Prevention of acute and chronic allograft rejection by a novel retinoic acid receptor-
-selective agonist
Ken-ichiro Seino1,2,3,
Toshihiko Yamauchi4,
Kohdoh Shikata4,
Seiichi Kobayashi5,
Mitsuo Nagai4,
Masaru Taniguchi1,6 and
Katashi Fukao3
1 RIKEN Research Center for Allergy and Immunology, Yokohama, Kanagawa 230-0045, Japan 2 PRESTO, JST, Kawaguchi, Saitama 332-0012, Japan 3 Department of Surgery, Institute of Clinical Medicine, University of Tsukuba, Tsukuba Science City, Ibaraki 305-8575, Japan 4 Tsukuba Research Laboratories, Eisai Co., Ltd, Tsukuba-shi, Ibaraki 300-2635, Japan 5 Eisai Research Institute, Eisai Co., Ltd, 100 Research Drive, Wilmington, MA 01887, USA 6 Department of Molecular Immunology, Chiba University, Chiba 260-8670, Japan
Correspondence to: K.-i. Seino, Laboratory for Immune Regulation, RIKEN Research Center for Allergy and Immunology, 1-7-22 Suehiro-cho, Tsurumi, Yokohama-City, Kanagawa 230-0045, Japan. E-mail: seinok{at}rcai.riken.jp
Transmitting editor: K. Okumura
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Abstract
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To investigate the involvement of retinoic acid receptor (RAR)-
in allograft rejection, we investigated the effect of a novel selective agonist to the receptor, ER-38925, in a mouse cardiac allograft model. Prophylactic treatment with ER-38925 inhibited the acute rejection of the mouse cardiac allograft (BALB/c
C3H/HeN) at 0.3 and 3 mg/kg, and its effect was enhanced in combination with tacrolimus. In this model, ER-38925 remarkably inhibited cytotoxic T lymphocyte induction and alloantigen-stimulated production of cytokines, i.e. IL-2, IL-12 and IFN-
. In the chronic rejection model, combined treatment with tacrolimus and ER-38925 reduced the grade and incidence of arteriosclerosis in the cardiac allografts significantly more potently than tacrolimus monotherapy. ER-38925 inhibited the proliferation of rat aortic smooth muscle cells stimulated in vitro, presumably through the induction of a cyclin-dependent kinase inhibitor, p27kip-1. Those results provide a rationale for using RAR-
agonists as immunosuppressants in human organ transplantation.
Keywords: arteriosclerosis, cell differentiation, retinoid, Th1/Th2, transplantation
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Introduction
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Retinoids are vitamin A derivatives used for the treatment of vitamin A deficiency and dermatological disorders, as well as in chemoprevention and therapy of certain cancers (1). Retinoids regulate gene expression through the action of retinoic acid receptors (RAR) and retinoid X receptors (RXR), which both belong to the nuclear hormone receptor family (2). Both RAR and RXR consist of three subtypes:
, ß and
. Those subtypes are expressed in various organs in a spatially and/or temporally regulated manner, and mediate the pleiotropic effects of retinoids, including beneficial and adverse effects. The overall effects of retinoids in particular organs are mainly dependent on what subtypes of RAR and/or RXR are expressed in those organs.
The influence of endogenous vitamin A and retinoic acid on the immune system has been widely investigated (3). Some synthetic retinoids were reported to ameliorate the symptoms in various immunological disease models. Retinoids such as 4-hydroxyretinamide, all-trans-retinoic acid (atRA) and 9-cis-retinoic acid (9-cis-RA) were reported to be effective in rat adjuvant arthritis (4) and experimental allergic encephalomyelitis (EAE) (5), both of which are believed to be caused by cell-mediated immunity. Recently, Am80, a specific RAR-
agonist, was reported to have more potent immunosuppressive effects than non-selective RAR agonists. This compound remarkably ameliorated various immunological diseases, such as type II collagen-induced arthritis in rats (6) and mice (7), and rat EAE (8). In these models, Am80 potently inhibited antibody production against the immunizing antigen. In accordance with these reports, we recently found that our newly discovered RAR-
-selective agonists had a strong immunosuppressive effect on either cell-mediated or humoral immunity (9) and one of those agonists remarkably ameliorated the autoimmune disorder in murine lupus nephritis model (submitted). These observations prompted us to investigate whether a RAR-
-selective ligand would be effective not only in autoimmune disease models, but also in an allograft rejection model. This strategy is in line with the fact that allograft rejection is mainly mediated by T cells in which RAR-
as well as other subtypes are expressed (10). In order to address this issue, we used a newly discovered RAR-
-selective agonist, ER-38925, and evaluated its effects in a mouse cardiac allograft model. The present study clearly showed that our RAR-
-selective retinoid agonist could inhibit not only acute, but also chronic rejection of a mouse cardiac allograft and remarkably prolonged its lifespan.
