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Platonin Improves Survival of Skin Allografts
Journal of Surgical Research 164, 146–154 (2010) doi:10.1016/j.jss.2009.05.045
Platonin Improves Survival of Skin Allografts Shih-Ping Cheng, M.D.,*,‡ Jie-Jen Lee, M.D., Ph.D.,‡,** Chin-Wen Chi, Ph.D.,*,{ Kuo-Ming Chang, M.D.,†† and Yu-Jen Chen, M.D., Ph.D.†,§,k,# ,1 *Department and Institute of Pharmacology, School of Medicine, National Yang-Ming University, Taipei, Taiwan; †Institute of Traditional Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan; ‡Department of Surgery, Mackay Memorial Hospital, Taipei, Taiwan; §Department of Radiation Oncology, Mackay Memorial Hospital, Taipei, Taiwan; kDepartment of Medical Research, Mackay Memorial Hospital, Taipei, Taiwan; {Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan; #Graduate Institute of Sport Coaching Science, Chinese Culture University, Taipei, Taiwan; **Institute of Pharmacology, Taipei Medical University, Taipei, Taiwan; and ††Department of Pathology and Laboratory Medicine, Hsinchu Mackay Memorial Hospital, Hsinchu, Taiwan Submitted for publication January 9, 2009
Background. Platonin is an immunomodulator with NF-kB inhibitory activity. It not only inhibits interleukin (IL)-1b, IL-6, and tumor necrosis factor (TNF)-a production in sepsis, but also attenuates heatstroke reactions. In addition, platonin redirects differentiation of dendritic cells toward an intermediate stage of maturation. The study was designed to examine whether platonin can reduce acute graft rejection. Materials and Methods. A C57BL/6 to BALB/c mice skin transplantation model was used. Platonin was given intraperitoneally to transplant recipients at various doses. Skin grafts were submitted to histologic analysis. NF-kB DNA binding activity and inducible nitric oxide synthase (iNOS) expression were determined in harvested draining lymph nodes. Leukocyte count, hepatic and renal functions were serially assessed. An array of serum cytokines was evaluated on d 1, 3, 5, and 7 after skin transplantation. Results. Platonin resulted in significantly prolonged skin allograft survival in a dose- and time-dependent manner. Histologic changes in the skin allografts paralleled the gross appearance of rejection. Serum cytokine analysis shows that platonin significantly suppressed the production of the proinflammatory cytokines IL-6 and TNF-a. However, no significant changes occurred in the serum levels of Th1-type and Th2-type cytokines. NF-kB activity and iNOS expression were remarkably suppressed in draining lymph nodes. In terms of toxicity, there were no significant differences in
1 To whom correspondence and reprint requests should be addressed at Department of Radiation Oncology, Mackay Memorial Hospital, 92, Sec. 2, Chung-Shan North Road, Taipei 10449, Taiwan. E-mail: [email protected].
0022-4804/$36.00 Ó 2010 Elsevier Inc. All rights reserved.
body weight, leukocyte count, plasma alanine aminotransferase, or creatinine between the platonintreated and control groups. Conclusion. Platonin effectively prolongs skin allograft survival without major toxicity. Ó 2010 Elsevier Inc. All rights reserved.
Key Words: platonin; skin allotransplantation; proinflammatory cytokine. INTRODUCTION
Organ transplantation is an important means of managing end-organ failure. Immunosuppressive agents, particularly the calcineurin inhibitors, have made a significant contribution to the success of transplantation. However, concerns about renal and cardiovascular toxicity have prompted growing interest in calcineurin inhibitor-sparing regimens, mostly involving a switch to a mammalian target-of-rapamycin (mTOR) inhibitor [1]. The antineoplastic activity and relatively non-nephrotoxic attributes of mTOR inhibitors are the most favorable properties, but a number of adverse effects have been reported, including hyperlipidemia, myelosuppression, and complications related to growth factor inhibition, such as impaired wound healing [2]. Therefore, it is of critical importance to search for new immunosuppressive drugs that are effective and not toxic to kidney or bone marrow. Platonin is a cyanine photosensitizing dye with a wide range of pharmacologic properties, including antimicrobial, antioxidant, antihistaminic, and anticancer activities [3]. Using in vitro and in vivo models, we have demonstrated that platonin inhibits nuclear
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factor-kB (NF-kB) activation and thereby reduces pyrogen expression in endotoxemia-induced fever [4–6]. Activation of NF-kB appears to play a major role in acute allograft rejection [7], is required for dendritic cell (DC) maturation and activation [8], and is crucial for activation and proliferation of lymphocytes [9]. Many of the immunosuppressive drugs currently used also inhibit NF-kB activation, which contributes partially to their mechanism of action [10]. Furthermore, up-regulation of proinflammatory mediators occurs rapidly after transplantation, an event that may then be shaped and amplified by a graft-specific adaptive response [11]. Platonin may favorably influence these events. For example, the elevated levels of interleukin (IL)1b, IL-6, and tumor necrosis factor (TNF)-a induced by systemic administration of lipopolysaccharide (LPS) in rabbits are attenuated by pretreatment with platonin [5]. In peripheral blood mononuclear cells, platonin suppresses the production of proinflammatory cytokines, nitric oxide (NO) production, and inducible NO synthase (iNOS) expression induced by LPS [12]. Targeting proinflammatory cytokines with anti-TNFa and anti-IL-1b antibodies has a beneficial effect on engraftment of islet grafts [13]. Taken together, platonin might benefit graft survival due to a suppressive effect on NF-kB and production of proinflammatory cytokines related to transplant rejection. Our earlier studies demonstrated that platonin modulates the differentiation, maturation, and functions of DC. With platonin treatment, human monocyte-derived DC have decreased CD83 expression, increased CD80 expression, and less stimulatory activity on allogeneic naive CD4þCD45RAþ T cells [14], indicating that platonin redirects DC differentiation toward an intermediate stage of maturation. CD80 has more productive interactions with CD152 (CTLA-4) than with CD28 [15], and engagement of CD152 by CD80 inhibits T cell receptorand CD28-mediated T cell proliferation. These actions of platonin on DC raise the possibility that this agent might be tolerogenic in transplant settings. Given that platonin inhibits NF-kB activation, suppresses proinflammatory cytokine release, and modulates DC development, we postulated that systemic administration of platonin might decrease transplant rejection. We therefore used a skin transplantation model to evaluate graft survival, serum cytokine levels, in vivo regulation of NF-kB activity and iNOS expression, and hepatic and renal toxicity in platonin-treated mice.
