Retinoic acids exert direct effects on T cells to suppress Th1 development and enhance Th2 development via retinoic acid receptors
Makoto Iwata1,
Yuko Eshima1 and
Hiroyuki Kagechika2
1 Mitsubishi Kagaku Institute of Life Sciences, 11 Minamiooya, Machida-shi, Tokyo 194-8511, Japan 2 Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyou-ku, Tokyo113-0033, Japan
Correspondence to: M. Iwata; E-mail: iwata{at}libra.ls.m-kagaku.co.jp
Transmitting editor: T. Saito
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Abstract
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The vitamin A metabolite, retinoic acid (RA), affects Th1 and Th2 development. The effect is partly exerted through the modulation of antigen-presenting cell functions, but it remains unclear whether RA directly exerts its effect on T cells to influence Th1/Th2 development. To clarify this problem, we used two experimental systems with isolated T cells in vitro. In one system, isolated CD4+CD8+ thymocytes differentiated into Th1 and Th2 by two transient stimulations with defined combinations of ionomycin and phorbol myristate acetate followed by treatment with IL-2 and IL-4 and/or IL-12. In the second system, functional differentiation was induced in purified naive CD4 T cells from DO-11.10 TCR-transgenic and RAG-2-deficient mice with cytokines and antibodies to CD3 and CD28. In both systems, all-trans-RA at
1 nM concentrations suppressed Th1 development, but enhanced Th2 development. 9-cis-RA elicited similar effects. The optimal enhancement of Th2 development in the second system, however, was achieved with a delayed addition of RA. The presence of RA during the initial stimulation period often suppressed Th2 development. The RA receptor (RAR) antagonists, LE540 and LE135, but not the retinoic X receptor (RXR) antagonist, PA452, inhibited the effect of RA on Th1/Th2 development. Accordingly, the RAR agonists, Am80 and Tp80, but not the RXR agonists, HX600 and TZ335, mimicked the effect of RA. The RXR agonists enhanced the effect of the RAR agonists only slightly, if at all. These results indicate that, via RAR, RA directly suppresses Th1 development and directly enhances Th2 development with its timely addition.
Keywords: cytokine, differentiation, nuclear receptor, T lymphocyte, vitamin
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Introduction
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Vitamin A deficiency causes immune dysfunction with IFN-
overproduction and impaired antibody responses. There is a Th cell imbalance with excess Th1 and insufficient Th2 function (1). On the other hand, high-level dietary vitamin A enhances Th2 cytokine production and IgA responses (2,3), and is likely to decrease Th1 cytokine production (2). The vitamin A retinol contributes to signal transduction in immune cells by serving as a cofactor to augment the activation of c-Raf and protein kinase C (4), and the vitamin A metabolite retinoic acid (RA) influences various cellular functions including proliferation, differentiation and apoptosis in a variety of cell types, including immune cells (57). RA bind to two families of receptors, the RA receptor (RAR) isotypes (
, ß and
) and the retinoid X receptor (RXR) isotypes (
, ß and
). 9-cis-RA binds to both RAR and RXR, whereas the major physiologic RA, all-trans-RA, binds to RAR and may bind to RXR at high concentrations (8,9). These receptors function as ligand-inducible transcription factors, mainly as RXR/RAR heterodimers (9).
Both all-trans-RA and 9-cis-RA inhibit IL-12 production in activated macrophages, and RA pretreatment of macrophages reduces IFN-
production and increases IL-4 production in antigen-primed CD4+ T cells (10). Since the antigen-presenting cell (APC)-derived cytokine IL-12 is critical for Th1 development (11), RA may indirectly affect Th cell differentiation. Accordingly, Hoag et al. (12) have recently shown that all-trans-RA enhanced Th2 development through an effect on APC function in the presence of exogenous IL-4. However, Stephensen et al. (13) have suggested that 9-cis-RA and the RXR agonist, AGN194204, but neither all-trans-RA nor a RAR agonist, enhanced Th2 development in DO-11.10 TCR-transgenic (Tg) mouse-derived T cells bearing CD4 and the clonotypic TCR upon antigenic stimulation with APC. Furthermore, AGN194204 also enhanced Th2 development in CD4+CD62Lhigh lymph node cells from normal C57BL/6 mice upon stimulation with antibodies to CD3 and CD28 (13), suggesting that RXR-mediated stimulation may directly enhance Th2 development. However, CD4+CD62Lhigh cells from normal mice or even from TCR-Tg mice are likely to contain not only naive T cells, but also memory and other types of T cells (1416). Thus, it remains unclear if RA exerts a direct effect on the development of T cells into Th1 and Th2 cells, and if RXR or RAR is involved in its effect.