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Methods
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Mice
Male C3H/HeN (H-2k) and BALB/c (H-2d) mice were purchased from Charles River Japan (Kanagawa, Japan), and used at 812 weeks old as recipients and donors respectively. All mice were maintained under standard conditions, and provided with pellet food and ultraviolet-sterilized water ad libitum.
Reagents
ER-38925 (9) (4-[5-(4,7-dimethylbenzo[b]furan-2-yl)-1H-2-pyrrolyl]benzoic acid) was synthesized at Eisai, Tsukuba Research Laboratories (Ibaraki, Japan). atRA and 9-cis-RA were purchased from Sigma (St Louis, MO). Am80 (11), a RAR-
selective agonist, was from Wako Pure Chemical (Osaka, Japan). CD417 (12), CD666 (12) and LGD1069 (13), which are RAR-ß-, RAR-
- and RXR-selective agonists respectively, were synthesized at Eisai Tsukuba Research Laboratories. Injectable solutions of tacrolimus (Prograf; Fujisawa Pharmaceuticals, Osaka, Japan) and cyclosporin A (CyA; Sandimmune; Novartis Pharma, Tokyo, Japan) were diluted with physiological saline before use. For cytokine ELISA, all capture and biotinylated anti-cytokine antibodies were purchased from PharMingen (San Diego, CA), except for anti-IL-4 (Endogen, St Woburn, MA). Horseradish peroxidase-labeled goat anti-biotin polyclonal antibody was from Vector (Burlingame, CA). Recombinant murine IL-2, IL-4, IL-12 (p40/p35 heterodimer) and IFN-
were purchased from R & D systems (Minneapolis, MN), and used as standards.
Cardiac allograft
Vascularized, heterotopic intra-abdominal cardiac transplantation was performed according to the Ono and Lindsey procedure (14). Under barbiturate and ether anesthesia, donor hearts were placed in the abdomens of the recipients, with aorta to aorta and pulmonary artery to vena cava anastomoses performed under magnification. Function of the transplanted hearts was assessed by daily palpation of ventricular contraction through the abdominal wall. Rejection was defined as loss of myocardial contraction and was confirmed at laparotomy under barbiturate anesthesia. After cardiac transplantation, different doses of ER-38925 were administered p.o. by gavage, once a day, from 0 to 14 days post-transplantation (DPT). Control mice were given vehicle only. In some experiments, mice with a cardiac allograft were treated with tacrolimus (0.1 mg/kg/day, i.p., 029 DPT) with or without simultaneous oral ER-38925 (0.3 mg/kg/day). Five recipients with the combination therapy were observed for >100 DPT for estimating their graft survivals (Fig. 1A). For examining post-transplant arteriosclerosis, all recipient mice treated with either tacrolimus alone or combination therapy were killed at 30 DPT to harvest their heart allografts for evaluation of graft arteriosclerosis (see Fig. 4). In tacrolimus monotherapy group shown in Fig. 4, six out of eight grafts were rejected before 30 DPT and were harvested for histopathology just after rejection was confirmed at daily palpation. At the biopsy, however, two out of those grafts were found to be so badly damaged that they were excluded from histopathological analysis.

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Fig. 1. ER-38925 prolonged the survival of mouse cardiac allografts. (A) Cardiac allograft survivals with treatment with ER-38925 at 0.3 (solid squares) or 3 mg/kg (solid triangles) from 0 to 14 DPT, and combination of ER-38925 (0.3 mg/kg) and tacrolimus (0.1 mg/kg, i.p., from 0 to 29 DPT) (solid circles). Vehicle-treated mice served as controls (open circles). Animal died of undefined cause without any sign of graft rejection. Significant difference from vehicle control, *P < 0.01 by log-rank test. (B and C) Histopathology of the cardiac allografts from untreated (B) and 3 mg/kg ER-38925-treated (C) animals. Original magnification x40.