MATERIALS AND METHODS Animals Six- to 8-wk-old male C57BL/6 (H-2b) and BALB/c (H-2d) mice were obtained from the Animal Resource Center of the National Science
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Council of Taiwan (Taipei, Taiwan). They were housed in a specific pathogen-free environment in compliance with the National Research Council’s Guide for the Care and Use of Laboratory Animals. All experiments were approved by the animal ethics committee of Mackay Memorial Hospital, Taipei, Taiwan (MMH-A-S-95059).
Skin Transplantation All operations were performed under anesthesia with a single intraperitoneal (i.p.) dose of ketamine hydrochloride (100 mg/kg body weight; Sigma, St. Louis, MO). Skin transplantation was conducted by a procedure modified from that described previously [16]. Briefly, a 2 3 1 cm full-thickness skin graft was removed from the flank of a euthanized C57BL/6 donor mouse, and the underside was gently scraped with a scalpel to remove fat and muscle. The right dorsal flank of anesthetized BALB/c recipient was shaved and washed with 70% ethanol. A graft bed was prepared with fine scissors by removing an area of epidermis and dermis down to the level of the intrinsic muscle. The graft was fixed to the graft bed with 10 interrupted sutures of 5-0 monofilament nylon thread (Dermalon; Davis and Geck, St. Louis, MO). No dressings or antibiotics were used. Skin graft survival was evaluated three times a week by visual and tactile examination. The percentage of viable graft was assessed using a transparent grid-template overlay. Rejection was defined as necrosis of more than 80% of the epidermal surface of the graft.
Drug Preparation and Treatment Platonin, 4,40 ,400 -thrimethyl-3,30 300 -triheptyl-7-(200 -thia-zolyl)-2,20 trimethinethiazolocyanine-3-300 -diiodide, (synthesized by Kankohsha Co., Osaka, Japan and obtained from Gwo Chyang Pharmaceutical Co., Tainan, Taiwan) was stored at 4 C without light exposure. Dilutions were prepared with normal saline immediately before i.p. injection. To minimize its photosensitizing effect, all procedures were performed under light-avoidance conditions. In the first set of experiments, 60 mice were randomly assigned to four groups of 15 mice each and treated according to one of the following regimens: (1) 1.0 mg/kg platonin i.p. per d, (2) 10 mg/kg platonin i.p. per d, (3) 100 mg/kg platonin i.p. per d, or (4) normal saline (as a vehicle control). All treatments were started on the day of transplantation and continued until rejection was observed. In the second set of experiments, 48 animals were randomly assigned to two groups receiving either 10 mg/kg platonin i.p. per d or normal saline. Skin grafting was performed in the same manner as described above. Pretransplantation blood samples were obtained via retro-orbital plexus. The weight of each mouse was determined every other day by a single observer. Six mice each were sacrificed by cervical dislocation on d 1, 3, 5, and 7 after operation, and blood samples were collected by cardiac puncture. Skin grafts and draining lymph nodes (axillary, brachial, and inguinal) were harvested at sacrifice.
Histologic Evaluation For histologic analysis, skin grafts were fixed in formalin and embedded in paraffin. Five-mm sections were cut, deparaffinized, rehydrated, and stained with hematoxylin and eosin. A pathologist blinded to which experimental group the specimens were from evaluated the slides to determine the presence of rejection, infection, and other histologic changes. This included a search for perivascular infiltrates, epidermal degeneration, and stromal changes. If perivascular infiltrates were present, the type of inflammatory cells involved (lymphocytes, eosinophils, or neutrophils) were noted. Changes in the stroma might include inflammation, edema, red blood cell extravasation, and necrosis.
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Serum Cytokine Analysis Serum obtained from blood samples were frozen at –80 C, then thawed to room temperature and analyzed using cytometric bead array kits according to the manufacturer’s instruction (BD Biosciences, San Jose, CA). IL-6, IL-10, IL-12p70, and interferon (IFN)-g were detected simultaneously using the mouse inflammation kit. IL-2, IL-4, IL-5, and TNF-a protein levels were measured using the mouse Th1/Th2 cytokine kit. For this assay, soluble cytokines were captured on various antibody-conjugated microparticles, and measured using flow cytometry as described previously [17]. Briefly, each sample (50 mL) was mixed with 50 mL of mixed capture beads and 50 mL of the mouse detection reagent consisting of PE-conjugated antibodies. The tubes were incubated at room temperature for 2 h in the dark. The samples were washed once and then resuspended in 300 mL of wash buffer before acquisition on a FACSCalibur flow cytometer (BD Biosciences). The concentration of each cytokine was determined by interpolation from the corresponding calibration curve generated from the mixed bead standard provided in the kit. Data were analyzed using BD Biosciences cytometric bead array software.
NF-kB DNA-Binding Assay Nuclear extraction was done with the Nuclear Extract kit (Active Motif, Carlsbad, CA) according to the protocol of the manufacturer. Briefly, at least 200 mg of each tissue was diced into small pieces. After addition of a hypotonic buffer supplemented with dithiothreitol and detergent, tissue was homogenized. The suspension was incubated on ice for 15 min, followed by a centrifugation at 4 C for 10 min at 850 g. Single cells were then resuspended in hypotonic buffer supplemented with detergent, and incubated on ice for 15 min to permeabilize the cell membranes. Nuclei were collected by centrifuging the suspension at 14,000 g in a precooled centrifuge at 4 C for 30 s. After removal of the supernatants containing cytoplasmic proteins, the pellet was again resuspended in a lysis buffer to break down the nuclei and extract the nuclear proteins. Suspension was then incubated on ice for 30 min on a rocking platform set at 150 rpm. Finally, nuclear proteins were collected by centrifuging the suspension at 14,000 g in a precooled centrifuge at 4 C for 10 min. Protein concentration was determined using a standard Bradford protein assay. The supernatant was stored at –80 C for NF-kB binding assay. The DNA-binding activity of NF-kB was quantified by enzyme linked immunosorbent assay (ELISA)-based TransAM NF-kB p65 transcription factor assay kit (Active Motif), which shows a good correlation and better sensitivity compared with an electrophoretic mobility shift assay (EMSA) [18]. The TransAm NF-kB ELISA kit consists of 96-well microtiter plates precoated with an oligonucleotide containing the appropriate NF-kB binding consensus sequence. The active form of NF-kB binding to the target oligonucleotide was detected by incubation with primary antibody specific for the activated form of p65, visualized by anti-IgG horseradish peroxidase conjugate and developing solution, and quantified at 450 nm with a reference wavelength of 655 nm. The results were expressed after subtraction of background binding obtained by incubation with a mutated consensus oligonucleotide which has no effect on NF-kB binding.
were washed three times in phosphate buffer solution, and further incubated with a biotinylated secondary antibody for 30 min at room temperature. Antigen-antibody complexes were detected by the avidin-biotin-peroxidase method. For negative controls, the primary antibody was omitted, and unspecific rabbit IgG was used at the same concentration as the primary antibody. Finally, the slides were counterstained with hematoxylin and then examined by light microscopy.