We focused on a possible direct effect of RA on Th cell differentiation, and used two independent culture systems to induce the functional differentiation of T cells into Th1 and Th2 cells in the absence of APC or other types of cells. We found that all-trans-RA, 9-cis-RA and the RAR agonists, but not the RXR agonists, suppressed Th1 development and enhanced Th2 development. The enhancement of Th2 development was, however, dependent on the timing of RA addition to the culture. Furthermore, RAR antagonists, but not a RXR antagonist, canceled the effect of RA on Th1 and Th2 development. These results clearly indicate that, through RAR, RA can directly inhibit Th1 development and enhance Th2 development with their timely addition.
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Methods
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Mice and reagents
DO-11.10
ßTCR-Tg mice with a RAG-2-deficient and B10.D2 background, and male MHC class I and II double-knockout (DKO) mice (C57BL/6 deficient in Aßb and ß2-microglobulin) were obtained from Taconic (Immuno-Biological Laboratories, Gunma, Japan). The mice were kept in our animal facility for at least 1 week before use. All-trans-RA, 9-cis-RA and phorbol myristate acetate (PMA) were obtained from Sigma (St Louis, MO). RA were dissolved in ethanol at 1 mM before dilution with medium and were handled under a small yellow light. Ionomycin was obtained from Calbiochem-Novabiochem (San Diego, CA). Mouse rIL-2, rIL-4 and rIL-12 were obtained from GenzymeTechne (Cambridge, MA). To express IL-2 and IL-4 activities, U was used by calculating 1 U/ml = 1 ED50 according to the manufacturers definition of ED50. Anti-transforming growth factor-ß mAb (clone 1D11) was obtained from R & D systems (Minneapolis, MN).
Thymocyte culture
DKO mouse thymocytes were used for in vitro induction of the differentiation of thymocytes into CD4 T cells and Th1/Th2 cells. T cell differentiation is arrested at the CD4+CD8+ stage in the thymus of these mice and most of the thymocytes are CD4+CD8+ (17). Further differentiation of T cells was induced in vitro as previously described (18). Briefly, CD4+CD8+ thymocytes were stimulated with 0.2 µg/ml ionomycin and 0.2 ng/ml PMA for 20 h at 37°C in 24-well tissue culture plates (25820; Corning, Corning, NY). The stimulated cells were washed and further cultured in the same volume of medium without the stimuli to obtain CD4+CD8low/ cells committed to the CD4 T cell lineage (19). For the secondary stimulation, the cells were cultured with 0.2 µg/ml ionomycin and 3 ng/ml PMA for 16 h. After culture, the cells were washed twice and further cultured at 1 x 106 cells/ml in medium containing 1050 U/ml IL-2 in the presence of the indicated concentrations of IL-4 and/or IL-12 with RA and/or antagonists or agonists of RAR or RXR for 4 days (Step D culture) (Fig. 1A) (20). For inducing exclusively Th1 development, 10 ng/ml IL-12, 1 µg/ml anti-IL-4 mAb and 50 U/ml IL-2 were added into the Step D culture (Th1 condition for cultured thymocytes). For inducing Th2 development, 100 U/ml IL-4, 1 µg/ml anti-IFN-
, 1 µg/ml anti-IL-12 and 10 U/ml IL-2 were added (Th2 condition for cultured thymocytes). For inducing both Th1 and Th2 development, 0.01 ng/ml IL-12, 100 U/ml IL-4 and 50 U/ml IL-2 were added (Th1/Th2 condition for cultured thymocytes). To assess the functional differentiation of the cells to Th1 or Th2, cytokines secreted upon stimulation were detected as previously described (18) using OptEIA mouse cytokine kits (BD PharMingen, San Diego, CA).