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Fig. 4. Prevention of cardiac allograft arteriosclerosis by combination therapy with ER-38925 and tacrolimus. (A) ER-38925 synergistically improved the survival of mouse cardiac allografts in combination with tacrolimus. There was significant difference between the two groups, *P < 0.05 by log-rank test. Extended observation of the survival for combination therapy was performed in another set of experiment, and the result is shown in Fig. 1(A). (B and C) Incidence (B) and grade (C) of graft coronary arteriosclerosis were measured as indicated in Methods. Significant difference between the two groups, *P < 0.05 by Students t-test, P < 0.05 by MannWhitney U-test. (D and E) Representative histopathology of the graft arteriole from the animal treated with tacrolimus alone (D) or tacrolimus + ER-38925 (E). Original magnification x40.
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Histology
Tissue samples were fixed in phosphate-buffered 10% formalin and embedded in paraffin. Five-micrometer sections were stained with Elastica van Gieson to show the internal elastic lamina. Changes in intimal thickness in arterioles and arteries were scored according to the following criteria originally reported by Billingham (15): grade 0, normal vessels with intact internal elastic lamina; grade 1, <10% occlusion of lumen by intimal thickening and proliferation, disruption of internal elastic lamina, with some form and vacuolated endothelial cells; grade 2, <50% occlusion of lumen; grade 3, 5099% occlusion of lumen; and grade 4, 100% occlusion of vessel lumen. All of the arteries and arterioles in each section were scored by an observer in a blinded fashion. Incidence of intimal thickening indicates percentage of vessels scored at grade 1 or more, and intimal thickness is expressed as an average score of all arteries and arterioles present in a graft.
Mixed lymphocyte reaction
Spleens from either BALB/c or C3H/HeN mice were aseptically removed and teased into a single-cell suspension. Spleen cells were layered onto Lympholite-M (Cedarlane, Hornby, Ontario, Canada) and centrifuged at room temperature. The resulting lymphocyte-enriched fraction was collected and 105 viable C3H/HeN lymphocytes were cultured with or without 105 mitomycin C (Sigma)-treated BALB/c splenocytes in 0.2 ml of complete RPMI 1640 medium (10% FCS, 50 µM 2-mercaptoethanol and antibiotics) at 37°C, 5% CO2 for 6 days. The cultures were pulsed with 0.5 µCi of [3H]thymidine (DuPont NEN, Boston, MA) for the last 16 h and radioactivity incorporated into the cells was counted with a ß-plate counter (Pharmacia Biotech, Uppsala, Sweden). In other experiments, the culture supernatants were harvested on days 13, and analyzed for IL-2, IL-4, IL-12 and IFN-
by capture ELISA.
Cytotoxic T lymphocyte (CTL) assay
C3H/HeN mice were i.p. injected with 8 x 107 BALB/c spleen cells. Five days later, recipients were anesthetized and killed by cervical dislocation, and their spleen cells were washed and resuspended in RPMI 1640 at various cell densities. 51Cr (DuPont NEN)-labeled target cells (P-815, mouse mastocytoma; H-2d) were plated at 1 x 104/well in 96-well round-bottomed plates with effector cells at various E:T ratios. After incubation at 37°C, 5% CO2 for 4 h, 51Cr radioactivity released into the culture supernatant was counted in a TopCount Microplate Scintillation Counter (Packard, Meriden, CT). Percent-specific lysis was defined as (a b)/(c b), where a and b are c.p.m. released by target cells incubated with or without effector cells respectively, and c is c.p.m. released by target cells incubated with 1% Triton X-100 (100% release).