Evaluation of Leukocyte Count, Hepatic, and Renal Functions The white blood cell counts of the blood samples were analyzed by an automatic Coulter counter (model Z1; Beckman Coulter Electronics, Fullerton, CA). Plasma levels of alanine aminotransferase (ALT) and creatinine were measured by a standard colorimetric method using a Synchron LX20 spectrophotometer (Beckman Coulter) and manufacturer-supplied reagents.
Statistical Analysis Comparison of means was performed using the Student’s two-tailed t-test. One-way analysis of variance (ANOVA) was used to compare skin graft survival among groups. The Bonferroni correction for multiple hypothesis testing was performed for post-hoc comparisons in pairs of groups. Bar graphs with error bars represent mean 6 standard error of the mean. A P value < 0.05 was considered to be statistically significant.
RESULTS Effects of Platonin on Graft Survival
For assessment of graft survival, we observed and recorded the area of viable graft of each recipient for consecutive 28 d. All allografts in the control group were rejected within 7 d with a median graft survival of 6 d after transplantation. Treatment with platonin
Immunohistochemistry Sections 5 mm thick were cut from representative tissue blocks, deparaffinized by rinsing with xylene, and rehydrated in distilled water through graded alcohol. Antigen retrieval was enhanced by autoclaving slides in sodium citrate buffer (pH 6.0) for 30 min. Endogenous peroxidase activity was quenched by 30 min of incubation in a 0.3% hydrogen peroxide-methanol buffer. The slides were then incubated overnight at 4 C in humid chambers with primary rabbit polyclonal anti-iNOS antibody (ab15323; Abcam, Cambridge, MA). The slides
FIG. 1. The effect of platonin on the survival of skin grafts. BALB/c mice, transplanted with skin grafts from C57BL/6 donors, were treated daily with platonin 1.0, 10, or 100 mg/kg starting on the day of transplantation. A control group was grafted with no additional treatment. At least 15 mice were tested in each group. There were significant differences in graft survival on d 1, 3, 5, and 7 among all groups (P < 0.001, one-way ANOVA) and between individual groups at all time points (Bonferroni post-hoc testing, P < 0.01).
CHENG ET AL.: PLATONIN PROLONGS SKIN GRAFT SURVIVAL
significantly prolonged skin graft survival (Fig. 1) in a dose-related manner, with a median survival of 8 d at a daily dose of 1.0 mg/kg, 14 d at 10 mg/kg, and 20 d at 100 mg/kg. The percentage of viable grafts at every time point after transplantation significantly differed among the various doses (one-way ANOVA, P < 0.001). Any two groups differed significantly throughout the experiment as well (Bonferroni posttest, P < 0.01 for all comparisons). A representative set of photographs showing dose- and time-dependent effect of platonin on the integrity of skin allografts is shown in Fig. 2A.
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neutrophils, with nuclear fragmentation, apoptosis, and vascular thrombosis. No eosinophils were observed. Epidermal changes included vacuolization, spongiosis, hyperkeratosis, and follicular plugging, eventually proceeding to extensive necrosis. By d 7, allografts in the control group had complete epidermal necrosis and necrotizing vasculitis. Platonin treatment clearly reduced the inflammatory reaction with better integrity of the epidermis and less marked inflammatory cell infiltrates (Fig. 2B). The microscopic changes correlated well with macroscopic observations. Changes in Body Weight and Leukocyte Count
Histologic Analysis
Histologic examination of the graft tissues showed increasingly severe vasculitis, epidermal degeneration, and dermal inflammation over time. The degree of perivascular inflammation and number of vessels involved increased along with progression of rejection. As rejection worsened, the infiltrate changed from predominantly mononuclear cells to polymorphonuclear
In the second set of experiments, the control group and platonin-treated recipients had statistically similar weights at various times after transplantation (Fig. 3A). The white blood cell count decreased after skin transplantation and then rose gradually in both groups. The leukocyte count after d 5 was slightly lower in platonin-treated mice without statistical significance (Fig. 3B).
FIG. 2. Comparison of gross and microscopic appearance of skin grafts between the platonin-treated and control mice. (A) Representative photographs of mouse skin allografts. Skin grafts were evaluated by visual and tactile inspection until necrosis. The necrotic graft was nonvascularized and had dried up or dropped off. (B) Representative hematoxylin and eosin stained allografts harvested on d 1, 3, 5, and 7 after grafting (original magnification, 3100). The extent of perivascular infiltration, epidermal degeneration, and dermal inflammation correlated with gross changes. (Color version of figure is available online.)
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Platonin thus suppressed the rise of proinflammatory cytokines after skin transplantation without changing the overall Th1/Th2 cytokine balance. In Vivo NF-kB DNA-Binding Activity
In direct allorecognition, rejection of organ allografts is a process initiated with the migration of donor passenger antigen-presenting cells to recipient lymphoid tissue. To determine if the regulation of proinflammatory cytokines was accompanied by inhibition of the activity of an upstream transcription factor NF-kB, we analyzed its DNA binding activity in the nuclear extracts from draining lymph nodes of grafted skin in recipients after transplantation. As shown in Fig. 5, platonin treatment significantly reduced binding of the p65 subunit to its target DNA sequence in draining lymph nodes. Assessment of iNOS Expression
FIG. 3. Body weight (A) and leukocyte count (B) of platonintreated mice compared with controls after skin transplantation. There were no significant differences in either variable between the two groups (n ¼ 6 in each group).
