- Email: [email protected]
Synergistic effects of Isatis tinctoria L. and tacrolimus in the prevention of acute heart rejection in mice
Transplant Immunology 22 (2009) 5–11
Contents lists available at ScienceDirect
Transplant Immunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t r i m
Synergistic effects of Isatis tinctoria L. and tacrolimus in the prevention of acute heart rejection in mice Yongzhi Wang a,1, Qing Qin b,c,1, Jibing Chen d,1, Xiaocong Kuang e, Junjie Xia a, Baiyi Xie a, Feng Wang a, Hua Liang f, Zhongquan Qi a,⁎ a
Organ Transplantation Institute, Xiamen University, Fujian Province, PR China School of Chemistry and Chemical Engineering, Guangxi University, Guangxi Province, PR China Dept. of Natural Products Chemistry, School of Pharmaceutical Sciences, Guangxi Medical University, Nanning, Guangxi Province, PR China d Basic Medical Department of Medical College, Xiamen University, Fujian Province, PR China e Pathophysiology Department of Basic Medical College, Guangxi Medical University, Guangxi Province, PR China f Zhongshan Hospital, Xiamen University, Fujian Province, PR China b c
a r t i c l e
i n f o
Article history: Received 1 July 2009 Received in revised form 9 August 2009 Accepted 1 September 2009 Keywords: Compound K FK-506 Lymphocyte T cells Cardiac transplantation Acute rejection
a b s t r a c t Although immunosuppressive treatments are available for acute cardiac rejection no viable treatment exists for long-term cardiac graft failure. Moreover, the extended use of calcineurin inhibitor immunosuppressants, the mainstay of current treatment for cardiac transplantation, leads to significant side effects such as nephrotoxicity and an increased risk of cardiac disease. Because some agents used in Traditional Chinese Medicine (TCM) have strong immunosuppressive effects coupled with low toxicity, we investigated the effect of Compound K (K), the synthesized analogue of highly unsaturated fatty acids from Isatis tinctoria L., either as a single treatment or combined with tacrolimus (FK-506) on acute cardiac allograft rejection. We compared the ability of K alone, or in combination with FK-506, to inhibit acute heart transplant rejection both in vitro and in vivo. We found that the inhibition of lymphocyte proliferation was positively correlated with K concentration. K significantly reduced IL-2 and IFN-γ expression levels and significantly inhibited lymphocyte proliferation in both a lymphocyte transformation test and a mixed lymphocyte reaction (MLR). We also found that the inhibitory effect of a combination of K and a sub-therapeutic dose of FK-506 (SubFK506) was stronger than that of full-dose FK-506 alone. Oral administration of K reduced acute cardiac allograft rejection in mice and had no apparent toxicity. In vivo, the immunosuppressive effect of K combined with a half-dose of FK-506 was equivalent to that of a full-dose of FK-506 alone. K combined with a half-dose of FK-506 reduced the expression levels of IL-2 and IFN-γ (both within the graft and in the recipients' serum) more effectively than a full-dose of FK-506. These results show that K has significant immunosuppressive effects both in vitro and in vivo. When used as a combination therapy with FK-506 we see a powerful inhibition of rejection with no obvious toxic side effects. The mechanism of action is postulated to involve the inhibition of IL-2 and IFN-γ expressions by lymphocytes, rather than the activation of Tr1 cells via the production of IL-10. © 2009 Elsevier B.V. All rights reserved.
1. Introduction In clinical organ transplantation, the calcineurin-inhibitors (such as CsA, RAPA and FK-506) are the first-line immunosuppressive agents of choice and have been used for many years. Studies have shown that these drugs clearly reduce the rate of acute rejection of kidney, heart and pancreatic islet transplants and prolong graft survival for many years [1–5]. However, side effects such as renal
⁎ Corresponding author. Organ Transplantation Institute, Xiamen University, 361005 Fujian Province, PR China. Tel./fax: +86 592 2180126. E-mail address: [email protected] (Z. Qi). 1 These authors contributed equally to the study and share the first authorship. 0966-3274/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.trim.2009.09.004
toxicity, neurotoxicity and the induction of diabetes limit the doses that can be used, thereby affecting the survival of the transplanted organ [6–9]. We are committed to the development of high efficiency, low toxicity immunosuppressants to replace the calmodulin-inhibitor immunosuppressants currently in use. Attention has been focused on plants and fungi that are associated with TCM as potential sources of new therapeutic agents. One such example is the parasitic fungus of insects called Isaria sinclairii that has recently been used by Novartis for the development of the novel immunosuppressive agent FTY720 [10]. The agent not only prevents rejection, but also reverses any signs of rejection that have already occurred [11]. This drug also has a synergistic effect when used in conjunction with calmodulin inhibitors and is currently completing Phase III trials for use in the treatment of multiple sclerosis [12,13].
6
Y. Wang et al. / Transplant Immunology 22 (2009) 5–11
We believe that there will certainly be a lot of high efficiency, low toxicity immunosuppressive drugs within TCM that have the potential for development and clinical use. Recently, we studied the immune-regulatory properties of K, an analogue of the highly unsaturated fatty acids from Isatis tinctoria L., and after testing, we found that K was the most promising. In this study, we compare the immunosuppressive effects of K and FK-506 (both individually and in combination) in the treatment of acute heart transplant rejection in a mouse model. We show that K can inhibit proliferation of lymphocytes in vitro and that it can enhance the effects of FK-506 in the prevention of acute rejection. Finally, we show that K acts via a mechanism that inhibits both IL-2 and IFN-γ expressions and production by lymphocytes rather than stimulating the production of IL-10, as what happens with FK-506. 2. Objectives The objectives of this study were four-fold. First, to determine whether K could reduce lymphocyte proliferation in a lymphocyte transformation test in vitro. Second, to determine whether K could reduce T lymphocyte proliferation and act synergistically with subtherapeutic doses of FK-506 in a mixed lymphocyte reaction (MLR). Third, to determine whether oral administration of K could reduce the acute rejection response in a mouse model and fourth, to investigate the mechanism of action of K both in vitro and in vivo. 3. Materials and methods 3.1. Animals Female C57BL/6 (B6) and BALB/c mice (8–12-weeks old) were purchased from Slac Laboratory Animal Co. Ltd. (Shanghai, China) and used as graft recipients and donors, respectively. Care and handling of animals were in accordance with the guidelines provided in the “Guide for the Care and Use of Laboratory Animals” published by the U.S. Department of Health and Human Services. 3.2. Drugs FK-506 was purchased from ALEXIS Co. Ltd. (Switzerland). The Compound K was obtained from Guangxi Medical University (Guangxi, China). 3.3. Lymphocyte isolation Lymphocytes were isolated from the spleens of B6 mice using mouse lymphocyte separation medium (Dakewei, China). Briefly, spleens were removed aseptically, placed in 4 ml mouse lymphocyte separation medium and gently ground through a nylon mesh using the top of the plunger from a 5 ml syringe. Debris was allowed to settle and the cell suspension isolated. The suspension was centrifuged at 200–400×g for 30 min and the white cell layer extracted and washed three times by centrifugation in RPMI 1640 (GIBCO, USA). After the final wash, the cell pellet was re-suspended in 10 ml of fresh RPMI 1640, the cells were counted and viability established by Trypan blue exclusion. Viability routinely exceeded 96%. 3.4. T lymphocyte isolation T lymphocytes were isolated from the spleens of B6 mice using nylon wool columns (Wako, Japan). Briefly, the nylon wool was washed three times in sterile PBS followed by washing three times in RPMI 1640 supplemented with 5% fetal bovine serum (FBS). Lymphocytes were suspended in 2 ml RPMI 1640/5% FBS and incubated with the nylon wool for 45 min at 37 °C. The nylon wool was then washed and the cell suspension centrifuged at 200–400×g for 5 min. The cell pellet was re-