ELISPOT assay
Cytokine-producing cells were detected by ELISPOT assay. Briefly, MaltiScreen plates with 96 wells (Millipore, Molsheim, France) were coated with capture antibodies and then blocked with PBS containing 1% BSA. Plates were washed with PBS, and then 1 x 105 cells of both recipient and mitomycin C-treated donor spleen cells were added to the wells, and incubated at 37°C, 5% CO2. Twelve hours later, spleen cell suspension was removed from each well and the plates were washed 3 times with PBS containing 0.5% Tween 20. Then biotinylated anti-IL-4, -IL-12 and -IFN-
were added at 1 µg/ml for 1 h at 37°C. The plates were washed and goat anti-biotin IgG at 1 µg/ml was added. After 1 h incubation at 37°C, the plates were washed then developed with TMB membrane peroxidase substrate (Kirkegaard & Perry, Gaithersburg, MD).
Rat smooth muscle cell (RSMC) culture
RSMC were prepared as previously described from abdominal aorta of normal SD rats (16), and maintained in DMEM (Gibco/BRL) supplemented with 10% FCS and antibiotics (complete DMEM). Cells were trypsinized and seeded onto 96-well flat-bottomed microtiter plates in complete DMEM at a concentration of 2 x 104/ml, and allowed to adhere to the wells and grow to confluence for 2 days. After 48 h of serum starvation (DMEM without FCS), the quiescent cells were pre-incubated with retinoids at various concentrations. After 24 h, growth stimuli, i.e. basic fibroblast growth factor (bFGF; PeproTech, London, UK) and 1% FCS, were added to the cultures for 24 h. RSMC proliferation was detected as [3H]thymidine uptake in the last 5 h.
Western blot analysis
For western blotting, 2 x 105 RSMC were seeded on six-well flat-bottomed plates in a volume of 2 ml complete DMEM and incubated for 2 days. After 48 h of serum starvation, the quiescent cells were pre-incubated with the drug for 24 h and then bFGF was added to the cultures at 10 ng/ml for 24 h. The resultant cells were rinsed with PBS and harvested after having been detached with trypsinEDTA. Cells were lysed in ice-cold lysis buffer (50 mM TrisHCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 1 mM EGTA, 1 mM PMSF, 1 mM DTT, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 1 mM glycerophosphate, 2 µg/ml aprotinin and 1 µg/ml leupeptin), and the lysates were centrifuged at 12,000 r.p.m. for 10 min at 4°C. An equal amount (30 µg) of protein in the supernatant was subjected to SDSPAGE using a 1020% gradient gel under reducing conditions and transferred onto Immobilon PVDF transfer membranes (Millipore, Bedford, MA). The membrane was blocked and probed with anti-p27kip1 antibody (1:500, Santa Cruz, CA) and then developed with peroxidase-conjugated second antibody. Immuno reactive bands were visualized by using the ECL western blotting detection reagents (Amersham Pharmacia Biotech, Uppsala, Sweden) followed by exposure to Hyperfilm ECL (Amersham Pharmacia). The bands were scanned by an image scanner (GT-8000; Epson, Tokyo, Japan) and relatively quantified by the use of Quantity One software (PDI, Huntington Station, NY).
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Results
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Anti-proliferative effect of retinoids on mouse spleen cells
The effects of retinoids on alloantigen-stimulated proliferation of C3H/HeN spleen cells were investigated. All retinoids used in this experiment inhibited mixed lymphocyte reactions of recipient splenocytes in a dose-dependent manner and their IC50 values in this assay are shown in Table 1, together with their retinoid receptor subtype selectivity determined as described in our previous report (9). As indicated, our newly synthesized retinoid, ER-38925, binds to and activates the retinoid receptors in a RAR-
dominant manner. Among the retinoids, ER-38925 and Am80 inhibited the proliferation with the lowest IC50 values (ER-38925: 0.2 nM, Am80: 0.4 nM), followed by 2.4 nM 9-cis-RA and 3.5 nM atRA. All these compounds also bound to and induced transactivation via RAR-
with high potency. Conversely, the retinoids with low affinity to RAR-
, such as CD417 (RAR-ß selective) (12), CD666 (RAR-
selective) (12) and LGD1069 (RXR selective) (13), inhibited the response with relatively high IC50 values. Overall, the suppressive effect of retinoids on the proliferation of alloantigen-stimulated murine lymphocytes seemed to correlate well with the magnitude of their affinity for RAR-
.