It is known that platonin suppresses iNOS expression in LPS-stimulated macrophages and peripheral blood mononuclear cells [12, 19]. To examine the in vivo effect of platonin on iNOS expression in our skin graft model, we harvested draining lymph nodes for protein expression of iNOS. As a result, the expression pattern of iNOS in draining lymph nodes shows a distribution from medullary sinus to paracortex from d 1 to 3 (Fig. 6). Treatment with platonin clearly reduced the intensity and distribution of iNOS signals in draining lymph nodes in a manner resembling the inhibition of NF-kB activity.
Serum Cytokine Levels
Evaluation of Hepatotoxicity and Nephrotoxicity
Serum levels of IL-2, IL-4, IL-5, and IL-12p70 were low and remained stable after transplantation, with no significant difference between the platonin-treated and control groups (Fig. 4). IL-10 and IFN-g levels were gradually up-regulated after grafting and, to a slightly lower but not statistically significant extent, in platonin-treated mice. In the control group, IL-6 levels were markedly elevated after transplantation. They dropped at d 5, although not to baseline. IL-6 levels in the control group again rose at d 7, the same time as rejection was apparent (Fig. 4D). A comparable rise was not seen in any of the platonin-treated mice, suggesting that platonin profoundly inhibits the production of IL-6. Levels of TNF-a demonstrated a pattern similar to IL-6 over time in the control group (Fig. 4 G), rising very evidently along with gross and histologic rejection. Serum TNF-a levels of the mice treated with platonin were lower than those in the control group, but the trend did not reach statistical significance except at d 7 (P < 0.1 at d 1, 3, and 5; P < 0.05 at d 7).
After skin transplantation, plasma ALT levels in both control and platonin-treated mice increased slowly (Fig. 7A), reaching approximately twice baseline levels at d 7. There were no significant differences between the control and platonin-treated mice in either ALT or creatinine levels (Fig. 7B). DISCUSSION
The present study demonstrates that treatment with platonin prolongs survival of skin allografts in major histocompatibility complex-incompatible mice. Concurrently, platonin suppresses production of the proinflammatory cytokines IL-6 and TNF-a, selectively inhibits NF-kB activation, and reduces iNOS expression in draining lymph nodes. No significant toxicity to bone marrow, liver, or kidney is noted. This is the first in vivo animal study to show the therapeutic potential of platonin in the field of transplantation. Recent cases of hand and face transplantation have renewed interest in studies on skin allograft rejection.
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FIG. 4. Serum cytokine levels of platonin-treated mice compared with those of controls. The levels of IL-2 (A), IL-4 (B), IL-5 (C), IL-6 (D), IL10 (E), IL-12p70 (F), TNF-a (G), and IFN-g (H) (pg/mL) were determined by cytometric bead array on d 1, 3, 5, and 7 after skin transplantation. *P < 0.05; **P < 0.005 versus respective control (n ¼ 6 in each group).
Acute rejection of skin grafts is an antigen-specific T cell-dependent response by which activated T cells specifically destroy major histocompatibility complexincompatible cells [20]. Skin transplantation is often referred to as a ‘‘strong’’ model of rejection, as skin allo-
FIG. 5. NF-kB activity in draining lymph nodes after skin transplantation. NF-kB activity was determined by measuring binding of the NF-kB subunit p65 to its NF-kB consensus binding sequence with an enzyme-linked immunosorbent assay-based technique. *P < 0.05; **P < 0.01 versus respective control (n ¼ 5 in each group).
grafts are highly susceptible to destruction [21]. Once a drug is effective against skin graft rejection, it is likely to be active, probably more so, in other rodent models of transplantation [20]. Thus, testing in skin transplantation may be more predictive of the clinical action of drugs. Our in vivo study clearly demonstrated, both grossly and histologically, the beneficial effect of platonin in limiting skin graft rejection in mice. The importance of cytokines has already been studied extensively in transplant immunology [22]. Viewed somewhat simplistically, Th1-type immunogenic cytokines are involved in cellular immunity and allograft rejection, whereas Th2-type regulatory, tolerogenic cytokines are involved in humoral immunity and graft acceptance [23]. The polarization of the immune response towards either Th1 or Th2 has been described in various animal transplantation models [24]. In the present study, however, platonin treatment did not significantly influence the levels of Th1 (IL-2, IL-12p70, and IFN-g) and Th2 (IL-4, IL-5, and IL-10) cytokines, implying that platonin’s suppression of rejection may not be related to a shift in the balance of Th1 and Th2 cytokines.
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FIG. 6. Representative photographs of iNOS expression in tissues from platonin-treated and control mice undergoing skin transplantation. After 1 and 3 d, draining lymph nodes were dissected and fixed in 4% formalin. Paraffin-embedded tissue sections were immunohistochemically stained with anti-iNOS antibody. Compared with controls, decreased iNOS expression was visualized by less intensive brown staining in the platonin-treated group (original magnification, 340). (Color version of figure is available online.)