suspended in 10 ml of fresh RPMI 1640, the cells counted and viability established by Trypan blue exclusion. Viability always exceeded 96%. Purity was always in excess of 90%. 3.5. Lymphocyte transformation test B6 spleen lymphocytes were plated in 96-well plates at a concentration of 3 × 105 cells/well. K was added to triplicate wells at concentrations of 5, 10, 20, 40, or 80 μg/ml followed by the addition of 5 μg/ml Con A (Sigma, USA) in RPMI 1640 supplemented with 10% FBS, penicillin, and streptomycin (total well volume = 200 μl). Plates were incubated at 37 °C in a 5% CO2 humidified atmosphere for 48 h. Cells cultured with K, but without Con A, were used as negative controls. Cell proliferation was quantified by the 5′-bromodeoxyuridine (BrdU) method using an enzyme linked immunosorbent assay (ELISA) kit (Millipore, USA), according to the manufacturer's instructions. Proliferation was measured using a microplate reader (BIO-RAD, USA). The level of inhibition was calculated with respect to the negative control and stimulated control values. 3.6. Mixed lymphocyte reaction T lymphocytes obtained from the spleens of female B6 mice were used as responder cells and spleen cells obtained from BALB/c mice were used as stimulator cells in a mixed lymphocyte reaction (MLR) assay. The responder cells (5 × 105 cells) were cultured in 96-well plates in the presence of stimulator cells (5 × 104 cells, pre-treated with mitomycin C, 40 μg/ml, Amresco, USA) in 200 μl RPMI 1640 supplemented with 10% FBS, penicillin, and streptomycin, and incubated at 37 °C in a 5% CO2 humidified atmosphere. K or FK-506 was added to four wells at the following concentrations: K alone (80 μg/ml), FK-506 alone (1 μg/ml) or a combination of K (80 μg/ml) and a sub-therapeutic dose of FK-506 (0.5 μg/ml). Responder cells cultured in medium without the stimulator cells were used as negative controls. After 72 h, 96 h or 120 h of culture, cell proliferation was quantified using the BrdU ELISA kit as outlined above. The results were expressed as the mean value of four wells. The percentage inhibition values were calculated with respect to the negative control and stimulated control values. 3.7. Heart transplantation Vascularized heterotopic heart transplantation from BALB/c donors to B6 recipients (n = 6) was performed by anastomosis to the vessels of the neck using a non-suture cuff technique as described previously [14]. Graft survival was monitored by palpation (twice daily) and body weight was recorded (daily) until complete graft rejection occurred. Rejection was defined as the loss of palpable cardiac contractions. B6 mice were treated with FK-506 (2 mg/kg/d), SubFK-506 (1 mg/kg/d), K (10 mg/kg/d) or SubFK-506 + K on Days 0– 10 post-transplant. The control group was given normal saline only. 3.8. Histological evaluation of rejection Heart grafts (n = 3) were removed on Day 6 post-transplant. Part of each graft was used for qRT-PCR, and the rest was used for histological evaluation. Paraffin-embedded transventricular tissue sections (5 μm thick) were stained with hematoxylin and eosin. Rejection was graded based on the extent of lymphocytic infiltration, the anatomical localization of leukocytes as per the International Society of Heart and Lung Transplantation (ISHLT) standards [15,16] and on the advice of an in-house pathologist. Briefly, heart tissue was scored as follows: 0 = no damage; 1 (mild) = evidence of interstitial edema and focal necrosis; 2 (moderate) = graft displayed diffuse myocardial cell swelling and necrosis; 3 (severe) = necrosis with the presence of contraction bands and neutrophil infiltrate, and 4 (highly
Y. Wang et al. / Transplant Immunology 22 (2009) 5–11
7
severe) = widespread necrosis with the presence of contraction bands, neutrophil infiltrate and hemorrhage. 3.9. Real-time quantitative reverse transcription-polymerase chain reaction Reverse transcription (RT) was performed using 1 μg of the total RNA extracted from the allografts harvested on Day 6 post-transplant using the Trizol-reagent. Real-time polymerase chain reaction (RTPCR) was carried out using 2 μl of the RT product on a StepOne RealTime PCR System (ABI, UK). Syber Green I and β-actin were used as controls. Each reaction was carried out in triplicate. The following primer sequences were used for the real-time RT-PCR: β-actin forward 5′-CATCCGTAAAGACCTCTATGCCAAC-3′ and reverse 5′-ATGGAGCCACCGATCCACA-3′; IFN-γ forward 5′-CGGCACAGTCATTGAAAGCCTA-3′ and reverse 5′-GTTGCTGATGGCCTGATTGTC-3′; IL-2 forward 5′-GGAGCAGCTGTTGATGGACCTAC-3′ and reverse 5′-AATCCAGAACATGCCGCAGAG-3′; IL-10 forward 5′-GACCAGCTGGACAACATACTGCTAA-3′ and reverse 5′-GATAAGGCTTGGCAACCCAAGTAA-3′. The results were expressed as the mean value of triplicate wells. 3.10. Cytokine ELISA Recipients' serum (n = 3) were isolated on Day 6 post-transplant. ELISA was performed using commercially available kits to detect IL-2, IFN-γ and IL-10 (Bender, USA) according to the manufacturer's instructions. Briefly for IFN-γ flat-bottomed 96-well ELISA plates were coated with an anti-mouse-IFN-γ antibody in PBS and incubated overnight at 4 °C. After washing (wash buffer) and blocking (assay buffer), sample, sample diluent and biotin-conjugate were applied to the wells and incubated for 2 h. After washing, the plates were incubated with Streptavidin-HRP for 1 h at room temperature. Samples were developed using a 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution and the reaction stopped with 2 M sulfuric acid. ELISA plates were read using a microplate reader at 450 nm. The results were expressed as the mean value of four wells. 3.11. Statistical analysis Survival curves and body weight curves were obtained using GraphPad Prism® (GraphPad Software, USA). The survival curves were analyzed using the log-rank test. The data from the MLR, ELISA and qRT-PCR experiments were analyzed using the Student's t test and expressed as mean values ± standard deviation (SD). A value of p < 0.05 was considered to be a significant statistical difference.
Fig. 1. The ability of K to inhibit lymphocyte proliferation in vitro. Positive control and treatment groups treated with 5 μg/ml Con A. Negative control without Con A. Maximal inhibition of T cell proliferation occurs at a K concentration of 80 μg/ml. Cell proliferation was quantified using the BrdU method. Each concentration was tested in triplicate. Data shown is mean ± SD and is representative of four separate experiments (⁎p < 0.05).
4.3. Mechanism of K-mediated inhibition of lymphocyte proliferation Lymphocytes were cultured for 48 h with 5 μg/ml Con A and K alone (80 μg/ml), FK-506 alone (1 μg/ml) or a combination treatment with FK-506 and K (0.5 μg/ml and 80 μg/ml, respectively). qRT-PCR was used to detect the expression levels of IL-2 and IFN-γ in each treatment group. Figs. 3a and b show that, compared with the control group, both IL-2 and IFN-γ expression levels are significantly reduced in all treatment groups. However, a combined treatment with K and FK-506 results in the greatest reduction in expression levels of both IL-2 and IFN-γ. These data clearly show that K inhibits the expressions of IL-2 and IFN-γ by lymphocytes and thus inhibits proliferation. Again, this effect is enhanced when K is used in combination with FK-506. 4.4. Cardiac allograft survival time and body weight changes in graft recipients The mice that received cardiac allografts were divided into the following treatment groups: K (10 mg/kg/d); FK-506 (2 mg/kg/d); SubFK-506 (1 mg/kg/d); SubFK-506 (1 mg/kg/d) + K (10 mg/kg/d) and a control group (normal saline). Treatment was given daily from Day 0 to Day 10 post-transplantation. As shown in Fig. 4a, the median survival time (MST) was 7 days in the control group, 8.5 days in the SubFK-506 group, 10 days in the K group, 16.5 days in the SubFK-506 + K group and 17.5 days in FK-506 group. Only minor changes in body weight were noted after transplantation, mainly because of the surgical trauma (Fig. 4b). These results suggest that treatment with K alone provides some protection against the acute rejection response, but when combined with SubFK-506 it can significantly prolong the graft survival time in a manner similar to a full-dose of FK-506. Importantly, no side effects of K treatment were noted. 4.5. K, combined with a sub-therapeutic dose of FK-506, can reduce acute rejection At 6 days post-heart transplantation, grafts were harvested and prepared for histology as outlined above. The ISHLT scores of the hearts harvested from the different
4. Results 4.1. Effect of K on lymphocyte transformation We looked at the ability of K to inhibit Con A-stimulated lymphocyte proliferation at a series of concentrations (10, 20, 40, and 80 μg/ml). Fig. 1 shows that, after a 48-hour incubation, K inhibited proliferation in a dose-dependent manner with a concentration of 80 μg/ml being statistically significant. These results clearly show that K inhibits lymphocyte proliferation in vitro. 4.2. Effect of K on mixed lymphocyte response To further investigate the inhibitory effect of K on T cell proliferation we undertook MLR experiments using K or FK-506 at the following concentrations: K alone (80 μg/ ml), FK-506 alone (1 μg/ml) or a combination of K (80 μg/ml) and a sub-therapeutic dose of FK-506 (0.5 μg/ml). T cell proliferation was assessed after 3, 4 or 5 days of culture using the BrdU method. We found that, compared with the control group, T cell proliferation was significantly inhibited by K alone, FK-506 alone and the combination treatment in assays carried out for 3, 4 or 5 days (Fig. 2; p < 0.05). Combination treatment with K and SubFK-506 resulted in a significant inhibition of T cell proliferation compared with FK-506 alone after 4 days of culture, but not after 3 or 5 days of culture. It is notable that, although K is able to inhibit T cell proliferation when compared with the control, it has an obvious synergistic effect when used in combination with FK-506.