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Fig. 1. Effects of all-trans-RA and 9-cis-RA on the functional differentiation of cultured thymocytes into Th1 and Th2. (A) CD4 lineage commitment and functional differentiation were induced in DKO mouse thymocytes in vitro as described in Methods. (B) Graded concentrations of all-trans-RA (open circles) or 9-cis-RA (closed circles) were added to the Step D culture in the presence of IL-4, IL-12 and IL-2 (Th1/Th2 condition). (C and D) All-trans-RA (10 nM) was added to the Step D culture either (C) in the presence of IL-12, IL-2 and anti-IL-4 mAb (Th1 condition) or (D) in the presence of IL-4, IL-2, anti-IFN- mAb and anti-IL-12 mAb (Th2 condition). After 4 days of culture, the cells were collected, washed, and re-stimulated with plate-bound antibodies to CD3 and CD28. Culture supernatants were collected after 48 h of culture, and were assessed for concentrations of IFN- , IL-4, IL-5 and IL-10 by ELISA. Vertical bars represent SD in triplicate cultures. Statistical analysis was performed with Students t-test and asterisks indicate that the inhibition or enhancement by RA is significant (P < 0.001).
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Naive T cells and culture conditions
Splenic and lymph node CD4 T cells were obtained from DO-11.10 TCR-Tg/RAG-2-deficient mice by using Dynabeads Mouse CD4 and DetachaBead Mouse CD4 (Dynal, Oslo, Norway) according to the manufacturers instructions, and were incubated with MACS CD62L Microbeads (Miltenyi Biotec, Bergish Gladbach, Germany). The cells were washed and applied to MS columns to obtain CD62Lhigh cells according to the manufacturers instructions. More than 99% were CD4+CD62Lhigh, indicating highly purified naive CD4 T cells were obtained. Naive CD4 T cells were suspended in DMEM supplemented with 10% heat-inactivated FCS (Intergen, Purchase, NY), 3 mM L-glutamine, 1 mM sodium pyruvate, 1 x MEM non-essential amino acids, 50 µM 2-mercaptoethanol, 20 mM HEPES (pH 7.2), 20 U/ml penicillin and 20 µg/ml streptomycin, and were plated at a density of 1.5 x 105 cells/0.4 ml in 48-well suspension culture plates (MS-8048R; Sumitomo Bakelite, Tokyo, Japan) that had been coated with 3 µg/ml anti-CD3 mAb (145-2C11) (21) and 3 µg/ml anti-CD28 mAb (37.51) (BD PharMingen). Cytokines were added to wells at the initiation of culture as indicated. For inducing exclusively Th1 development, 4 ng/ml IL-12 and 5 µg/ml anti-IL-4 mAb were added (Th1 condition for naive T cells). For inducing exclusively Th2 development, 50 U/ml IL-4 and 2 µg/ml anti-IL-12 mAb were added (Th2 condition for naive T cells). For inducing both Th1 and Th2 development, 4 ng/ml IL-12 and 50 U/ml IL-4 were added (Th1/Th2 condition for naive T cells). After 2 days, cells were harvested and transferred to new 24-well culture plates (BD Falcon, Franklin Lakes, NJ) without antibodies, and were expanded 3 times with medium containing the same concentrations of cytokines and 50 U/ml IL-2 for 4 days. One milliliter of fresh medium containing 20 U of IL-2 with or without RA was added for the last 24 h to avoid cell death from starvation if necessary. In some experiments, supernatants were removed after the initial 2 days of culture with antibodies, and the cells were resuspended and cultured in 1.2 ml of fresh medium containing the same concentrations of cytokines and 50 U/ml IL-2. RA and/or antagonists or agonists of RAR or RXR were added as indicated. After culture, cells were harvested and washed twice, and re-stimulated for the measurement of cytokine production in the culture supernatant or intracellular cytokine production as previously described (18).