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Table 1. Retinoid receptor selectivity and antiproliferative effect of retinoids on allo-antigen-stimulated murine splenocytes
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Prolongation of the survival of mouse cardiac allografts by ER-38925
Since RAR-
selective retinoids inhibited allo-specific murine T lymphocyte activation more effectively than the other subtype-selective retinoids, we next examined ER-38925, our RAR-
-selective ligand, in a mouse cardiac allograft model. When 0.3 or 1 mg/kg ER-38925 was orally administered, the maximal blood concentration was 0.129 µg/ml (40 nM) or 0.548 µg/ml (170 nM) respectively, by which substantial biological effect was expected according to the in vitro data: the IC50 value of ER-38925 in inhibiting mixed lymphocyte reaction was 0.2 nM (Table 1). In untreated C3H/HeN control mice, all of the allografts were rejected within 10 DPT. ER-38925 at 0.3 and 3 mg/kg prolonged the allograft survival in a dose-dependent manner, and these effects were statistically significant (Fig. 1A). In the groups with 3 mg/kg of the drug for only 2 weeks, 75% of the cardiac allografts survived for >100 DPT, although two recipients died of undefined causes without any sign of graft rejection and infection. The histopathology of grafted hearts from animals treated with ER-38925 at 3 mg/kg or untreated is also shown. Figure 1(B) shows the myocardium from an untreated allograft, and reveals intense myocardial inflammation with diffuse mononuclear cell infiltration, hemorrhage and destruction of myocardial architecture. In contrast, the graft treated with ER-38925 at 3 mg/kg was almost free from these lesions (Fig. 1C).
In another experiment, combination therapy with ER-38925 and tacrolimus was examined. In the mice treated with both a suboptimal dose of tacrolimus and 0.3 mg/kg ER-38925, 80% of the allografts (four out of five) survived for >100 DPT (Fig. 1A). This survival rate was significantly better than that of the 0.3 mg/kg ER-38925 monotherapy group (P < 0.01). In addition, when hosts were treated with the sub-optimal dose of tacrolimus alone (tacrolimus monotherapy group in Fig. 4), only 20% of the allografts (two out of eight) survived for 30 DPT (Fig. 4A). Taken together, ER-38925 seemed to work synergistically with tacrolimus to suppress mouse cardiac allograft rejection.
Effect of ER-38925 on the induction of allo-specific CTL in recipients
In order to investigate the mechanisms by which ER-38925 suppresses cardiac allograft rejection, we next examined CTL induction in mice treated with the drug. As shown in Fig. 2(A), ER-38925 remarkably suppressed H-2d-specific CTL induction in C3H/HeN recipients. Even at 0.3 mg/kg, it inhibited the response by >90%. The effect of ER-38925 at 0.3 mg/kg (p.o.) was comparable to that of CyA (10 mg/kg, i.p.) and more potent than that of tacrolimus (1 mg/kg, i.p.) (Fig. 2B).

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Fig. 2. Effect of ER-38925 and other immunosuppressants on CTL induction in allograft recipients. (A) Recipient mice were orally administered ER-38925 at 0.3 (solid diamonds) or 3 mg/kg (solid triangles) for 5 days after immunization. Vehicle-treated mice served as controls (open circles). CTL activity in the recipient spleen cells was determined as described in Methods. (B) Recipients were treated with CyA (solid squares; 10 mg/kg, i.p.), tacrolimus (solid triangles; 1 mg/kg, i.p.), ER-38925 (solid diamonds; 0.3 mg/kg, p.o.) or vehicle (open circles) for 5 days. At >10:1 E:T ratio, the values of ER-38925 were statistically different from those of vehicle control (P < 0.05 by Students t-test).
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Effect of ER-38925 on cytokine production by the recipients spleen cells
C3H/HeN mouse spleen cells were obtained 5 days after immunization with donor spleen cells and re-stimulated with the donor antigen in vitro to evaluate cytokine production. Various cytokines, i.e. IL-2, IL-4, IL-12 and IFN-
, were produced in supernatants of allo-stimulated recipient splenocytes in a time-dependent manner (Fig. 3A). Of those, Th1-related cytokines (IL-2, IL-12, and IFN-
) were dominantly produced in this study, while little IL-4, a Th2 cytokine, was detected. ER-38925 at 0.3 mg/kg significantly inhibited IL-2, IL-12 and IFN-
production, when compared with the vehicle-treated control. The results of ELISPOT assay with the same cell preparations indicate a significant reduction in the number of cytokine-producing cells in ER-38925-treated recipients (Fig. 3B). Although the amount of IL-4 and the frequency of IL-4-producing cells in ER-38925-treated mice were also reduced, neither effect was statistically significant.