TNF-a is a proinflammatory cytokine with various immunologic functions affecting the activation of antigen-presenting and T cells, and altering the production of other cytokines. TNF-a plays an important role in early graft injury [25], and serum TNF-a levels are increased in liver, kidney, and pancreas transplant rejection [26]. Preliminary studies of anti-TNF-a monoclonal antibody in renal transplantation have yielded encouraging results [27]. Our previous studies demonstrated that platonin suppressed TNF-a production in rats before or after onset of heatstroke [28, 29]. In this study, although TNF-a production in platonin-treated mice was significantly lower than in controls only on d 7 after transplantation, the levels did not appear to rise in the treated animals as they did in controls. Differences in TNF- a levels could not be solely responsible for platonin’s effects. Regardless, these results suggest a plausible partial explanation for the prolonged skin graft survival we demonstrated in the platonin-treated mice. IL-6 is another pleiotropic cytokine of great importance in the induction of acute phase reactions and immune responses [30]. Consistent with our findings, He and coworkers [31] found that serum levels of IL-6 increased on d 1 following cardiac transplantation, indicating that initial IL-6 up-regulation did not require an adaptive immune response. They found that IL-6 levels decreased on d 3 and 5 but rose again on d 7 in mice with allogeneic transplants, suggesting that a second phase of IL-6 production was induced during the rejection process. Interestingly, Liang et al. [32] found that IL-6-deficient cardiac grafts transplanted into
allogeneic wild-type recipients had significantly longer survival than in controls, but allogeneic grafts transplanted into IL-6-deficient recipients did not survive better than in controls. The evidence to date for the role of IL-6 thus suggests it has a complex relationship with other factors in the rejection process. We found that the serum IL-6 levels were lower in the platonin-treated group, mice whose skin grafts lasted much longer than those of the controls. Part of platonin’s effect in prolonging graft survival, then, may involve suppression of IL-6. With respect to molecular mechanism, selective NFkB inhibition may be responsible for the extended survival. NF-kB functions as a master switch in a variety of processes including reperfusion injury and graft rejection [7, 33]. However, NF-kB is also involved in normal cellular physiology, and global NF-kB inhibition might result in serious side effects, such as hepatotoxicity and opportunistic infections [34]. It is therefore of interest that there are no significant changes in Th1 cytokines in our study. As aforementioned, skin allografts are so immunogenic that interventions that can successfully prevent rejection of heart or pancreatic islets may fail to elicit acceptance of skin transplants. In this regard, Zhou and colleagues [35] have shown that inhibition of T-cell-intrinsic NF-kB activation attenuates cardiac but not skin allograft rejection. Generally, after skin transplantation, Langerhans cells (a type of epidermal DC) migrate out of the donor skin into the lymph node of the recipient, where they can activate T cells. Allogeneic Langerhans cells have been shown to allow NF-kB-impaired T cells to reach an activation
CHENG ET AL.: PLATONIN PROLONGS SKIN GRAFT SURVIVAL
FIG. 7. Plasma alanine aminotransferase (ALT) (A) and creatinine (B) of levels platonin-treated mice compared with those of controls after skin transplantation. There were no significant differences in either variable between the two groups (n ¼ 6 in each group).
threshold necessary for T-cell differentiation and allograft rejection [36]. As depicted in this study, selective NF-kB inhibition was observed in draining lymph nodes. We previously demonstrated that platonin modulates the differentiation, maturation, and functions of DC toward an intermediate stage of maturation [14]. Ex vivo treatment by incubating pretransplant grafts with platonin also delayed the rejection of fully allogeneic skin transplants in mice (preliminary unpublished observations). These results together suggest that selective NF-kB inhibition may involve the effect of platonin against rejection. NF-kB is required for maximal transcription of many cytokines, including TNF-a and IL-6 [37]. Classic activation of macrophages requires IFN-g and the engagement of receptors, which recognize exogenous molecules such as the toll-like receptors [38]. Exposure of macrophages to the ligands of these receptors activates the downstream cascades, particularly NF-kB,which rapidly induces the expression of inflammation-associated genes, resulting in the production of proinflammatory cytokines, Th1 type inflammation, and release of NO
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by virtue of iNOS activity [39, 40]. In line with the observations that platonin suppressed the production of the proinflammatory cytokines, platonin attenuates NO production in LPS-stimulated macrophages and peripheral blood mononuclear cells through inhibiting iNOS expression [12, 19]. In our study, platonin significantly suppressed iNOS expression in draining lymph nodes. Blockade of NO production by iNOS inhibitors has been shown to prolong skin xenograft survival by down-regulation of the expressions of proinflammatory cytokines [41]. On the other hand, it is reported that NO derived from iNOS has a dual role in cardiac transplantation: detrimental in acute rejection and beneficial in chronic rejection [42]. Causal relationships between platonin-induced iNOS inhibition and attenuated skin graft rejection deserve further investigation. Platonin in our study caused no obvious hepatic or renal toxicity. If further investigation confirms the apparent safety of this substance, this would add to the attractiveness of platonin as an immunosuppressive agent. Infection is a common adverse complication of immunosuppression, yet platonin has antibacterial activity, and has been used for treatment of periodontal disease [3]. An agent that simultaneously suppresses graft rejection and bacterial infection would be a tremendous addition to our current armamentarium. Platonin is a water-soluble agent suitable for intravenous injection [29], making it suitable for clinical trials without a need to do drug formulation. No significant effect on leukocyte counts by platonin further renders this agent falling in a new category of immunosuppressants possessing a better pharmacologic profile than that of mTOR inhibitors. Furthermore, platonin possesses free radical-scavenging activities, and acts as an antioxidant [4, 43]. Recent data suggest that concurrent treatment with antioxidants may attenuate the renal dysfunction associated with cyclosporine [44]. In summary, our data provide clear evidence that platonin effectively prolongs skin allograft survival in mice without major toxicity. This effect appears to involve suppression of the up-regulation of proinflammatory cytokines, selective inhibition of NF-kB activity, and reduced iNOS expression, but without changing the overall Th1/Th2 cytokine balance. Our results along with the other evidence available to date on platonin’s effects, suggest that this agent may have unique benefits in transplant medicine. Clearly, this promising agent is worthy of further investigation.
ACKNOWLEDGMENTS The authors thank Dr. Mary Jeanne Buttrey and Dr. Hsin-Chen Lee for their critical reading and helpful editorial comments on the manuscript. They also thank Hui-Ru Shieh and Chien-Ying Huang for technical assistance and help.