Semi-quantitative RT-PCR
Total RNA was prepared from cultured cells by using the lysis buffer Isogen (Wako Pure Chemical, Tokyo, Japan) as previously described (22). Oligo-dT primed first-strand cDNA was prepared from 5 µg total RNA using Superscript II reverse transcriptase (Life Technologies, Grand Island, NY) and the resulting cDNA was serially diluted. PCR was performed in a total volume of 30 µl containing 0.5 µl of appropriately diluted cDNA, 0.2 mM of dNTP, 0.6 µM of each primer and 1 x PCR buffer (Roche Molecular Systems, Branchburg, NJ), and 0.5 U of AmpliTaq DNA polymerase (Roche Molecular Systems) by using a Takara PCR thermal cycler (TP480). After the first denaturation step (94°C for 3 min), the amplification was performed for 25 or 30 cycles at 94°C for 30 s, 60°C for 30 s and 72°C for 1 min (25 cycles for ß-actin, IL-12Rß2, GATA-3 and IL-4R
; 30 cycles for T-bet and c-maf). The final cycle was followed by an extension step of 5 min at 72°C. Sequences of the sense and anti-sense primers were as follows: ß-actin, 5'-CGTGGGCCGCCCTAGGCACCA-3' and 5'-TTGGCCTTAGGGTTCAGGGGGG-3' (35); T-bet, 5'-TGCC TGCAGTGCTTCTAACA-3' and 5'-GCTGTGAGATCATATCC TTGGG-3'; 12Rß2, 5'-TCTGCGAAATTCAGTACCGACG-3' and 5'-CATGCCATCAGGAGATTATCCG-3'; GATA-3, 5'-CTC CAAGTGTGCGAAGAGTTCCTC-3' and 5'-GTGCCCATTTGG ACATCAGACT-3'; c-maf, 5'-GACGAGCAGTTGGTGACC ATG-3' and 5'-GCTCACCAGCTTCTCGTATTTC-3'; IL-4R
, 5'-GCTCCGTGCCCTTATTTACTTTCG-3' and 5'-GGTTGG CTTCTGGTGGTATTCC-3'. PCR products were resolved by electrophoresis on a 1.3% agarose gel containing ethidium bromide. T-bet, IL-12Rß2, GATA-3, c-maf, IL-4R
, and ß-actin cDNA yielded PCR products of 305, 376, 423, 273, 382 and 243 bp respectively. Fluorescence images were captured by a video monitor system (Image Freezer; ATTO, Tokyo, Japan) and band intensities were quantified using the analytical program Densitograph 4.0 (ATTO). The bands that corresponded to the exponential amplification phases were analyzed, and the intensities of cDNA-derived bands were normalized with those of ß-actin cDNA-derived bands.
Statistical analysis
The significance of mean values was determined by Students t-test. Values of P > 0.05 were considered insignificant.
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Results
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RA directly influence the functional differentiation of T cells into Th1 and Th2 in vitro
To examine the direct effect of RA on T cell differentiation, we first used our in vitro T cell differentiation system starting from CD4+CD8+ thymocytes of DKO mice. Thymocyte differentiation was induced by 2 times transient stimulation with ionomycin/PMA in the absence of other types of cells such as APC and thus provided >108 T cells per mouse ready for differentiation into Th1 or Th2 (Fig. 1A) (18,20). The thymocytes stimulated twice with ionomycin/PMA were washed, and cultured with IL-2 and IL-4 and/or IL-12 in the presence of all-trans-RA or 9-cis-RA for 4 days. The cultured cells were re-stimulated with plate-bound antibodies to CD3 and CD28 to assess their ability to produce IFN-
, IL-4, IL-5 and IL-10. In the presence of both IL-12 and IL-4 (Th1/Th2 condition), all-trans-RA and 9-cis-RA suppressed IFN-
production and enhanced IL-4 and IL-5 production dose-dependently (Fig. 1B). The effect of all-trans-RA at 1 nM on IFN-
and IL-4 production was more significant than that of 9-cis-RA at 1 nM, but the two RA exerted similar effects on the cytokine production. Thus, all-trans-RA at physiological or near-physiological concentrations (23) appears to affect Th1/Th2 development, although the effective concentrations of RA in vitro and in vivo should be different. IL-10 production was slightly enhanced by RA at high concentrations (Fig. 1B). In the presence of IL-12 without IL-4 (Th1 condition), all-trans-RA also suppressed the production of IFN-
(Fig. 1C), and in the presence of IL-4 without IL-12 (Th2 condition), all-trans-RA enhanced IL-4 production (Fig. 1D). These results suggest that both all-trans-RA and 9-cis-RA inhibit Th1 development and enhance Th2 development.