Fig. 2. K reduces T cell proliferation in an in vitro MLR assay responder and stimulator cells were mixed for 3, 4 or 5 days. Cell proliferation was quantified using the BrdU method. Each drug concentration was tested in quadruple wells. Data shown is mean ± SD and is representative of three separate experiments. Drug treatment inhibited T cell proliferation at all time points tested compared with the control group (p < 0.05). On Day 4, combination treatment inhibited T cell proliferation to a greater extent than FK-506 alone (p < 0.05).
8
Y. Wang et al. / Transplant Immunology 22 (2009) 5–11
Fig. 3. K inhibits lymphocyte IL-2 and IFN-γ gene expressions in vitro. Lymphocytes stimulated with 5 μg/ml Con A were treated with K alone (80 μg/ml), FK-506 alone (1 μg/ml) or a combination of K with a sub-therapeutic dose of FK-506 (0.5 μg/ml) and cultured for 48 h. qRT-PCR was used to detect the expressions of IL-2 and IFN-γ in each group. Each treatment was done in triplicate wells. Data shown is the mean ± SD and is representative of three separate experiments. Expressions of IFN-γ and IL-2 were inhibited in all treatment groups relative to the control group, with combination therapy being the most effective (p < 0.05, ⁎p < 0.05, ⁎⁎p < 0.01).
Fig. 4. Cardiac allograft survival time and body weight changes in graft recipients. Vascularized heterotopic heart transplants from BALB/c mice to B6 mice were monitored twice a day by manual palpation and body weight was recorded daily until complete graft rejection occurred. Recipient mice were treated on Days 0–10 posttransplant with FK-506 (2 mg/kg/d), half-dose (Sub) FK-506 (1 mg/kg/d), K (10 mg/kg/ d) or SubFK-506 + K. The control group was given normal saline. (a) Kaplan–Meier curve of palpation scores for the five groups; and (b) body weight changes in recipients. Each data point represents the mean values obtained from six individuals.
treatment groups are shown in Figs. 5a–e. The grafts from the control group showed highly severe infiltration and tissue damage and the grafts from both the K and the SubFK-506 groups showed moderate infiltration and tissue damage. The grafts from the full-dose FK-506 and the combined treatment groups, however, showed only mild infiltration and tissue damage. These results suggest that treatment with K alone provides limited protection against acute rejection responses, but that this protection is increased when K is combined with a sub-therapeutic dose of FK-506.
in the circulating levels of the pro-inflammatory cytokine IFN-γ, and that the effect is enhanced when combined with FK-506.
4.6. The relative expressions of key genes in rejected cardiac allografts After the cardiac grafts were harvested, mRNA was prepared and reverse transcribed. The RT products were analyzed by real-time qPCR. The gene expression profiles of the treatment groups were normalized relative to the control group profiles. As shown in Figs. 6a and b, compared with the control group, the relative expressions of IL-2 and IFN-γ are significantly reduced in all the treatment groups (p < 0.01). Interestingly, both IL-2 and IFN-γ expressions are significantly less in the combined treatment group than in the full-dose FK-506 group (p < 0.05). As seen in Fig. 6c, the expression of IL-10 is enhanced in the full-dose FK-506 group but its expression actually reduced in the K-treated group (p < 0.01). These data suggest that K works by reducing the expressions of both IL-2 and IFN-γ within the graft rather than enhancing the expression of IL-10, resulting in prolonged graft survival times. 4.7. The relative expression cytokine levels in the recipients' blood serum Cytokine levels within the recipients' serum were analyzed by ELISA. As seen in Fig. 7b, compared with control group, the relative expression levels of IFN-γ are significantly reduced in all the treatment groups (p < 0.05), and are significantly lower in the combined treatment group than in the full-dose FK-506 group (p < 0.05). No significant differences are seen in the relative expressions of IL-2 and IL-10 between the treatment groups (Figs. 7a, c). These data indicate that K treatment results in a decrease
5. Discussion The use of immunosuppressants that exhibit high efficiency and low toxicity is important to prolong graft survival time and to improve the quality of life in clinical situations. The use of combined treatments with a variety of different immunosuppressive agents not only inhibits the rejection response but can also reduce the side effects of the drugs. This is an important principle in clinical immunosuppressive therapy. We have previously synthesized K, the analogue of highly unsaturated fatty acids from I. tinctoria L, and using different in vitro methods such as lymphocyte transformation tests, MLR, qRT-PCR and ELISA we have shown that this monomer has strong immunosuppressive properties and has the potential to be developed as a new immunosuppressant. We found that the degree of immunosuppression was positively correlated with drug concentration in a manner similar to that seen with other clinical immunosuppressive agents such as FK-506 [17]. T cells are the main mediators of acute cardiac rejection [18–20] . We have attempted to use an MLR assay [21] to provide evidence of the inhibition of the acute rejection response by K. We found that K could indeed inhibit T cell proliferation in in vitro MLR experiments but the effect was not significantly different from that of FK-506.
Y. Wang et al. / Transplant Immunology 22 (2009) 5–11
9
Fig. 5. K in combination with a sub-therapeutic dose of FK-506 can reduce acute rejection. On post-transplant Day 6, grafts were harvested and prepared for histology. H & E stained heart tissue from: (a) an untreated animal; b) an animal treated with a sub-therapeutic dose of FK-506 (1 mg/kg/d); (c) an animal treated with K (10 mg/kg/d); (d) an animal treated with full-dose of FK-506 (2 mg/kg/d); (e) an animal treated with K plus a sub-therapeutic dose of FK-506; and (f) ISHLT scoring of the hearts. The dot plot represents the scores for each animal in each group. The line represents the mean scores (p = 0.12, n = 3).
However, a combination of K and FK-506 was more effective than either K or FK-506 alone. This led us to explore the mechanism of action of K, as it has an obvious synergistic effect when used in combination with FK-506. We studied this interaction with reference to other works that have studied the mechanism of action of conventional calmodulin antagonists and analyzed IL-2 and IFN-γ gene expressions after different drug treatments [22,23]. We found that K significantly inhibits the expressions of both the IL-2 and the IFN-γ genes by proliferating T cells in the MLR assay. Compared with FK-506 alone, K provides limited immunosuppression, but combined with sub-therapeutic doses of FK-506 it provides stronger immunosuppression than treatment with full-dose FK-506 alone. This suggests that combination therapy may be a powerful treatment for acute cardiac rejection. To explore the prospects for the clinical application of K, our experiments focused on treatment with K, either alone or in combination with a sub-therapeutic dose of FK-506, and we compared the effects of this treatment with those of a full-dose of FK-506 in an acute cardiac allograft rejection model. As shown in Fig. 4a, treatment with a maximum dose of K alone, extends cardiac allograft survival for only a short time over non-treated controls (9 days versus 7 days).
This is clearly not satisfactory. Combined therapy (K + SubFK-506) shows immunosuppressive effects comparable to those of a full-dose of FK-506 (16.5 days versus 17.5 days). This is supported by other studies that have looked at the effects of combined drug treatments on cardiac allograft survival [24–29]. Next, we investigated the similarities and differences between the inhibitory mechanisms induced by combined therapy and full-dose FK-506 by examining the histology of the rejected allografts and by analyzing the levels of Th1 and Tr1 cytokines in both the graft and the serum of the recipients. Looking at the Th1 and Tr1 cytokine expression levels within the rejected grafts, we found that IL-2, IFNγ and IL-10 levels are all significantly lower after combined treatment than after treatment with full-dose FK-506 alone. It is known that FK506 suppresses the transcription of both the IL-2 and the IFN-γ genes by lymphocytes and it is possible that it also enhances the expression of IL-10 [21]. K appears not to induce IL-10 gene expression, but does inhibit IL-2 and IFN-γ gene expressions, and the combination of K and SubFK-506 has a synergistic effect on Th1 cytokine production that is more powerful than full-dose FK-506 alone. Our results suggest that there may be a K-induced tolerance mechanism that does not induce the proliferation of IL-10-secreting Tr1 cells [30].
