal increases in iNOS mRNA. Unstimulated RAW 264.7 cells expressed a low but detectable level of iNOS mRNA, and cells that had been maintained in culture for longer times had a higher basal level of iNOS mRNA. Unstimulated RAW 264.7 cells also expressed a low basal level of tumor necrosis factor {alpha} mRNA, indicative of the partially activated state of this cell line (data not shown).



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  		Figure 6. Induction of iNOS mRNA after exposure of RAW 264.7 cells to IFN-{gamma} with and without treatment with all-trans-RA. Cells were pretreated for 24 h with 10-8 M all-trans-RA or vehicle (0.1% ethanol, EtOH). Then the medium was changed to medium containing IFN-{gamma} (3 U/mL) and all-trans-RA or vehicle, all-trans-RA alone, or vehicle alone. After 0.5, 2, 4, and 6 h, RNA was extracted and quantified in triplicate by real-time PCR analysis for iNOS mRNA. A representative experiment of three independent experiments is shown.

 
Despite the lack of effect of RA on iNOS mRNA, both the expression of iNOS protein and the production of NO were potentiated to similar extents by RA in IFN-{gamma}- and LPS-treated RAW 264.7 cells (Fig. 7 A ). The concentration of nitrite in the cell culture medium and the expression of iNOS protein in cell lysates were strongly correlated (r =0.94, P <0.002). The potentiation by RA was greatest (nearly 10-fold) in the first 6 h after IFN-{gamma} stimulation, during which time iNOS protein increased most rapidly (Fig. 7B) . iNOS protein was at or below the limit of detection in cells stimulated with IFN-{gamma} only (therefore not shown in Fig. 7B insert). Similarly, iNOS protein was also enhanced by RA in cells stimulated with LPS in both the presence and absence of RA (data not shown).



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  		Figure 7. Expression of iNOS protein after exposure of RAW 264.7 cells to IFN-{gamma} or LPS with and without treatment with all-trans-RA. (A) Cells were pretreated for 24 h with 10-8 M all-trans-RA or vehicle. Then the medium was changed to medium containing IFN-{gamma} (3 or 6 U/mL), LPS (1 ng/mL), or LPS (100 ng/mL) plus IFN-{gamma} (50 U/mL) for maximal stimulation, 10-7 M all-trans-RA or vehicle (0.1% ethanol, EtOH), all-trans-RA alone, or vehicle alone. After 24 h, cell culture medium samples were taken for nitrite analysis (black bars), and the cells were lysed for Western blot analysis for iNOS protein and quantified by densitometry (open bars). The data are expressed as the fold difference relative to results for IFN-{gamma} at 3 U/mL. The Western blot analysis for iNOS protein illustrates the results for the conditions shown directly above in the bar graph. (B) Cells were pretreated with 10-8 M all-trans-RA or vehicle for 24 h. Then the medium was changed to medium containing IFN-{gamma} (3 U/mL) and 10-8 M all-trans-RA or vehicle, all-trans-RA alone, or vehicle alone. After 3, 6, 18, and 24 h, cells were lysed for Western blot analysis. Densitometric analysis of the blot was performed followed by normalization to the Coomassie blue-stained gel. Since iNOS protein was nearly undetectable without IFN-{gamma} treatment, the y-axis represents the fold difference in iNOS protein relative to the level of iNOS protein after treatment with IFN-{gamma} at 3 U/mL for 3 h.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Treatment of RAW 264.7 cells with physiological concentrations of all-trans-RA for 18–24 h prior to induction of iNOS with IFN-{gamma} or LPS resulted in enhanced NO production relative to NO production by cells cultured in a low-retinoid medium. It is important to note that RA pretreatment did not alter maximum NO production but rendered RAW 264.7 cells more sensitive to submaximal concentrations of these iNOS-inducing agents (Fig. 1) . The increase in NO production by RA was not related to a change in the number of cells, and iNOS specific activity was higher in RA-treated cells. RAW 264.7 cells expressed all of the RAR and RXR receptors except the RXR{gamma} isoform, a finding similar to a previous study in isolated Kupffer cells [36 ], in which all the RAR and RXR isoforms were present but RXR{gamma} was expressed at significantly lower levels. The results of our studies using several synthetic retinoids with selectivity for the RAR or RXR receptors are consistent with a model in which the RA-induced enhancement of NO production is mediated through an RAR pathway, because all of the RAR-selective retinoids tested enhanced NO production and because naturally occurring all-trans-RA was more effective than 9-cis-RA. Of the retinoid analogues tested, only the RXR-selective retinoids Ro25-7386 and 9-cis-UAB30 did not enhance IFN-{gamma}-stimulated NO production. The ability of all-trans-RA to enhance NO production in RAW 264.7 cells was strongly correlated with an increase in iNOS protein (Fig. 7A) . This positive correlation suggests that RA pretreatment did not affect the catalytic activity of iNOS and that a higher rate of new iNOS protein synthesis was indeed responsible for the higher level of NO production in RA-treated cells. The level of iNOS protein expression increased rapidly during the first 6 h of IFN-{gamma} stimulation in RA-pretreated cells and remained high for the next 18 h.