Th1 and Th2 development from naive T cells is affected variably depending on the timing of RA addition
To confirm the results obtained with the thymocyte differentiation system, we chose DO-11.10 TCR-Tg mice to prepare naive CD4+ T cells. Since these mice were RAG-2-deficient, they lacked endogenous TCR
chains and expressed the transgenic TCR
ß exclusively. Thus, few memory or other types of cells should contaminate the CD4+CD62Lhigh naive T cell preparation in the absence of ovalbumin peptide. Purified CD4+CD62Lhigh naive T cells were stimulated with plate-bound anti-CD3 and anti-CD28 in the presence of IL-12 or IL-4 to induce differentiation into Th1 and Th2 cells respectively. After 2 days of stimulation, the cells were further cultured in the presence of the same cytokine plus IL-2, but without antibodies, for 4 days, and were assessed for their ability to produce IFN-
and IL-4 (Fig. 2A). When 10 nM of all-trans-RA was added at the beginning of culture, both IFN-
production in the Th1 condition and IL-4 production in the Th2 condition were suppressed (Fig. 2B and C). However, when all-trans-RA was added after the initial stimulation, IL-4 production in the Th2 condition was significantly enhanced (Fig. 2C). The effect of all-trans-RA was not significant when it was added 3 or 4 days after the start of culture. In the absence of exogenous cytokines, IFN-
production was suppressed by 10 nM of all-trans-RA, but an increase in IL-4 production was not detected even with a delayed addition of RA (data not shown).
We then studied the effect of RA on Th1 and Th2 development in naive CD4 T cells in the presence of both IL-12 and IL-4 (Th1/Th2 condition) by determining their ability to produce intracellular IFN-
and IL-4. All-trans-RA at 10 nM enhanced the induction of IL-4+ cells, but suppressed the induction of IFN-
+ cells, when it was added during the first 3 days of culture (Fig. 3). The enhancement of IL-4+ cells was most efficient when all-trans-RA was added 1 or 2 days after the start of culture in repeated experiments (Fig. 3A and data not shown). The suppression of IFN-
+ cells was most efficient when all-trans-RA was added 1 day after the start of culture, not at the start of culture (Fig. 3A and B). However, we found that, with RA added at the start of culture, the majority of the induced IFN-
+ cells bore low levels of IFN-
and that the ability to produce IFN-
into the supernatant upon re-stimulation was equally low if all-trans-RA was added within 2 or 3 days after the start of culture (data not shown). The cells that received RA 1 day after the start of culture produced 151 ± 11 ng/ml IFN-
and 63 ± 5 ng/ml IL-4 upon re-stimulation, while those without RA addition produced 393 ± 52 ng/ml IFN-
and 44 ± 5 ng/ml IL-4 in triplicate cultures, indicating that the delayed addition of RA also modulated the ability of T cells to produce IFN-
and IL-4 in this culture condition. The optimal concentrations of all-trans-RA to increase IL-4+ cells and to decrease IFN-
+ cells were 10100 nM when it was added 1 day after the start of culture (Fig. 3C). These results indicate that all-trans-RA suppresses Th1 development if it is added during or immediately after the TCR-mediated stimulation period and that all-trans-RA enhances Th2 development if it is added 1 day after the start of culture or immediately after the stimulation period. A delayed addition of 10 nM 9-cis-RA also suppressed Th1 development and enhanced Th2 development (data not shown).