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REFERENCES 1. Scherer MN, Banas B, Mantouvalou K, et al. Current concepts and perspectives of immunosuppression in organ transplantation. Langenbecks Arch Surg 2007;392:511. 2. Augustine JJ, Bodziak KA, Hricik DE. Use of sirolimus in solid organ transplantation. Drugs 2007;67:369. 3. Ishihara M, Fujisawa S. Photo-oxygenation, photodegradation, and antioxidative activity of platonin, a cyanine photosensitizing dye. In Vivo 2007;21:163. 4. Hsiao G, Lee JJ, Chou DS, et al. Platonin, a photosensitizing dye, improves circulatory failure and mortality in rat models of endotoxemia. Biol Pharm Bull 2002;25:995. 5. Lee JJ, Huang WT, Shao DZ, et al. Platonin, a cyanine photosensitizing dye, inhibits pyrogen release and results in antipyresis. J Pharmacol Sci 2003;93:376. 6. Shao DZ, Lee JJ, Huang WT, et al. Inhibition of nuclear factorkB prevents staphylococcal enterotoxin A-induced fever. Mol Cell Biochem 2004;262:177. 7. Finn PW, He H, Ma C, et al. Molecular profiling of the role of the NF-kB family of transcription factors during alloimmunity. J Leukoc Biol 2002;72:1054. 8. Rescigno M, Martino M, Sutherland CL, Gold MR, RicciardiCastagnoli P. Dendritic cell survival and maturation are regulated by different signaling pathways. J Exp Med 1998;188:2175. 9. Ghosh S, Karin M. Missing pieces in the NF-kB puzzle. Cell 2002; 109(Suppl):S81. 10. Karin M, Yamamoto Y, Wang QM. The IKK NF-kB system: A treasure trove for drug development. Nat Rev Drug Discov 2004;3:17. 11. LaRosa DF, Rahman AH, Turka LA. The innate immune system in allograft rejection and tolerance. J Immunol 2007;178:7503. 12. Shao DZ, Lin M. Platonin inhibits LPS-induced NF-kB by preventing activation of Akt and IKKb in human PBMC. Inflamm Res 2008;57:601. 13. Satoh M, Yasunami Y, Matsuoka N, et al. Successful islet transplantation to two recipients from a single donor by targeting proinflammatory cytokines in mice. Transplantation 2007; 83:1085. 14. Lee JJ, Liao HF, Yang YC, et al. Platonin modulates differentiation and maturation of human monocyte-derived dendritic cells. Int Immunopharmacol 2006;6:287. 15. Manzotti CN, Liu MK, Burke F, et al. Integration of CD28 and CTLA-4 function results in differential responses of T cells to CD80 and CD86. Eur J Immunol 2006;36:1413. 16. Tomita Y, Mayumi H, Eto M, et al. Importance of suppressor T cells in cyclophosphamide-induced tolerance to the non-H-2encoded alloantigens. Is mixed chimerism really required in maintaining a skin allograft tolerance? J Immunol 1990; 144:463. 17. Soldan SS, Alvarez Retuerto AI, et al. Immune modulation in multiple sclerosis patients treated with the pregnancy hormone estriol. J Immunol 2003;171:6267. 18. Renard P, Ernest I, Houbion A, et al. Development of a sensitive multi-well colorimetric assay for active NFkB. Nucleic Acids Res 2001;29:E21. 19. Chen CC, Lee JJ, Tsai PS, et al. Platonin attenuates LPS-induced CAT-2 and CAT-2B induction in stimulated murine macrophages. Acta Anaesthesiol Scand 2006;50:604. 20. Gardner CR. The pharmacology of immunosuppressant drugs in skin transplant rejection in mice and other rodents. Gen Pharmacol 1995;26:245. 21. Binet I, Wood KJ. In vivo models of inflammation: Immune rejection and skin transplantation in vivo. Methods Mol Biol 2003; 225:239.
22. Walsh PT, Strom TB, Turka LA. Routes to transplant tolerance versus rejection; The role of cytokines. Immunity 2004;20:121. 23. Reding R, Gras J, Truong DQ, et al. The immunologic monitoring of alloreactive responses in liver transplant recipients: A review. Liver Transpl 2006;12:373. 24. Strom TB, Roy-Chaudhury P, Manfro R, et al. The Th1/Th2 paradigm and the allograft response. Curr Opin Immunol 1996; 8:688. 25. Goto M, Takei Y, Kawano S, et al. Tumor necrosis factor and endotoxin in the pathogenesis of liver and pulmonary injuries after orthotopic liver transplantation in the rat. Hepatology 1992;16:487. 26. Pascher A, Klupp J. Biologics in the treatment of transplant rejection and ischemia/reperfusion injury: New applications for TNFa inhibitors? BioDrugs 2005;19:211. 27. Buhaescu I, Segall L, Goldsmith D, et al. New immunosuppressive therapies in renal transplantation: Monoclonal antibodies. J Nephrol 2005;18:529. 28. Lee JJ, Lin MT, Wang NL, et al. Platonin, a cyanine photosensitizing dye, causes attenuation of circulatory shock, hypercoagulable state, and tissue ischemia during heat stroke. Shock 2005; 24:577. 29. Tsai CC, Lin MT, Yang CC, et al. Platonin, a cyanine photosensitizing dye, is effective for attenuation of heatstroke in rats. Shock 2006;26:601. 30. Kopf M, Baumann H, Freer G, et al. Impaired immune and acute-phase responses in interleukin-6-deficient mice. Nature 1994;368:339. 31. He H, Stone JR, Perkins DL. Analysis of differential immune responses induced by innate and adaptive immunity following transplantation. Immunology 2003;109:185. 32. Liang Y, Christopher K, Finn PW, et al. Graft produced interleukin-6 functions as a danger signal and promotes rejection after transplantation. Transplantation 2007;84:771. 33. Tsoulfas G, Geller DA. NF-kB in transplantation: Friend or foe? Transpl Infect Dis 2001;3:212. 34. Li Q, Verma IM. NF-kB regulation in the immune system. Nat Rev Immunol 2002;2:725. 35. Zhou P, Hwang KW, Palucki DA, et al. Impaired NF-kB activation in T cells permits tolerance to primary heart allografts and to secondary donor skin grafts. Am J Transplant 2003;3:139. 36. Molinero LL, Zhou P, Wang Y, et al. Epidermal Langerhans cells promote skin allograft rejection in mice with NF-kB-impaired T cells. Am J Transplant 2008;8:21. 37. Blackwell TS, Christman JW. The role of nuclear factor-kB in cytokine gene regulation. Am J Respir Cell Mol Biol 1997;17:3. 38. Martinez FO, Sica A, Mantovani A, et al. Macrophage activation and polarization. Front Biosci 2008;13:453. 39. Wyburn KR, Jose MD, Wu H, et al. The role of macrophages in allograft rejection. Transplantation 2005;80:1641. 40. Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell 2006;124:783. 41. Kim JY, Kim D, Lee EM, et al. Inducible nitric oxide synthase inhibitors prolonged the survival of skin xenografts through selective down-regulation of pro-inflammatory cytokine and CC-chemokine expressions. Transpl Immunol 2003;12:63. 42. Pieper GM, Roza AM. The complex role of iNOS in acutely rejecting cardiac transplants. Free Radic Biol Med 2008;44:1536. 43. Ishihara M, Kadoma Y, Fujisawa S. Kinetic radical-scavenging activity of platonin, a cyanine photosensitizing dye. In Vivo 2006;20:845. 44. Zal F, Mostafavi-Pour Z, Vessal M. Comparison of the effects of vitamin E and/or quercetin in attenuating chronic cyclosporine A-induced nephrotoxicity in male rats. Clin Exp Pharmacol Physiol 2007;34:720.