10
Y. Wang et al. / Transplant Immunology 22 (2009) 5–11
Fig. 6. The relative expression of some key genes within the allografts. IL-2, IFN-γ and IL-10 mRNA were extracted from the grafts and used for real-time qPCR. (a) and (b): compared with the control group, the relative expressions of IL-2 and IFN-γ are significantly reduced in all the treatment groups (p < 0.01), and is significantly less in the combination treatment group than in the full-dose FK-506 group (p < 0.05). (c) The expression of IL-10 is enhanced in the full-dose FK-506 group, but its expression is reduced in the K-treated group (p < 0.01). Data shown is the mean ± SD and is representative of three separate experiments (⁎p < 0.05).
Analysis of the Th1-type cytokine expression levels in the recipients' serum shows that IFN-γ expression in the combination therapy group was significantly lower than in the full-dose FK-506 group, in line with the levels of gene expression seen within the graft. This evidence further validates the potential use of combined K/ SubFK-506 therapy as a replacement for full-dose FK-506 for the treatment of organ transplant rejection and long-term maintenance of graft survival. Our in vivo studies of acute cardiac allograft rejection showed no differences in body weight between the control and treatment groups during the post-transplant period. These data suggest that K has little effect on body weight and so is not likely to be toxic. The minor changes in body weight seen after transplantation were most likely to be the result of surgical trauma rather than K-mediated toxicity. In summary, K has low toxicity and, combined with subtherapeutic doses of FK-506, can strongly inhibit acute cardiac allograft rejection. Although K was able to inhibit IL-10 expression by Tr1 cells, combination therapy with SubFK-506 shows a strong synergy and significant inhibition of Th1-type cytokine expression
Fig. 7. The relative levels of cytokine in the recipient's blood. Recipients' serum was analyzed by ELISA. Compared with the control group, the relative levels of IFN-γ are significantly reduced in all the treatment groups (p < 0.05), but are significantly lower in the combined treatment group than in the full-dose FK-506 group (p < 0.05). There is no significant difference in the levels of IL-2 and IL-10 between any of the groups. Data shown is the mean± SD and is representative of three separate experiments (⁎p < 0.05).
within the graft leading to prolonged graft survival. This may be because K and FK-506 affect different signal pathways that regulate lymphocyte proliferation. Further studies are planned to investigate this possibility. References [1] Fung JJ, Abu-Elmagd K, Todo S, et al. Overview of FK506 in transplantation. Clin Transpl 1990:115–21. [2] Jordan JL, Hirsch GM, Lee TDG. C. sinensis ablates allograft vasculopathy when used as an adjuvant therapy with cyclosporin A. Transpl Immunol 2008;19:159–66. [3] Peddi VR, Kamath S, Munda R, Demmy AM, Alexander JW, First MR. Use of tacrolimus eliminates acute rejection as a major complication following simultaneous kidney and pancreas transplantation. Clin Transplant 1998;12:401–5. [4] Onsager DR, Canver CC, Jahania MS, et al. Efficacy of tacrolimus in the treatment of refractory rejection in heart and lung transplant recipients. J Heart Lung Transplant 1999;18:448–55. [5] Haeryfar SM, Lan Z, Leon-Ponte M, et al. Prolongation of cardiac allograft survival by rapamycin and the invariant natural killer T cell glycolipid agonist OCH. Transplantation 2008;86:460–8. [6] Condé SA, Aarestrup FM, Vieira BJ, Bastos MG. Roxithromycin reduces cyclosporine-induced gingival hyperplasia in renal transplant patients. Transplant Proc 2008;40(5):1435–8.
Y. Wang et al. / Transplant Immunology 22 (2009) 5–11 [7] Mueller AR, Platz KP, Bechstein WO, et al. Neurotoxicity after orthotopic liver transplantation. A comparison between cyclosporine and FK506. Transplantation 1994;58:155–70. [8] Kamar N, Mariat C, Delahousse M, et al. Diabetes mellitus after kidney transplantation: a French multicentre observational study. Nephrol Dial Transplant 2005;22: 1986–93. [9] Molinari M, Al-Saif F, Ryan EA, et al. Sirolimus-induced ulceration of the small bowel in islet transplant recipients: report of two cases. Am J Transplant 2005;5: 2799–804. [10] Brinkmann V. FTY720: mechanism of action and potential benefit in organ transplantation. Yonsei Med J 2004;45:991–7. [11] Chiba K. FTY720, a new class of immunomodulator, inhibits lymphocyte egress from secondary lymphoid tissues and thymus by agonistic activity at sphingosine 1-phosphate receptor. Pharmacol Ther 2005;108:308–19. [12] Schuurman HJ, Menninger K, Audet M, et al. Oral efficacy of the new immunomodulator FTY720 in cynomolgus monkey kidney allotransplantation, given alone or in combination with cyclosporine or RAD. Transplantation 2002;74: 951–60. [13] Baumruker T, Billich A, Brinkmann V. FTY720, an immunomodulatory sphingolipid mimetic: translation of a novel mechanism into clinical benefit in multiple sclerosis. Expert Opin Investig Drugs 2007;16:283–9. [14] Matsuura A, Abe T, Yasuura K. Simplified mouse cervical heart transplantation using a cuff technique. Transplantation 1991;51:896–8. [15] Billingham ME, Cary NR, et al. A working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection: Heart Rejection Study Group. The International Society for Heart Transplantation. J Heart Transplant 1990;9:587–93. [16] Aneja R, Hake PW, Burroughs TJ, Denenberg AG, Wong HR, Zingarelli B. Epigallocatechin, a green tea polyphenol, attenuates myocardial ischemia reperfusion injury in rats. Mol Med 2004;10:55–62. [17] Almawi WY, Assi JW, Chudzik DM, Jaoude MM, Rieder MJ. Inhibition of cytokine production and cytokine-stimulated T-cell activation by FK506 (tacrolimus). Cell Transplant 2001;10:615–23. [18] Lafferty KJ, Simeonovic CJ. Immunology of graft rejection. Transplant Proc 1984;16:927–30.
11
[19] Krieger NR, Yin DP, Fathman CG. CD4+ but not CD8+ cells are essential for allorejection. J Exp Med 1996;184:2013–8. [20] Pietra BA, Wiseman A, Bolwerk A, Rizeq M, Gill RG. CD4 T cell-mediated cardiac allograft rejection requires donor but not host MHC class II. J Clin Invest 2000;106:1003–10. [21] Morikawa K, Oseko F, Morikawa S. Immunosuppressive activity of bromocriptine on human T lymphocyte function in vitro. Clin Exp Immunol 1994;95:514–8. [22] Yoshimura N, Matsui S, Hamashima T, Oka T. Effect of a new immunosuppressive agent, FK506, on human lymphocyte responses in vitro. II. Inhibition of the production of IL-2 and gamma-IFN, but not B cell-stimulating factor 2. Transplantation 1989;47:356–9. [23] Banerji SS, Parsons JN, Tocci MJ. The immunosuppressant FK-506 specifically inhibits mitogen-induced activation of the interleukin-2 promoter and the isolated enhancer elements NFIL-2A and NF-AT1. Mol Cell Biol 1991;11:4074–87. [24] Vu MD, Qi S, Xu D, et al. Tacrolimus (FK506) and sirolimus (rapamycin) in combination are not antagonistic but produce extended graft survival in cardiac transplantation in the rat. Transplantation 1997;64(12):1853–6. [25] Qi Z, Simanaitis M, Ekberg H. Malononitrilamides and tacrolimus additively prevent acute rejection in rat cardiac allografts. Transpl Immunol 1999;7:169–75. [26] Shirouzu Y, Hikida S, Tanigawa H, Shirouzu K. Inhibition of acute rejection after skin or cardiac transplantation by administration of donor antigens in the rat. Surg Today 2004;34:341–8. [27] Deuse T, Schrepfer S, Pelletier MP, Fischbein MP, Robbins RC, Reichenspurner H. Is the malononitrilamide FK778 better for the prevention of acute or chronic rejection? Transplant Proc 2007;39:569–72. [28] Qi S, Zhao H, Ma A, et al. Effect of baohuoside-1 aglycone and tacrolimus monotherapy and combination therapy on prevention of acute heart allograft rejection in the rat. Microsurgery 2007;27:268–70. [29] Deuse T, Velotta JB, Hoyt G, et al. Novel immunosuppression: R348, a JAK3- and Syk-inhibitor attenuates acute cardiac allograft rejection. Transplantation 2008;85:885–92. [30] Roncarolo MG, Bacchetta R, Bordignon C, Narula S, Levings MK. Type 1 T regulatory cells. Immunol Rev 2001;182:68–79.