All-trans-RA inhibits the expression of Th1-associated genes and enhances that of Th2-associated genes
To examine if all-trans-RA also regulates the expression of genes essential for Th1 and Th2 development, mRNA levels of T-bet, IL-12Rß2, GATA-3, c-maf and IL-4R
were semi-quantitatively assessed by RT-PCR. CD4+CD8+ thymocytes of DKO mice were treated with ionomycin/PMA 2 times and cultured in the Th1/Th2 condition. Both Th1- and Th2-associated genes were expressed with or without RA (Fig. 4). However, all-trans-RA at 10 nM suppressed the expression of the Th1-associated genes, T-bet (24) and IL-12Rß2 (25), and enhanced the expression of the Th2-associated genes, c-maf (26) and IL-4R
(27) (Fig. 4). The suppression of IL-12Rß2 was consistently observed, but was mostly moderate. The expression of GATA-3, a gene expressed in naive T cells and Th2 cells, but marginally in Th1 cells (28), was enhanced by RA (Fig. 4). The results further support the notion that all-trans-RA directly regulates Th1/Th2 development.

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Fig. 4. All-trans-RA affects the mRNA expression levels of Th1-associated genes and Th2-associated genes during Th cell differentiation. DKO mouse thymocytes were stimulated twice as shown in Fig. 1 and vehicle or 10 nM all-trans-RA was added to the Step D culture in the Th1/Th2 condition for 4 days. The reverse-transcribed cDNA from each sample was serially diluted, and each diluted sample was subjected to PCR amplification for T-bet and c-maf for 30 cycles, and IL-12Rß2, GATA-3 and ß-actin for 25 cycles. (A) The PCR products were resolved by electrophoresis on a 1.3% agarose gel containing ethidium bromide. (B) Relative expression levels of mRNA of each gene were calculated. A representative result of three experiments is shown.
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RA regulate Th1/Th2 development through RAR
9-cis-RA binds to both RAR and RXR, whereas all-trans-RA binds to RAR and may bind to RXR at high concentrations. The doseresponse analysis with all-trans-RA and 9-cis-RA in Fig. 1 suggested that RAR rather than RXR might be responsible for the effect of RA on Th1/Th2 development. However, isomerization of all-trans-RA to 9-cis-RA might occur within the cells (29). We thus examined the effect of antagonists of RAR and RXR on Th1/Th2 development. CD4+CD8+ thymocytes of DKO mice were treated with ionomycin/PMA 2 times and cultured with an antagonist or agonist in the presence or absence of RA. The cells were collected, and their ability to produce IFN-
and IL-4 was determined. As shown in Fig. 5(A and D), 1 µM of the RAR pan-antagonist LE540 (30) partially canceled the suppressive effect of 1 nM all-trans-RA on IFN-
production, and partially or completely canceled the enhancing effect of all-trans-RA and 9-cis-RA on IL-4 production, although LE540 by itself at 1 µM slightly inhibited IFN-
production and slightly enhanced IL-4 production. LE135 is a potent antagonist of RARß and a less potent antagonist of RAR
(30). LE135 also canceled the effect of 1 nM all-trans-RA on IL-4 and IFN-
production (Fig. 5B and E). On the other hand, the RXR pan-antagonist PA452 (31) at up to 1 µM failed to cancel the effects of 1 nM all-trans-RA (Fig. 5C and F).
We then determined the effect of agonists of RAR and RXR on Th1/Th2 development in the same culture system. Am80 is a potent RAR
agonist, and is a less potent agonist of RARß (32). Am80 dose-dependently suppressed IFN-
production and enhanced IL-4 production (Fig. 6A). The RAR pan-agonist Tp80 (33) suppressed IFN-
production and enhanced IL-4 production at 10 nM (Fig. 6B). On the other hand, the RXR pan-agonists HX600 (30), TZ335 (34) and PA024 (35) only slightly affected the production of IFN-
and IL-4 (Fig. 6C). The RXR pan-agonists at 100 nM exerted little effect if any on the Am80- or Tp80-induced modulation of IFN-
and IL-4 production (Fig. 6A and B). These results collectively indicate that RAR
and/or ß are involved in the direct effect of RA on Th1/Th2 development, but that RXR activation may not play an important role in the effect.