Platonin Improves Survival of Skin Allografts Shih-Ping Cheng, M.D.,*,‡ Jie-Jen Lee, M.D., Ph.D.,‡,** Chin-Wen Chi, Ph.D.,*,{ Kuo-Ming Chang, M.D.,†† and Yu-Jen Chen, M.D., Ph.D.†,§,k,# ,1 *Department and Institute of Pharmacology, School of Medicine, National Yang-Ming University, Taipei, Taiwan; †Institute of Traditional Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan; ‡Department of Surgery, Mackay Memorial Hospital, Taipei, Taiwan; §Department of Radiation Oncology, Mackay Memorial Hospital, Taipei, Taiwan; kDepartment of Medical Research, Mackay Memorial Hospital, Taipei, Taiwan; {Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan; #Graduate Institute of Sport Coaching Science, Chinese Culture University, Taipei, Taiwan; **Institute of Pharmacology, Taipei Medical University, Taipei, Taiwan; and ††Department of Pathology and Laboratory Medicine, Hsinchu Mackay Memorial Hospital, Hsinchu, Taiwan Submitted for publication January 9, 2009
Background. Platonin is an immunomodulator with NF-kB inhibitory activity. It not only inhibits interleukin (IL)-1b, IL-6, and tumor necrosis factor (TNF)-a production in sepsis, but also attenuates heatstroke reactions. In addition, platonin redirects differentiation of dendritic cells toward an intermediate stage of maturation. The study was designed to examine whether platonin can reduce acute graft rejection. Materials and Methods. A C57BL/6 to BALB/c mice skin transplantation model was used. Platonin was given intraperitoneally to transplant recipients at various doses. Skin grafts were submitted to histologic analysis. NF-kB DNA binding activity and inducible nitric oxide synthase (iNOS) expression were determined in harvested draining lymph nodes. Leukocyte count, hepatic and renal functions were serially assessed. An array of serum cytokines was evaluated on d 1, 3, 5, and 7 after skin transplantation. Results. Platonin resulted in significantly prolonged skin allograft survival in a dose- and time-dependent manner. Histologic changes in the skin allografts paralleled the gross appearance of rejection. Serum cytokine analysis shows that platonin significantly suppressed the production of the proinflammatory cytokines IL-6 and TNF-a. However, no significant changes occurred in the serum levels of Th1-type and Th2-type cytokines. NF-kB activity and iNOS expression were remarkably suppressed in draining lymph nodes. In terms of toxicity, there were no significant differences in
1 To whom correspondence and reprint requests should be addressed at Department of Radiation Oncology, Mackay Memorial Hospital, 92, Sec. 2, Chung-Shan North Road, Taipei 10449, Taiwan. E-mail: [email protected].
0022-4804/$36.00 Ó 2010 Elsevier Inc. All rights reserved.
body weight, leukocyte count, plasma alanine aminotransferase, or creatinine between the platonintreated and control groups. Conclusion. Platonin effectively prolongs skin allograft survival without major toxicity. Ó 2010 Elsevier Inc. All rights reserved.
Key Words: platonin; skin allotransplantation; proinflammatory cytokine. INTRODUCTION
Organ transplantation is an important means of managing end-organ failure. Immunosuppressive agents, particularly the calcineurin inhibitors, have made a significant contribution to the success of transplantation. However, concerns about renal and cardiovascular toxicity have prompted growing interest in calcineurin inhibitor-sparing regimens, mostly involving a switch to a mammalian target-of-rapamycin (mTOR) inhibitor [1]. The antineoplastic activity and relatively non-nephrotoxic attributes of mTOR inhibitors are the most favorable properties, but a number of adverse effects have been reported, including hyperlipidemia, myelosuppression, and complications related to growth factor inhibition, such as impaired wound healing [2]. Therefore, it is of critical importance to search for new immunosuppressive drugs that are effective and not toxic to kidney or bone marrow. Platonin is a cyanine photosensitizing dye with a wide range of pharmacologic properties, including antimicrobial, antioxidant, antihistaminic, and anticancer activities [3]. Using in vitro and in vivo models, we have demonstrated that platonin inhibits nuclear
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factor-kB (NF-kB) activation and thereby reduces pyrogen expression in endotoxemia-induced fever [4–6]. Activation of NF-kB appears to play a major role in acute allograft rejection [7], is required for dendritic cell (DC) maturation and activation [8], and is crucial for activation and proliferation of lymphocytes [9]. Many of the immunosuppressive drugs currently used also inhibit NF-kB activation, which contributes partially to their mechanism of action [10]. Furthermore, up-regulation of proinflammatory mediators occurs rapidly after transplantation, an event that may then be shaped and amplified by a graft-specific adaptive response [11]. Platonin may favorably influence these events. For example, the elevated levels of interleukin (IL)1b, IL-6, and tumor necrosis factor (TNF)-a induced by systemic administration of lipopolysaccharide (LPS) in rabbits are attenuated by pretreatment with platonin [5]. In peripheral blood mononuclear cells, platonin suppresses the production of proinflammatory cytokines, nitric oxide (NO) production, and inducible NO synthase (iNOS) expression induced by LPS [12]. Targeting proinflammatory cytokines with anti-TNFa and anti-IL-1b antibodies has a beneficial effect on engraftment of islet grafts [13]. Taken together, platonin might benefit graft survival due to a suppressive effect on NF-kB and production of proinflammatory cytokines related to transplant rejection. Our earlier studies demonstrated that platonin modulates the differentiation, maturation, and functions of DC. With platonin treatment, human monocyte-derived DC have decreased CD83 expression, increased CD80 expression, and less stimulatory activity on allogeneic naive CD4þCD45RAþ T cells [14], indicating that platonin redirects DC differentiation toward an intermediate stage of maturation. CD80 has more productive interactions with CD152 (CTLA-4) than with CD28 [15], and engagement of CD152 by CD80 inhibits T cell receptorand CD28-mediated T cell proliferation. These actions of platonin on DC raise the possibility that this agent might be tolerogenic in transplant settings. Given that platonin inhibits NF-kB activation, suppresses proinflammatory cytokine release, and modulates DC development, we postulated that systemic administration of platonin might decrease transplant rejection. We therefore used a skin transplantation model to evaluate graft survival, serum cytokine levels, in vivo regulation of NF-kB activity and iNOS expression, and hepatic and renal toxicity in platonin-treated mice.