Contents lists available at ScienceDirect
Transplant Immunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t r i m
Synergistic effects of Isatis tinctoria L. and tacrolimus in the prevention of acute heart rejection in mice Yongzhi Wang a,1, Qing Qin b,c,1, Jibing Chen d,1, Xiaocong Kuang e, Junjie Xia a, Baiyi Xie a, Feng Wang a, Hua Liang f, Zhongquan Qi a,⁎ a
Organ Transplantation Institute, Xiamen University, Fujian Province, PR China School of Chemistry and Chemical Engineering, Guangxi University, Guangxi Province, PR China Dept. of Natural Products Chemistry, School of Pharmaceutical Sciences, Guangxi Medical University, Nanning, Guangxi Province, PR China d Basic Medical Department of Medical College, Xiamen University, Fujian Province, PR China e Pathophysiology Department of Basic Medical College, Guangxi Medical University, Guangxi Province, PR China f Zhongshan Hospital, Xiamen University, Fujian Province, PR China b c
a r t i c l e
i n f o
Article history: Received 1 July 2009 Received in revised form 9 August 2009 Accepted 1 September 2009 Keywords: Compound K FK-506 Lymphocyte T cells Cardiac transplantation Acute rejection
a b s t r a c t Although immunosuppressive treatments are available for acute cardiac rejection no viable treatment exists for long-term cardiac graft failure. Moreover, the extended use of calcineurin inhibitor immunosuppressants, the mainstay of current treatment for cardiac transplantation, leads to significant side effects such as nephrotoxicity and an increased risk of cardiac disease. Because some agents used in Traditional Chinese Medicine (TCM) have strong immunosuppressive effects coupled with low toxicity, we investigated the effect of Compound K (K), the synthesized analogue of highly unsaturated fatty acids from Isatis tinctoria L., either as a single treatment or combined with tacrolimus (FK-506) on acute cardiac allograft rejection. We compared the ability of K alone, or in combination with FK-506, to inhibit acute heart transplant rejection both in vitro and in vivo. We found that the inhibition of lymphocyte proliferation was positively correlated with K concentration. K significantly reduced IL-2 and IFN-γ expression levels and significantly inhibited lymphocyte proliferation in both a lymphocyte transformation test and a mixed lymphocyte reaction (MLR). We also found that the inhibitory effect of a combination of K and a sub-therapeutic dose of FK-506 (SubFK506) was stronger than that of full-dose FK-506 alone. Oral administration of K reduced acute cardiac allograft rejection in mice and had no apparent toxicity. In vivo, the immunosuppressive effect of K combined with a half-dose of FK-506 was equivalent to that of a full-dose of FK-506 alone. K combined with a half-dose of FK-506 reduced the expression levels of IL-2 and IFN-γ (both within the graft and in the recipients' serum) more effectively than a full-dose of FK-506. These results show that K has significant immunosuppressive effects both in vitro and in vivo. When used as a combination therapy with FK-506 we see a powerful inhibition of rejection with no obvious toxic side effects. The mechanism of action is postulated to involve the inhibition of IL-2 and IFN-γ expressions by lymphocytes, rather than the activation of Tr1 cells via the production of IL-10. © 2009 Elsevier B.V. All rights reserved.
1. Introduction In clinical organ transplantation, the calcineurin-inhibitors (such as CsA, RAPA and FK-506) are the first-line immunosuppressive agents of choice and have been used for many years. Studies have shown that these drugs clearly reduce the rate of acute rejection of kidney, heart and pancreatic islet transplants and prolong graft survival for many years [1–5]. However, side effects such as renal
⁎ Corresponding author. Organ Transplantation Institute, Xiamen University, 361005 Fujian Province, PR China. Tel./fax: +86 592 2180126. E-mail address: [email protected] (Z. Qi). 1 These authors contributed equally to the study and share the first authorship. 0966-3274/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.trim.2009.09.004
toxicity, neurotoxicity and the induction of diabetes limit the doses that can be used, thereby affecting the survival of the transplanted organ [6–9]. We are committed to the development of high efficiency, low toxicity immunosuppressants to replace the calmodulin-inhibitor immunosuppressants currently in use. Attention has been focused on plants and fungi that are associated with TCM as potential sources of new therapeutic agents. One such example is the parasitic fungus of insects called Isaria sinclairii that has recently been used by Novartis for the development of the novel immunosuppressive agent FTY720 [10]. The agent not only prevents rejection, but also reverses any signs of rejection that have already occurred [11]. This drug also has a synergistic effect when used in conjunction with calmodulin inhibitors and is currently completing Phase III trials for use in the treatment of multiple sclerosis [12,13].
6
Y. Wang et al. / Transplant Immunology 22 (2009) 5–11
We believe that there will certainly be a lot of high efficiency, low toxicity immunosuppressive drugs within TCM that have the potential for development and clinical use. Recently, we studied the immune-regulatory properties of K, an analogue of the highly unsaturated fatty acids from Isatis tinctoria L., and after testing, we found that K was the most promising. In this study, we compare the immunosuppressive effects of K and FK-506 (both individually and in combination) in the treatment of acute heart transplant rejection in a mouse model. We show that K can inhibit proliferation of lymphocytes in vitro and that it can enhance the effects of FK-506 in the prevention of acute rejection. Finally, we show that K acts via a mechanism that inhibits both IL-2 and IFN-γ expressions and production by lymphocytes rather than stimulating the production of IL-10, as what happens with FK-506. 2. Objectives The objectives of this study were four-fold. First, to determine whether K could reduce lymphocyte proliferation in a lymphocyte transformation test in vitro. Second, to determine whether K could reduce T lymphocyte proliferation and act synergistically with subtherapeutic doses of FK-506 in a mixed lymphocyte reaction (MLR). Third, to determine whether oral administration of K could reduce the acute rejection response in a mouse model and fourth, to investigate the mechanism of action of K both in vitro and in vivo. 3. Materials and methods 3.1. Animals Female C57BL/6 (B6) and BALB/c mice (8–12-weeks old) were purchased from Slac Laboratory Animal Co. Ltd. (Shanghai, China) and used as graft recipients and donors, respectively. Care and handling of animals were in accordance with the guidelines provided in the “Guide for the Care and Use of Laboratory Animals” published by the U.S. Department of Health and Human Services. 3.2. Drugs FK-506 was purchased from ALEXIS Co. Ltd. (Switzerland). The Compound K was obtained from Guangxi Medical University (Guangxi, China). 3.3. Lymphocyte isolation Lymphocytes were isolated from the spleens of B6 mice using mouse lymphocyte separation medium (Dakewei, China). Briefly, spleens were removed aseptically, placed in 4 ml mouse lymphocyte separation medium and gently ground through a nylon mesh using the top of the plunger from a 5 ml syringe. Debris was allowed to settle and the cell suspension isolated. The suspension was centrifuged at 200–400×g for 30 min and the white cell layer extracted and washed three times by centrifugation in RPMI 1640 (GIBCO, USA). After the final wash, the cell pellet was re-suspended in 10 ml of fresh RPMI 1640, the cells were counted and viability established by Trypan blue exclusion. Viability routinely exceeded 96%. 3.4. T lymphocyte isolation T lymphocytes were isolated from the spleens of B6 mice using nylon wool columns (Wako, Japan). Briefly, the nylon wool was washed three times in sterile PBS followed by washing three times in RPMI 1640 supplemented with 5% fetal bovine serum (FBS). Lymphocytes were suspended in 2 ml RPMI 1640/5% FBS and incubated with the nylon wool for 45 min at 37 °C. The nylon wool was then washed and the cell suspension centrifuged at 200–400×g for 5 min. The cell pellet was re-