RAR, but not RXR, is critical for RA to exert their effects on the differentiation of naive T cells to Th1 and Th2 cells
Effects of antagonists and agonists of RAR and RXR on Th1 and Th2 development in naive CD4+ T cells from DO-11.10 mice were also examined. An antagonist, an agonist and/or 10 nM all-trans-RA were added 1 day after the start of culture in the Th1/Th2 condition. The RARß (and
) antagonist LE135, but not the RXR pan-antagonist PA452, canceled the effect of all-trans-RA on the induction of IL-4+ cells and IFN-
+ cells (Fig. 7). On the other hand, the RAR
(and ß) agonist Am80, but not the RXR pan-agonist HX600, exerted effects similar to those of all-trans-RA on IL-4 and IFN-
production (Fig. 8). HX600 failed to enhance the effects of Am80. Similar results were obtained in the analyses of intracellular cytokine production (data not shown). These results confirm that RAR
and/or ß, but not RXR, are involved in the RA-induced regulation of Th1/Th2 development.

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Fig. 8. Effects of agonists of RAR and RXR on Th1 and Th2 development in naive T cells. CD4+ naive T cells from DO-11.10 mice were stimulated and cultured in the Th1/Th2 condition as shown in Fig. 3. All-trans-RA (10 nM), Am80 (100 nM) and/or HX600 (100 nM) were added 24 h after the start of culture. The cells were then re-stimulated for 48 h with plate-bound antibodies to CD3 and CD28, and the production of IFN- (A) and IL-4 (B) was determined. The data are expressed as means ± SD of triplicate cultures.
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Discussion
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RA modulates Th1/Th2 responses in vivo. Previous reports indicate that APC is one of the target sites of RA. RA also directly down-regulates IFN-
production in an established Th1 cell line upon stimulation with antibodies to CD3 and CD28 (36). However, it remained unclear whether RA directly exerts its effect on naive T cells to influence Th1/Th2 development. In the present study, we demonstrated that both all-trans-RA and 9-cis-RA directly suppressed Th1 development and enhanced Th2 development in naive T cells in the absence of APC or other types of cells. For the enhancement of Th2 development, however, the timing of RA addition was critical. RA can be suppressive of Th2 development if it is given at the initiation of TCR stimulation. This phenomenon may contribute to the lack of a primary IgE response in mice treated with RA together with antigen in spite of a potentiated secondary IgE response in these mice (37). TCR-mediated T cell activation involves AP-1 activity and RA negatively regulate AP-1-responsive genes in a retinoid receptor-dependent fashion (38). Thus, the suppression of both Th1 and Th2 development by RA given at the initiation of culture may involve the retinoid receptor-mediated inhibition of AP-1 activity.
9-cis-RA binds to both RAR and RXR, whereas all-trans-RA binds well to RAR, but not to RXR (8,9). Since isomerization of all-trans-RA to 9-cis-RA might occur within the cells (29), to clarify which receptor family is critically involved in the direct effect of RA on the functional differentiation of T cells, we used several antagonists and agonists of RAR and RXR. The results indicated that RAR
and/or ß are involved in the direct effect of RA on Th1/Th2 development. Although RAR function as ligand-inducible transcription factors as RXR/RAR heterodimers (9), RXR activation does not appear to play an important role in the direct effect of RA on Th1/Th2 development. In contrast to our results, Stephensen et al. (13) have recently suggested that the RXR agonist AGN194024 enhanced Th2 development upon stimulation with antibodies to CD3 and CD28 without exogenous IL-4. However, they used CD4+CD62Lhigh lymph node cells from normal C57BL/6 mice as naive T cells. It is likely that these cell preparations not only contained naive T cells, but also contained memory and other types of T cells (1416). Thus, an indirect effect through the activation of RXR in the latter cell populations might be involved in the enhanced Th2 development. Nonetheless, some RXR agonists may induce weak effects on Th cell differentiation, since TZ335, one of the RXR agonists we used in Fig. 6(C), exerted a weak effect on IL-4 production at a high concentration (1 µM) in the presence of IL-4 and IL-12. It might be possible that heterodimers of RXR with its permissive partners including peroxisome proliferator activator receptors, LXR
and NGFI-B (9) directly affect Th1/Th2 development through the ligation of RXR under some conditions.