The results of various studies have shown that the ultimate expression of iNOS activity and NO production is regulated by several mechanisms, including transcriptional and post-transcriptional control. The transcriptional response to inflammatory and immunomodulatory factors such as IFN-{gamma}, LPS, IL-6, and tumor necrosis factor {alpha} is mediated through the binding of specific factors to DNA response elements in the region up to ~1.6 kb 5' of the transcription start site of the iNOS gene [37 , 38 ]. Although binding to these and several other response elements generally confers positive regulation of iNOS gene expression [38 ], negative regulation also has been reported [39 ], an example being transcription factor binding to an activator protein 1 site in the iNOS promoter [37 ]. Regulation may also occur via changes in iNOS mRNA stability, rate of mRNA translation, and protein degradation [40 41 42 43 44 ]. We had anticipated that the effect of RA on NO production would involve increases in both iNOS mRNA and iNOS protein. Such changes were observed in rats in vivo after treatment with RA and LPS [22 ]. However, in three independent time-course experiments, iNOS mRNA was not changed as a function of RA treatment, although its production was significantly induced by IFN-{gamma} (Fig. 6) . Nevertheless, iNOS protein expression was increased within 6 h of IFN-{gamma} stimulation to a much greater extent in RA-treated than in vehicle-treated cells (Fig. 7) . These results suggest that RA’s effect on iNOS expression may involve post-transcriptional mechanisms, possibly enhanced translational efficiency and stability, owing to the fact that iNOS protein increased rapidly, within 6 h, in RA-treated IFN-{gamma}-stimulated cells and, once induced, remained elevated for several hours. Although this significant increase in iNOS protein without a prior or concomitant change in iNOS mRNA was unanticipated, it was not surprising, given the known complexity of iNOS regulation.

Some previous studies have demonstrated the potential of RA to reduce iNOS gene expression or activity, implying an anti-inflammatory effect of RA treatment, while others have demonstrated RA’s potential to increase NO production [22–26 and this study], suggesting a proinflammatory action. Although these results seem opposed, we suggest that several potentially important factors may account for the differences that have been observed among studies with respect to the ability of RA to modulate iNOS expression and NO production. The potentiation of NO production by RA in our RAW 264.7 cells cultured under low serum retinoid conditions suggests that RA at physiological doses is necessary for an efficient response to low levels of immune or proinflammatory stimuli. In several other studies [16 17 18 , 21 ], the culture medium contained 5–10% FBS, which might have supplied sufficient vitamin A (retinol) to maintain cells in a state of vitamin A adequacy. In our studies with RAW 264.7 cells cultured in 1% FBS, the potentiation of NO production by RA occurred within a physiological range of RA concentrations, with greatest potentiation between 1 and 100 nM all-trans-RA. At 10 µM all-trans-RA, NO production was abolished. In previous cell studies [16 17 18 19 20 21 ], the higher the concentration of RA added, the greater was the inhibition of NO synthesis, with the greatest inhibition in the high nanomolar to micromolar range. These RA concentrations are ~10- to 50-fold above those present in normal plasma, and their use in cell studies is likely to reveal RA’s pharmacological potential. We also titrated a broad dose range of the iNOS inducing agents. This experimental design revealed that the potentiation of NO production by RA occurs in response to submaximal doses of iNOS-inducing stimuli. In contrast, several studies employed high doses of the iNOS-inducing agent [16 17 18 19 20 21 ] without dose titration [16 17 18 19 20 21 ]. Hence, it is not known whether the NO response to submaximal iNOS-inducing stimuli would be enhanced by RA in other cell types, similar to our results in RAW 264.7 cells. The states of cell differentiation, maturation, and prior activation have also varied among studies. In some studies that demonstrated RA’s potential to increase NO production [23 24 25 26 ], this was seen as a part of a program of RA-induced cell differentiation occurring over a course of several days, during which time there was a concomitant increase in both iNOS mRNA and protein levels [23 , 25 ]. Taking all of these studies together, it is apparent that RA’s effects on NO production are mediated in a context-dependent manner by several complex mechanisms that may include changes in iNOS gene expression [17 , 19 , 21 , 23 , 25 ] or altered iNOS protein levels without altered mRNA levels, as in this report. The variety of responses to RA among cells is reflective of the overall complexity of iNOS regulation and NO production. In vivo, differences in the number of iNOS-producing cells would also be expected to modulate outcome. It is still uncertain how NO responses in rodent models and humans compare quantitatively with one another. NO production in rodent macrophages may exceed production in human cells, but evidence for high-level NO production by human monocytes/macrophages has been presented [45 ]; MacMicking et al. [2 ] concluded, based on a review of studies with patients with infectious or inflammatory diseases, that human monocyte and macrophage iNOS (NOS2) is functionally expressed.