suspended in 10 ml of fresh RPMI 1640, the cells counted and viability established by Trypan blue exclusion. Viability always exceeded 96%. Purity was always in excess of 90%. 3.5. Lymphocyte transformation test B6 spleen lymphocytes were plated in 96-well plates at a concentration of 3 × 105 cells/well. K was added to triplicate wells at concentrations of 5, 10, 20, 40, or 80 μg/ml followed by the addition of 5 μg/ml Con A (Sigma, USA) in RPMI 1640 supplemented with 10% FBS, penicillin, and streptomycin (total well volume = 200 μl). Plates were incubated at 37 °C in a 5% CO2 humidified atmosphere for 48 h. Cells cultured with K, but without Con A, were used as negative controls. Cell proliferation was quantified by the 5′-bromodeoxyuridine (BrdU) method using an enzyme linked immunosorbent assay (ELISA) kit (Millipore, USA), according to the manufacturer's instructions. Proliferation was measured using a microplate reader (BIO-RAD, USA). The level of inhibition was calculated with respect to the negative control and stimulated control values. 3.6. Mixed lymphocyte reaction T lymphocytes obtained from the spleens of female B6 mice were used as responder cells and spleen cells obtained from BALB/c mice were used as stimulator cells in a mixed lymphocyte reaction (MLR) assay. The responder cells (5 × 105 cells) were cultured in 96-well plates in the presence of stimulator cells (5 × 104 cells, pre-treated with mitomycin C, 40 μg/ml, Amresco, USA) in 200 μl RPMI 1640 supplemented with 10% FBS, penicillin, and streptomycin, and incubated at 37 °C in a 5% CO2 humidified atmosphere. K or FK-506 was added to four wells at the following concentrations: K alone (80 μg/ml), FK-506 alone (1 μg/ml) or a combination of K (80 μg/ml) and a sub-therapeutic dose of FK-506 (0.5 μg/ml). Responder cells cultured in medium without the stimulator cells were used as negative controls. After 72 h, 96 h or 120 h of culture, cell proliferation was quantified using the BrdU ELISA kit as outlined above. The results were expressed as the mean value of four wells. The percentage inhibition values were calculated with respect to the negative control and stimulated control values. 3.7. Heart transplantation Vascularized heterotopic heart transplantation from BALB/c donors to B6 recipients (n = 6) was performed by anastomosis to the vessels of the neck using a non-suture cuff technique as described previously [14]. Graft survival was monitored by palpation (twice daily) and body weight was recorded (daily) until complete graft rejection occurred. Rejection was defined as the loss of palpable cardiac contractions. B6 mice were treated with FK-506 (2 mg/kg/d), SubFK-506 (1 mg/kg/d), K (10 mg/kg/d) or SubFK-506 + K on Days 0– 10 post-transplant. The control group was given normal saline only. 3.8. Histological evaluation of rejection Heart grafts (n = 3) were removed on Day 6 post-transplant. Part of each graft was used for qRT-PCR, and the rest was used for histological evaluation. Paraffin-embedded transventricular tissue sections (5 μm thick) were stained with hematoxylin and eosin. Rejection was graded based on the extent of lymphocytic infiltration, the anatomical localization of leukocytes as per the International Society of Heart and Lung Transplantation (ISHLT) standards [15,16] and on the advice of an in-house pathologist. Briefly, heart tissue was scored as follows: 0 = no damage; 1 (mild) = evidence of interstitial edema and focal necrosis; 2 (moderate) = graft displayed diffuse myocardial cell swelling and necrosis; 3 (severe) = necrosis with the presence of contraction bands and neutrophil infiltrate, and 4 (highly
Y. Wang et al. / Transplant Immunology 22 (2009) 5–11
7
severe) = widespread necrosis with the presence of contraction bands, neutrophil infiltrate and hemorrhage. 3.9. Real-time quantitative reverse transcription-polymerase chain reaction Reverse transcription (RT) was performed using 1 μg of the total RNA extracted from the allografts harvested on Day 6 post-transplant using the Trizol-reagent. Real-time polymerase chain reaction (RTPCR) was carried out using 2 μl of the RT product on a StepOne RealTime PCR System (ABI, UK). Syber Green I and β-actin were used as controls. Each reaction was carried out in triplicate. The following primer sequences were used for the real-time RT-PCR: β-actin forward 5′-CATCCGTAAAGACCTCTATGCCAAC-3′ and reverse 5′-ATGGAGCCACCGATCCACA-3′; IFN-γ forward 5′-CGGCACAGTCATTGAAAGCCTA-3′ and reverse 5′-GTTGCTGATGGCCTGATTGTC-3′; IL-2 forward 5′-GGAGCAGCTGTTGATGGACCTAC-3′ and reverse 5′-AATCCAGAACATGCCGCAGAG-3′; IL-10 forward 5′-GACCAGCTGGACAACATACTGCTAA-3′ and reverse 5′-GATAAGGCTTGGCAACCCAAGTAA-3′. The results were expressed as the mean value of triplicate wells. 3.10. Cytokine ELISA Recipients' serum (n = 3) were isolated on Day 6 post-transplant. ELISA was performed using commercially available kits to detect IL-2, IFN-γ and IL-10 (Bender, USA) according to the manufacturer's instructions. Briefly for IFN-γ flat-bottomed 96-well ELISA plates were coated with an anti-mouse-IFN-γ antibody in PBS and incubated overnight at 4 °C. After washing (wash buffer) and blocking (assay buffer), sample, sample diluent and biotin-conjugate were applied to the wells and incubated for 2 h. After washing, the plates were incubated with Streptavidin-HRP for 1 h at room temperature. Samples were developed using a 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution and the reaction stopped with 2 M sulfuric acid. ELISA plates were read using a microplate reader at 450 nm. The results were expressed as the mean value of four wells. 3.11. Statistical analysis Survival curves and body weight curves were obtained using GraphPad Prism® (GraphPad Software, USA). The survival curves were analyzed using the log-rank test. The data from the MLR, ELISA and qRT-PCR experiments were analyzed using the Student's t test and expressed as mean values ± standard deviation (SD). A value of p < 0.05 was considered to be a significant statistical difference.
Fig. 1. The ability of K to inhibit lymphocyte proliferation in vitro. Positive control and treatment groups treated with 5 μg/ml Con A. Negative control without Con A. Maximal inhibition of T cell proliferation occurs at a K concentration of 80 μg/ml. Cell proliferation was quantified using the BrdU method. Each concentration was tested in triplicate. Data shown is mean ± SD and is representative of four separate experiments (⁎p < 0.05).
4.3. Mechanism of K-mediated inhibition of lymphocyte proliferation Lymphocytes were cultured for 48 h with 5 μg/ml Con A and K alone (80 μg/ml), FK-506 alone (1 μg/ml) or a combination treatment with FK-506 and K (0.5 μg/ml and 80 μg/ml, respectively). qRT-PCR was used to detect the expression levels of IL-2 and IFN-γ in each treatment group. Figs. 3a and b show that, compared with the control group, both IL-2 and IFN-γ expression levels are significantly reduced in all treatment groups. However, a combined treatment with K and FK-506 results in the greatest reduction in expression levels of both IL-2 and IFN-γ. These data clearly show that K inhibits the expressions of IL-2 and IFN-γ by lymphocytes and thus inhibits proliferation. Again, this effect is enhanced when K is used in combination with FK-506. 4.4. Cardiac allograft survival time and body weight changes in graft recipients The mice that received cardiac allografts were divided into the following treatment groups: K (10 mg/kg/d); FK-506 (2 mg/kg/d); SubFK-506 (1 mg/kg/d); SubFK-506 (1 mg/kg/d) + K (10 mg/kg/d) and a control group (normal saline). Treatment was given daily from Day 0 to Day 10 post-transplantation. As shown in Fig. 4a, the median survival time (MST) was 7 days in the control group, 8.5 days in the SubFK-506 group, 10 days in the K group, 16.5 days in the SubFK-506 + K group and 17.5 days in FK-506 group. Only minor changes in body weight were noted after transplantation, mainly because of the surgical trauma (Fig. 4b). These results suggest that treatment with K alone provides some protection against the acute rejection response, but when combined with SubFK-506 it can significantly prolong the graft survival time in a manner similar to a full-dose of FK-506. Importantly, no side effects of K treatment were noted. 4.5. K, combined with a sub-therapeutic dose of FK-506, can reduce acute rejection At 6 days post-heart transplantation, grafts were harvested and prepared for histology as outlined above. The ISHLT scores of the hearts harvested from the different
4. Results 4.1. Effect of K on lymphocyte transformation We looked at the ability of K to inhibit Con A-stimulated lymphocyte proliferation at a series of concentrations (10, 20, 40, and 80 μg/ml). Fig. 1 shows that, after a 48-hour incubation, K inhibited proliferation in a dose-dependent manner with a concentration of 80 μg/ml being statistically significant. These results clearly show that K inhibits lymphocyte proliferation in vitro. 4.2. Effect of K on mixed lymphocyte response To further investigate the inhibitory effect of K on T cell proliferation we undertook MLR experiments using K or FK-506 at the following concentrations: K alone (80 μg/ ml), FK-506 alone (1 μg/ml) or a combination of K (80 μg/ml) and a sub-therapeutic dose of FK-506 (0.5 μg/ml). T cell proliferation was assessed after 3, 4 or 5 days of culture using the BrdU method. We found that, compared with the control group, T cell proliferation was significantly inhibited by K alone, FK-506 alone and the combination treatment in assays carried out for 3, 4 or 5 days (Fig. 2; p < 0.05). Combination treatment with K and SubFK-506 resulted in a significant inhibition of T cell proliferation compared with FK-506 alone after 4 days of culture, but not after 3 or 5 days of culture. It is notable that, although K is able to inhibit T cell proliferation when compared with the control, it has an obvious synergistic effect when used in combination with FK-506.