Kang et al. (10) reported that pretreatment with the RAR agonist 4-[E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid (TTNPB) inhibited IL-12 production by macrophages stimulated with lipopolysaccharide or heat-killed Listeria monocytogenes, although pretreatment with all-trans-RA or 9-cis-RA was more effective. TTNPB- or retinoid-pretreated macrophages reduced their ability to induce IFN-
and increased the ability to induce IL-4 in antigen-primed CD4+ T cells. However, the same group (39) also suggested a possibility that retinoids inhibit IL-12 production in macrophages through physical association of RXR and NF-
B. Indeed, Stephensen et al. (13) have shown that the RXR agonist AGN194204 enhanced Th2 development upon antigenic stimulation with APC in repeated experiments and that TTNPB exerted little effect. Thus, retinoid-induced modulation of the APC function may largely involve the regulation of RXR activity.
RA has been shown to induce or enhance transforming growth factor (TGF)-ß activity in some types of cells (40). TGF-ß blocks Th1 development by inhibiting the expression of IL-12Rß2 or T-bet (41,42). It may be possible that RA-induced TGF-ß suppresses Th1 development, although TGF-ß also blocks Th2 development through inhibition of GATA-3 expression (43,44). RA, however, did not enhance TGF-ß production during the stimulation of DO-11.10 mouse-derived naive T cells (data not shown). Furthermore, addition of an anti-TGF-ß mAb enhanced the induction of both Th1 and Th2 development, and failed to cancel the effect of RA (data not show). We noticed, however, that RA enhanced Th2 development in naive CD4+ T cells more efficiently when the cells were resuspended in fresh medium after 2 days of stimulation with antibodies than when simply expanded with fresh medium, suggesting that some soluble factor(s) produced by the cells themselves may disturb the differentiation. The suppression of IL-12Rß2 expression by RA was consistently observed, but was mostly moderate (Fig. 4), suggesting that it may not be a main cause of the suppression of Th1 development.
RA down-regulates IFN-
production in an established Th1 cell line (36). We found that RA also down-regulated IFN-
production in naive T cells after 2 days of stimulation with antibodies to CD3 and CD28 (data not shown). It may partly contribute to the suppression of Th1 development. The molecular mechanism of the RAR-dependent effect of RA on Th1/Th2 development still remains to be elucidated.
The modernization of life styles is often accompanied by an increase in allergic diseases. Because the increase has been so rapid, it is likely that changes in environmental rather than genetic factors are mainly involved. Although vitamin A deficiency still causes serious problems in many developing areas of the world (45), vitamin A intake levels in modernized areas may tend to skew the Th cell balance toward Th2. It may be also possible that some environmental chemicals mimic the effect of RA through binding to RAR. Hind-limb malformations found in amphibians across North America within the last decade can be mimicked with an exposure of embryos to RA, although xenobiotic disturbance has not been proved to cause the malformations in the field (46). RA may be beneficial, however, in the treatment of multiple sclerosis or experimental allergic encephalomyelitis (2) and other Th1-mediated diseases. By clarifying the mechanism by which RA modulates Th1/Th2 development, better methodologies or the cure of various immune diseases may be discovered.
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Acknowledgements
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We thank S. Kamijo and coworkers for their help with animal care, A. Hirakiyama for technical assistance and K. Hirose for secretarial assistance.
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Abbreviations
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APCantigen-presenting cell
DKOMHC class I and class II double knockout
PMAphorbol myristate acetate
RAretinoic acid
RARretinoic acid receptor
RXRretinoic X receptor
Tgtransgenic
TGFtransforming growth factor
TTNPB4-[E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid
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