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Fig. 3. Effect of ER-38925 on cytokine production from alloantigen-stimulated recipient spleen cells. (A) Time course of cytokine production of alloantigen-stimulated spleen cells. ER-38925-treated spleen cells (solid triangles) produced less cytokines than the vehicle-treated control (open circles). Significant difference from the time-matched vehicle control, *P < 0.05, and **P < 0.01 by Students t-test. (B) Cytokine-producing cells were enumerated in ELISPOT assay after 24 h stimulation. ER-38925 reduced cytokine-producing cells in the recipients. Significant difference from the vehicle control, *P < 0.05, by Students t-test.
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Effect of ER-38925 on arteriosclerosis in murine cardiac allografts
In another set of experiments, heart allografts were harvested at 30 days after transplantation for evaluation of graft coronary arteriosclerosis in recipients treated with tacrolimus (0.1 mg/kg/day, i.p.) alone or tacrolimus plus ER-38925 (0.3 mg/kg/day, p.o.). In the tacrolimus monotherapy group, six out of eight mice rejected their cardiac allograft before 30 DPT. These grafts were harvested immediately after rejection and subjected to histopathological evaluation. In contrast, all of the grafts treated with the drug combination survived and functioned well until 30 DPT in this experiment (Fig. 4A). The total numbers of cardiac arteries and arterioles evaluated in both groups were almost the same (14.2 + 1.7 for tacrolimus monotherapy and 15.5 + 2.5 for tacrolimus + ER-38925 combination therapy). In cardiac allografts from recipients treated with tacrolimus alone, 91 + 9% of vessels were affected by intimal thickening with the mean grade of 2.7 + 0.5. Combined treatment with tacrolimus and ER-38925 reduced the percentage of affected vessels to 49 + 12% (Fig. 4B) and the mean grade of intimal thickening to 0.8 + 0.2 (Fig. 4C). In accordance with these results, graft coronary arterioles from the animals treated with tacrolimus alone revealed severe intimal thickening lesions (Fig. 4D), while those from the combined treatment group did not (Fig. 4E). The incidence and grade of transplant arteriosclerosis in long-term surviving (>80 DPT) recipients treated with ER-38925 alone was also minimal (data not shown).
Effect of ER-38925 on in vitro growth of RSMC
In order to examine the mechanism through which our retinoid inhibited the intimal thickening of graft coronary arterioles, we next investigated its effect on in vitro proliferation of RSMC. When stimulated either with bFGF (3 ng/ml) or FCS (1%), RSMC incorporated 2.5-fold more [3H]thymidine than the unstimulated control culture. ER-38925 potently inhibited bFGF-induced and FCS-induced proliferation of RSMC with IC50 values of 61 and 36 nM respectively (Fig. 5A).

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Fig. 5. Involvement of CDKI/p27kip1 induction by ER-38925 in its inhibition of bFGF-stimulated RSMC proliferation. (A) ER-38925 inhibited proliferation of RSMC, stimulated with bFGF (3 ng/ml) or FCS (1%) for 24 h, in a concentration-dependent manner. Representative results from three independent experiments. (B) Upper panel shows [3H]thymidine incorporation into RSMC stimulated with bFGF (10 ng/ml) for 24 h in the presence or the absence of 1 µM of ER-38925. Lower inset indicates p27kip1 protein expression measured by western blotting in the corresponding cultures. Representative results from three independent experiments.
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Up-regulation of cyclin-dependent kinase (CDK)-inhibitor (CKI), p27kip1 in RSMC by ER-38925
To date, retinoids have been reported to inhibit the growth of various cell lines, by up-regulating numbers of the Cip/Kip CKI family including p21 and p27 (1721), those which bind and inhibit all G1 cyclin/CDK complexes. Moreover, RSMC can be growth-arrested by up-regulating various CKI (22). Accordingly, we examined the ability of our retinoid to induce CKI in RSMC. Western blot analysis of p27kip1 expression in RSMC revealed that the expression of p27kip1 was greatly reduced in the cells after 24 h stimulation with bFGF, whereas this reduction was almost completely blocked by 1 µM ER-38925 (Fig. 5B, lower inset). ER-38925-induced up-regulation of p27kip1 was consistent with reduced [3H]thymidine incorporation into these cells (Fig. 5B, upper panel). In addition, ER-38925 up-regulated p27kip1 expression in RSMC even in the absence of bFGF, accompanied by slight inhibition of RSMC proliferation. In contrast to p27kip1, no consistent change in the expression of p21waf-1 was observed in RSMC, even after treatment with bFGF and/or ER-38925 for 24 h (data not shown).