Fig. 2. K reduces T cell proliferation in an in vitro MLR assay responder and stimulator cells were mixed for 3, 4 or 5 days. Cell proliferation was quantified using the BrdU method. Each drug concentration was tested in quadruple wells. Data shown is mean ± SD and is representative of three separate experiments. Drug treatment inhibited T cell proliferation at all time points tested compared with the control group (p < 0.05). On Day 4, combination treatment inhibited T cell proliferation to a greater extent than FK-506 alone (p < 0.05).
8
Y. Wang et al. / Transplant Immunology 22 (2009) 5–11
Fig. 3. K inhibits lymphocyte IL-2 and IFN-γ gene expressions in vitro. Lymphocytes stimulated with 5 μg/ml Con A were treated with K alone (80 μg/ml), FK-506 alone (1 μg/ml) or a combination of K with a sub-therapeutic dose of FK-506 (0.5 μg/ml) and cultured for 48 h. qRT-PCR was used to detect the expressions of IL-2 and IFN-γ in each group. Each treatment was done in triplicate wells. Data shown is the mean ± SD and is representative of three separate experiments. Expressions of IFN-γ and IL-2 were inhibited in all treatment groups relative to the control group, with combination therapy being the most effective (p < 0.05, ⁎p < 0.05, ⁎⁎p < 0.01).
Fig. 4. Cardiac allograft survival time and body weight changes in graft recipients. Vascularized heterotopic heart transplants from BALB/c mice to B6 mice were monitored twice a day by manual palpation and body weight was recorded daily until complete graft rejection occurred. Recipient mice were treated on Days 0–10 posttransplant with FK-506 (2 mg/kg/d), half-dose (Sub) FK-506 (1 mg/kg/d), K (10 mg/kg/ d) or SubFK-506 + K. The control group was given normal saline. (a) Kaplan–Meier curve of palpation scores for the five groups; and (b) body weight changes in recipients. Each data point represents the mean values obtained from six individuals.
treatment groups are shown in Figs. 5a–e. The grafts from the control group showed highly severe infiltration and tissue damage and the grafts from both the K and the SubFK-506 groups showed moderate infiltration and tissue damage. The grafts from the full-dose FK-506 and the combined treatment groups, however, showed only mild infiltration and tissue damage. These results suggest that treatment with K alone provides limited protection against acute rejection responses, but that this protection is increased when K is combined with a sub-therapeutic dose of FK-506.
in the circulating levels of the pro-inflammatory cytokine IFN-γ, and that the effect is enhanced when combined with FK-506.
4.6. The relative expressions of key genes in rejected cardiac allografts After the cardiac grafts were harvested, mRNA was prepared and reverse transcribed. The RT products were analyzed by real-time qPCR. The gene expression profiles of the treatment groups were normalized relative to the control group profiles. As shown in Figs. 6a and b, compared with the control group, the relative expressions of IL-2 and IFN-γ are significantly reduced in all the treatment groups (p < 0.01). Interestingly, both IL-2 and IFN-γ expressions are significantly less in the combined treatment group than in the full-dose FK-506 group (p < 0.05). As seen in Fig. 6c, the expression of IL-10 is enhanced in the full-dose FK-506 group but its expression actually reduced in the K-treated group (p < 0.01). These data suggest that K works by reducing the expressions of both IL-2 and IFN-γ within the graft rather than enhancing the expression of IL-10, resulting in prolonged graft survival times. 4.7. The relative expression cytokine levels in the recipients' blood serum Cytokine levels within the recipients' serum were analyzed by ELISA. As seen in Fig. 7b, compared with control group, the relative expression levels of IFN-γ are significantly reduced in all the treatment groups (p < 0.05), and are significantly lower in the combined treatment group than in the full-dose FK-506 group (p < 0.05). No significant differences are seen in the relative expressions of IL-2 and IL-10 between the treatment groups (Figs. 7a, c). These data indicate that K treatment results in a decrease
5. Discussion The use of immunosuppressants that exhibit high efficiency and low toxicity is important to prolong graft survival time and to improve the quality of life in clinical situations. The use of combined treatments with a variety of different immunosuppressive agents not only inhibits the rejection response but can also reduce the side effects of the drugs. This is an important principle in clinical immunosuppressive therapy. We have previously synthesized K, the analogue of highly unsaturated fatty acids from I. tinctoria L, and using different in vitro methods such as lymphocyte transformation tests, MLR, qRT-PCR and ELISA we have shown that this monomer has strong immunosuppressive properties and has the potential to be developed as a new immunosuppressant. We found that the degree of immunosuppression was positively correlated with drug concentration in a manner similar to that seen with other clinical immunosuppressive agents such as FK-506 [17]. T cells are the main mediators of acute cardiac rejection [18–20] . We have attempted to use an MLR assay [21] to provide evidence of the inhibition of the acute rejection response by K. We found that K could indeed inhibit T cell proliferation in in vitro MLR experiments but the effect was not significantly different from that of FK-506.
Y. Wang et al. / Transplant Immunology 22 (2009) 5–11
9
Fig. 5. K in combination with a sub-therapeutic dose of FK-506 can reduce acute rejection. On post-transplant Day 6, grafts were harvested and prepared for histology. H & E stained heart tissue from: (a) an untreated animal; b) an animal treated with a sub-therapeutic dose of FK-506 (1 mg/kg/d); (c) an animal treated with K (10 mg/kg/d); (d) an animal treated with full-dose of FK-506 (2 mg/kg/d); (e) an animal treated with K plus a sub-therapeutic dose of FK-506; and (f) ISHLT scoring of the hearts. The dot plot represents the scores for each animal in each group. The line represents the mean scores (p = 0.12, n = 3).
However, a combination of K and FK-506 was more effective than either K or FK-506 alone. This led us to explore the mechanism of action of K, as it has an obvious synergistic effect when used in combination with FK-506. We studied this interaction with reference to other works that have studied the mechanism of action of conventional calmodulin antagonists and analyzed IL-2 and IFN-γ gene expressions after different drug treatments [22,23]. We found that K significantly inhibits the expressions of both the IL-2 and the IFN-γ genes by proliferating T cells in the MLR assay. Compared with FK-506 alone, K provides limited immunosuppression, but combined with sub-therapeutic doses of FK-506 it provides stronger immunosuppression than treatment with full-dose FK-506 alone. This suggests that combination therapy may be a powerful treatment for acute cardiac rejection. To explore the prospects for the clinical application of K, our experiments focused on treatment with K, either alone or in combination with a sub-therapeutic dose of FK-506, and we compared the effects of this treatment with those of a full-dose of FK-506 in an acute cardiac allograft rejection model. As shown in Fig. 4a, treatment with a maximum dose of K alone, extends cardiac allograft survival for only a short time over non-treated controls (9 days versus 7 days).
This is clearly not satisfactory. Combined therapy (K + SubFK-506) shows immunosuppressive effects comparable to those of a full-dose of FK-506 (16.5 days versus 17.5 days). This is supported by other studies that have looked at the effects of combined drug treatments on cardiac allograft survival [24–29]. Next, we investigated the similarities and differences between the inhibitory mechanisms induced by combined therapy and full-dose FK-506 by examining the histology of the rejected allografts and by analyzing the levels of Th1 and Tr1 cytokines in both the graft and the serum of the recipients. Looking at the Th1 and Tr1 cytokine expression levels within the rejected grafts, we found that IL-2, IFNγ and IL-10 levels are all significantly lower after combined treatment than after treatment with full-dose FK-506 alone. It is known that FK506 suppresses the transcription of both the IL-2 and the IFN-γ genes by lymphocytes and it is possible that it also enhances the expression of IL-10 [21]. K appears not to induce IL-10 gene expression, but does inhibit IL-2 and IFN-γ gene expressions, and the combination of K and SubFK-506 has a synergistic effect on Th1 cytokine production that is more powerful than full-dose FK-506 alone. Our results suggest that there may be a K-induced tolerance mechanism that does not induce the proliferation of IL-10-secreting Tr1 cells [30].