The results of these studies may have implications for understanding how vitamin A deficiency versus RA sufficiency shapes the immune response to infectious diseases. Vitamin A deficiency is known to be associated with poor cell-mediated and humoral immune responses and with increased severity of infectious diseases (see ref 9 and 46 for reviews). Vitamin A deficiency is thought to cause an imbalance in the development of Th1 and Th2 responses, with a relative expansion of Th1 cell production and Th1-type cytokine production and with a down-regulation of Th2 cell functions [11 , 12 , 47 ]. NO is reported to restrict Th1 cell proliferation and inhibit Th1 cell cytokine production [4 ]. Based on the results in the present study showing that physiological concentrations of RA potentiate the macrophage NO response to suboptimal concentrations of immune stimuli, it may be postulated (Fig. 8 ) that during the early stage of an infection, when amounts of the infectious or inflammatory agent are still low (submaximal), a deficiency of vitamin A (RA) would compromise the macrophage’s ability to produce NO in response to immune stimuli such as IFN-{gamma} and LPS. A low macrophage NO response, favorable to Th1 cell development, could be an initiating factor in the imbalance between the Th1 and Th2 cell functions observed in vitamin A deficiency in vivo [11 , 48 ]. It is interesting that a requirement for NO for optimal DNA synthesis by human peripheral-blood lymphocytes has been reported [49 ]. It also has been reported that antigen-activated Th1 cells, but not Th2 cells, are capable of a high output of NO on their own [50 ]. Therefore, if it is the case that Th2 cells are dependent on macrophages (or other cells) for NO production to initiate their differentiation, whereas Th1 cells are able to produce NO autonomously, the differential in macrophage-derived NO production due to RA may be especially important in initiating Th2 cell responses. An abnormally strong Th1 response due to vitamin A deficiency might predict that, later in the immune response, NO production would increase. However, iNOS expression and NO production in response to proinflammatory immune stimuli in vitamin A-deficient animals have not to our knowledge been reported. Further studies are needed to determine whether the response threshold to proinflammatory stimuli is elevated in vitamin A deficiency in vivo and whether decreased NO synthesis contributes to a microenvironment that is favorable to Th1 cell expansion and its sequelae.



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  		Figure 8. Model of the potential effect of vitamin A deficiency on nitric oxide (NO) production and its sequelae for the development of T helper cell (Th1/Th2) immunity. Provision of RA prior to macrophage stimulation is proposed to correct the initial low NO response, helping in turn to normalize the balance between the development of the Th1 response and that of the Th2 response.

 


   ACKNOWLEDGEMENTS
 
Support was provided by NIH grant R02 DK-41479 and funds from the Howard Heinz Endowment and the Dorothy Foehr Huck Chair. We thank Dr. Deborah Grove for quantitative real-time PCR analysis.

Received September 19, 2000; revised January 29, 2001; accepted January 31, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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