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Discussion
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As observed in this study (Table 1) and reported elsewhere (9), ER-38925 shows substantial RAR-
selectivity in both binding and transactivation assays. In addition, all of the retinoids used in this study inhibited allo-specific in vitro lymphocyte proliferation in a concentration-dependent manner. Among them, compounds with high affinity to RAR-
, including atRA, 9-cis-RA, Am80 and ER-38925, were more potent than those selective for the other types of RAR (RAR-ß and
) or RXR. These findings suggest that RAR-
plays a pivotal role in the inhibitory effect of retinoids on allo-antigen-stimulated murine lymphocytes. Actually, in addition to the present data, Am80 as well as ER-38925 significantly inhibited the induction of allo-specific CTL in an allogenic graft-versus host disease model, which was 10100 times more potent than a non-selective agonist did (submitted). Furthermore, in the allogenic graft versus host disease model, 0.3 mg/kg ER-38925 significantly reduced serum levels of IL-12 and IFN-
(submitted). We have also found that the anti-proliferative effect of atRA on allo-reactive T lymphocytes was completely reversed by our RAR-specific antagonist (unpublished data). On the other hand, it was indicated that the inhibition of murine B lymphocyte proliferation by retinoids was mainly mediated through RAR-
, by means of a specific antagonist for this receptor (23). Furthermore, in a murine lupus nephritis model, our RAR-
-selective agonist significantly suppressed antibody production and ameliorated the disease (submitted). Also, ER-38925 significantly inhibited alloantigen-specific antibody production (our unpublished data). Taking these findings altogether, retinoids seem to inhibit both T and B lymphocyte proliferation and function, mainly in a RAR-
-dependent manner.
Up to now, various retinoid derivatives have been reported to be immunosuppressive in a range of immunological disease models (48). In addition, we have shown here that our novel RAR-
-selective retinoid, ER-38925, remarkably suppressed allo-specific immune response and significantly prolonged the survival of mouse cardiac allografts (Fig.1). We have already found that another RAR-
-selective retinoid, ER-34617, can prolong graft survival in a stringent mouse skin allograft model (24) and E6060 can ameliorate lupus nephritis in lupus-prone mice, NZB/NZW F1 (submitted). All these findings indicate that RAR-
-selective retinoids, including ours, may represent a novel approach to immunosuppressive therapy for various immune-compromised conditions, such as autoimmune diseases, or graft rejection after organ transplantation. Regarding the toxicity, retinoids primarily possess teratogenicity. Because of it, the clinical development of our retinoids has been suspended. However, the general toxicity of ER-38925 seemed very low, as we have administered up to 30 mg/kg ER-38925 to normal mice for 7 days and observed no toxic effect: there was no weight loss or alteration in complete blood count, suggesting that it induced no bone marrow suppression or pan-immunosuppression.