10
Y. Wang et al. / Transplant Immunology 22 (2009) 5–11
Fig. 6. The relative expression of some key genes within the allografts. IL-2, IFN-γ and IL-10 mRNA were extracted from the grafts and used for real-time qPCR. (a) and (b): compared with the control group, the relative expressions of IL-2 and IFN-γ are significantly reduced in all the treatment groups (p < 0.01), and is significantly less in the combination treatment group than in the full-dose FK-506 group (p < 0.05). (c) The expression of IL-10 is enhanced in the full-dose FK-506 group, but its expression is reduced in the K-treated group (p < 0.01). Data shown is the mean ± SD and is representative of three separate experiments (⁎p < 0.05).
Analysis of the Th1-type cytokine expression levels in the recipients' serum shows that IFN-γ expression in the combination therapy group was significantly lower than in the full-dose FK-506 group, in line with the levels of gene expression seen within the graft. This evidence further validates the potential use of combined K/ SubFK-506 therapy as a replacement for full-dose FK-506 for the treatment of organ transplant rejection and long-term maintenance of graft survival. Our in vivo studies of acute cardiac allograft rejection showed no differences in body weight between the control and treatment groups during the post-transplant period. These data suggest that K has little effect on body weight and so is not likely to be toxic. The minor changes in body weight seen after transplantation were most likely to be the result of surgical trauma rather than K-mediated toxicity. In summary, K has low toxicity and, combined with subtherapeutic doses of FK-506, can strongly inhibit acute cardiac allograft rejection. Although K was able to inhibit IL-10 expression by Tr1 cells, combination therapy with SubFK-506 shows a strong synergy and significant inhibition of Th1-type cytokine expression
Fig. 7. The relative levels of cytokine in the recipient's blood. Recipients' serum was analyzed by ELISA. Compared with the control group, the relative levels of IFN-γ are significantly reduced in all the treatment groups (p < 0.05), but are significantly lower in the combined treatment group than in the full-dose FK-506 group (p < 0.05). There is no significant difference in the levels of IL-2 and IL-10 between any of the groups. Data shown is the mean± SD and is representative of three separate experiments (⁎p < 0.05).
within the graft leading to prolonged graft survival. This may be because K and FK-506 affect different signal pathways that regulate lymphocyte proliferation. Further studies are planned to investigate this possibility. References [1] Fung JJ, Abu-Elmagd K, Todo S, et al. Overview of FK506 in transplantation. Clin Transpl 1990:115–21. [2] Jordan JL, Hirsch GM, Lee TDG. C. sinensis ablates allograft vasculopathy when used as an adjuvant therapy with cyclosporin A. Transpl Immunol 2008;19:159–66. [3] Peddi VR, Kamath S, Munda R, Demmy AM, Alexander JW, First MR. Use of tacrolimus eliminates acute rejection as a major complication following simultaneous kidney and pancreas transplantation. Clin Transplant 1998;12:401–5. [4] Onsager DR, Canver CC, Jahania MS, et al. Efficacy of tacrolimus in the treatment of refractory rejection in heart and lung transplant recipients. J Heart Lung Transplant 1999;18:448–55. [5] Haeryfar SM, Lan Z, Leon-Ponte M, et al. Prolongation of cardiac allograft survival by rapamycin and the invariant natural killer T cell glycolipid agonist OCH. Transplantation 2008;86:460–8. [6] Condé SA, Aarestrup FM, Vieira BJ, Bastos MG. Roxithromycin reduces cyclosporine-induced gingival hyperplasia in renal transplant patients. Transplant Proc 2008;40(5):1435–8.
Y. Wang et al. / Transplant Immunology 22 (2009) 5–11 [7] Mueller AR, Platz KP, Bechstein WO, et al. Neurotoxicity after orthotopic liver transplantation. A comparison between cyclosporine and FK506. Transplantation 1994;58:155–70. [8] Kamar N, Mariat C, Delahousse M, et al. Diabetes mellitus after kidney transplantation: a French multicentre observational study. Nephrol Dial Transplant 2005;22: 1986–93. [9] Molinari M, Al-Saif F, Ryan EA, et al. Sirolimus-induced ulceration of the small bowel in islet transplant recipients: report of two cases. Am J Transplant 2005;5: 2799–804. [10] Brinkmann V. FTY720: mechanism of action and potential benefit in organ transplantation. Yonsei Med J 2004;45:991–7. [11] Chiba K. FTY720, a new class of immunomodulator, inhibits lymphocyte egress from secondary lymphoid tissues and thymus by agonistic activity at sphingosine 1-phosphate receptor. Pharmacol Ther 2005;108:308–19. [12] Schuurman HJ, Menninger K, Audet M, et al. Oral efficacy of the new immunomodulator FTY720 in cynomolgus monkey kidney allotransplantation, given alone or in combination with cyclosporine or RAD. Transplantation 2002;74: 951–60. [13] Baumruker T, Billich A, Brinkmann V. FTY720, an immunomodulatory sphingolipid mimetic: translation of a novel mechanism into clinical benefit in multiple sclerosis. Expert Opin Investig Drugs 2007;16:283–9. [14] Matsuura A, Abe T, Yasuura K. Simplified mouse cervical heart transplantation using a cuff technique. Transplantation 1991;51:896–8. [15] Billingham ME, Cary NR, et al. A working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection: Heart Rejection Study Group. The International Society for Heart Transplantation. J Heart Transplant 1990;9:587–93. [16] Aneja R, Hake PW, Burroughs TJ, Denenberg AG, Wong HR, Zingarelli B. Epigallocatechin, a green tea polyphenol, attenuates myocardial ischemia reperfusion injury in rats. Mol Med 2004;10:55–62. [17] Almawi WY, Assi JW, Chudzik DM, Jaoude MM, Rieder MJ. Inhibition of cytokine production and cytokine-stimulated T-cell activation by FK506 (tacrolimus). Cell Transplant 2001;10:615–23. [18] Lafferty KJ, Simeonovic CJ. Immunology of graft rejection. Transplant Proc 1984;16:927–30.
11
[19] Krieger NR, Yin DP, Fathman CG. CD4+ but not CD8+ cells are essential for allorejection. J Exp Med 1996;184:2013–8. [20] Pietra BA, Wiseman A, Bolwerk A, Rizeq M, Gill RG. CD4 T cell-mediated cardiac allograft rejection requires donor but not host MHC class II. J Clin Invest 2000;106:1003–10. [21] Morikawa K, Oseko F, Morikawa S. Immunosuppressive activity of bromocriptine on human T lymphocyte function in vitro. Clin Exp Immunol 1994;95:514–8. [22] Yoshimura N, Matsui S, Hamashima T, Oka T. Effect of a new immunosuppressive agent, FK506, on human lymphocyte responses in vitro. II. Inhibition of the production of IL-2 and gamma-IFN, but not B cell-stimulating factor 2. Transplantation 1989;47:356–9. [23] Banerji SS, Parsons JN, Tocci MJ. The immunosuppressant FK-506 specifically inhibits mitogen-induced activation of the interleukin-2 promoter and the isolated enhancer elements NFIL-2A and NF-AT1. Mol Cell Biol 1991;11:4074–87. [24] Vu MD, Qi S, Xu D, et al. Tacrolimus (FK506) and sirolimus (rapamycin) in combination are not antagonistic but produce extended graft survival in cardiac transplantation in the rat. Transplantation 1997;64(12):1853–6. [25] Qi Z, Simanaitis M, Ekberg H. Malononitrilamides and tacrolimus additively prevent acute rejection in rat cardiac allografts. Transpl Immunol 1999;7:169–75. [26] Shirouzu Y, Hikida S, Tanigawa H, Shirouzu K. Inhibition of acute rejection after skin or cardiac transplantation by administration of donor antigens in the rat. Surg Today 2004;34:341–8. [27] Deuse T, Schrepfer S, Pelletier MP, Fischbein MP, Robbins RC, Reichenspurner H. Is the malononitrilamide FK778 better for the prevention of acute or chronic rejection? Transplant Proc 2007;39:569–72. [28] Qi S, Zhao H, Ma A, et al. Effect of baohuoside-1 aglycone and tacrolimus monotherapy and combination therapy on prevention of acute heart allograft rejection in the rat. Microsurgery 2007;27:268–70. [29] Deuse T, Velotta JB, Hoyt G, et al. Novel immunosuppression: R348, a JAK3- and Syk-inhibitor attenuates acute cardiac allograft rejection. Transplantation 2008;85:885–92. [30] Roncarolo MG, Bacchetta R, Bordignon C, Narula S, Levings MK. Type 1 T regulatory cells. Immunol Rev 2001;182:68–79.