Recently, it has been reported that retinoids preferentially inhibit the development of Th1 and skew the host immune responses to Th2 dominant. Hayes and colleagues observed increased IFN-
and decreased IL-4 and IL-5 secretion in vitamin A-deficient mice, and such an imbalance was reversed by the administration of retinoic acid (2527). They also reported that retinoic acid inhibits IFN-
production from a Th1 cell line via CD28 co-stimulatory signal blockade (28). In addition, Racke et al. (5) reported that retinoic acid ameliorated neurological disease in the murine EAE model, by suppressing Th1 cytokine production, i.e. IL-2, tumor necrosis factor-
and IFN-
, but up-regulating IL-4 production from encephalogenic T cells. More recently, Morinobu indicated that retinoic acid inhibited IL-12-induced differentiation of human CD4+CD45RO T cells to Th1, but promoted differentiation of those cells to Th2 in the presence of IL-4 (29). Retinoid-mediated suppression of murine (28) and human (30) IFN-
production is exerted at the transcriptional level. Additionally, retinoic acid inhibits IL-2 production from a human T cell line activated with phorbol ester and calcium ionophore at the transcriptional level (31). Moreover, it was recently reported that retinoids inhibit IL-12 production from activated macrophages through functional interactions between their receptors (RXR and RAR) and NF
B, a crucial transcription factor for IL-12 gene expression (32). Since IL-12 is a potent promoter of Th1 differentiation (33), and it was also reported that decreased IL-12 production causes overexpression of Th2 cytokines (34), retinoids must skew the host immune response toward Th2 dominant, at least in part, through the inhibition of IL-12 production. In accordance with these reports, production of Th1-type cytokines, such as IL-2, IL-12 and IFN-
, was significantly suppressed in the spleen cells from ER-38925-treated recipients (Fig. 3A). Inhibition by ER-38925 was probably due to the reduction in the number of these cytokine-producing cells (Fig. 3B). Overall, these findings suggest that our retinoid inhibited Th1 differentiation in the recipients and, thereby, secretion of Th1 cytokines. These effects would contribute to the potent suppression of CTL induction (Fig. 2), since all those cytokines have been reported to be critical for allo-specific CTL induction (3537). On the other hand, we could not detect any significant elevation of IL-4 production in ER-38925-treated recipients (Fig. 3A and B). Consequently, it remains to be elucidated if this compound also skews the host immune response toward Th2 dominant in this model, as other retinoids did in other experimental systems.
Chronic rejection of mouse cardiac allografts, characterized by graft coronary arteriosclerosis, was significantly inhibited by the combination of tacrolimus and ER-38925, compared to the tacrolimus monotherapy controls (Fig. 4B and C). In addition, ER-38925 inhibited RSMC proliferation induced by bFGF or FCS (Fig. 5A). This is consistent with the recent reports that retinoic acid inhibited the proliferation of RSMC (38) and limited arterial restenosis in rats (39) or rabbits (40) after balloon angioplasty. Therefore, inhibition of graft coronary arteriopathy by ER-38925 is possibly mediated, at least in part, through its direct inhibition of vascular smooth muscle cell proliferation in the setting of chronic graft rejection. Actually, RAR-
, -ß, and -
, and RXR-
and -ß have been reported to be expressed in vascular smooth muscle cells (41). Furthermore, as recent reports indicated, intimal thickening of arteries in chronic rejection may be assignable to aberrant differentiation of recipient-derived stem cells (42,43). Because retinoids are powerful differentiation inducers, it should be of great interest to study the mechanism by which ER-38925 inhibited the arteriopathy in this view.
CDK are indispensable factors for cell growth and proliferation. The activity of CDK is regulated by various mechanisms, including association with inhibitory proteins called CKI. We observed that ER-38925 remarkably up-regulated one CKI, p27kip1, in RSMC cultured with or without bFGF (Fig. 5C) and this effect would have contributed to its anti-proliferative effect on these cells. Recently, CDK2, which mediates smooth muscle cell proliferation, was reported to play a pivotal role in the establishment of coronary arteriosclerosis of murine cardiac allografts and an antisense oligonucleotide of CDK2 was shown to be a powerful preventive measure against chronic graft rejection (44). Moreover, p27kip1 was recently reported to inhibit transcription of IL-2 and induce clonal anergy (45). Overall, agents with potency to up-regulate CKI and, thereby, inhibit the activity of CDK, like ER-38925, are candidates for novel therapeutic drugs to prevent acute and chronic allograft rejection.
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Acknowledgements
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This work was supported in part by Japan Foundation of Cardiovascular Research and The Ichiro Kanehara Foundation. The authors thank Junko Seki for her secretarial assistance.
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Abbreviations
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9-cis-RA9-cis-retinoic acid
atRAall-trans-retinoic acid
bFGFbasic fibroblast growth factor
CDKcyclin-dependent kinase
CKICDK-inhibitor
CTLcytotoxic T lymphocyte
CyAcyclosporin A
DPTdays post-transplantation
EAEexperimental allergic encephalomyelitis
RARretinoic acid receptor
RSMCrat smooth muscle cell
RXRretinoid X receptor
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