MATERIALS AND METHODS Animals Six- to 8-wk-old male C57BL/6 (H-2b) and BALB/c (H-2d) mice were obtained from the Animal Resource Center of the National Science
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Council of Taiwan (Taipei, Taiwan). They were housed in a specific pathogen-free environment in compliance with the National Research Council’s Guide for the Care and Use of Laboratory Animals. All experiments were approved by the animal ethics committee of Mackay Memorial Hospital, Taipei, Taiwan (MMH-A-S-95059).
Skin Transplantation All operations were performed under anesthesia with a single intraperitoneal (i.p.) dose of ketamine hydrochloride (100 mg/kg body weight; Sigma, St. Louis, MO). Skin transplantation was conducted by a procedure modified from that described previously [16]. Briefly, a 2 3 1 cm full-thickness skin graft was removed from the flank of a euthanized C57BL/6 donor mouse, and the underside was gently scraped with a scalpel to remove fat and muscle. The right dorsal flank of anesthetized BALB/c recipient was shaved and washed with 70% ethanol. A graft bed was prepared with fine scissors by removing an area of epidermis and dermis down to the level of the intrinsic muscle. The graft was fixed to the graft bed with 10 interrupted sutures of 5-0 monofilament nylon thread (Dermalon; Davis and Geck, St. Louis, MO). No dressings or antibiotics were used. Skin graft survival was evaluated three times a week by visual and tactile examination. The percentage of viable graft was assessed using a transparent grid-template overlay. Rejection was defined as necrosis of more than 80% of the epidermal surface of the graft.
Drug Preparation and Treatment Platonin, 4,40 ,400 -thrimethyl-3,30 300 -triheptyl-7-(200 -thia-zolyl)-2,20 trimethinethiazolocyanine-3-300 -diiodide, (synthesized by Kankohsha Co., Osaka, Japan and obtained from Gwo Chyang Pharmaceutical Co., Tainan, Taiwan) was stored at 4 C without light exposure. Dilutions were prepared with normal saline immediately before i.p. injection. To minimize its photosensitizing effect, all procedures were performed under light-avoidance conditions. In the first set of experiments, 60 mice were randomly assigned to four groups of 15 mice each and treated according to one of the following regimens: (1) 1.0 mg/kg platonin i.p. per d, (2) 10 mg/kg platonin i.p. per d, (3) 100 mg/kg platonin i.p. per d, or (4) normal saline (as a vehicle control). All treatments were started on the day of transplantation and continued until rejection was observed. In the second set of experiments, 48 animals were randomly assigned to two groups receiving either 10 mg/kg platonin i.p. per d or normal saline. Skin grafting was performed in the same manner as described above. Pretransplantation blood samples were obtained via retro-orbital plexus. The weight of each mouse was determined every other day by a single observer. Six mice each were sacrificed by cervical dislocation on d 1, 3, 5, and 7 after operation, and blood samples were collected by cardiac puncture. Skin grafts and draining lymph nodes (axillary, brachial, and inguinal) were harvested at sacrifice.
Histologic Evaluation For histologic analysis, skin grafts were fixed in formalin and embedded in paraffin. Five-mm sections were cut, deparaffinized, rehydrated, and stained with hematoxylin and eosin. A pathologist blinded to which experimental group the specimens were from evaluated the slides to determine the presence of rejection, infection, and other histologic changes. This included a search for perivascular infiltrates, epidermal degeneration, and stromal changes. If perivascular infiltrates were present, the type of inflammatory cells involved (lymphocytes, eosinophils, or neutrophils) were noted. Changes in the stroma might include inflammation, edema, red blood cell extravasation, and necrosis.
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Serum Cytokine Analysis Serum obtained from blood samples were frozen at –80 C, then thawed to room temperature and analyzed using cytometric bead array kits according to the manufacturer’s instruction (BD Biosciences, San Jose, CA). IL-6, IL-10, IL-12p70, and interferon (IFN)-g were detected simultaneously using the mouse inflammation kit. IL-2, IL-4, IL-5, and TNF-a protein levels were measured using the mouse Th1/Th2 cytokine kit. For this assay, soluble cytokines were captured on various antibody-conjugated microparticles, and measured using flow cytometry as described previously [17]. Briefly, each sample (50 mL) was mixed with 50 mL of mixed capture beads and 50 mL of the mouse detection reagent consisting of PE-conjugated antibodies. The tubes were incubated at room temperature for 2 h in the dark. The samples were washed once and then resuspended in 300 mL of wash buffer before acquisition on a FACSCalibur flow cytometer (BD Biosciences). The concentration of each cytokine was determined by interpolation from the corresponding calibration curve generated from the mixed bead standard provided in the kit. Data were analyzed using BD Biosciences cytometric bead array software.
NF-kB DNA-Binding Assay Nuclear extraction was done with the Nuclear Extract kit (Active Motif, Carlsbad, CA) according to the protocol of the manufacturer. Briefly, at least 200 mg of each tissue was diced into small pieces. After addition of a hypotonic buffer supplemented with dithiothreitol and detergent, tissue was homogenized. The suspension was incubated on ice for 15 min, followed by a centrifugation at 4 C for 10 min at 850 g. Single cells were then resuspended in hypotonic buffer supplemented with detergent, and incubated on ice for 15 min to permeabilize the cell membranes. Nuclei were collected by centrifuging the suspension at 14,000 g in a precooled centrifuge at 4 C for 30 s. After removal of the supernatants containing cytoplasmic proteins, the pellet was again resuspended in a lysis buffer to break down the nuclei and extract the nuclear proteins. Suspension was then incubated on ice for 30 min on a rocking platform set at 150 rpm. Finally, nuclear proteins were collected by centrifuging the suspension at 14,000 g in a precooled centrifuge at 4 C for 10 min. Protein concentration was determined using a standard Bradford protein assay. The supernatant was stored at –80 C for NF-kB binding assay. The DNA-binding activity of NF-kB was quantified by enzyme linked immunosorbent assay (ELISA)-based TransAM NF-kB p65 transcription factor assay kit (Active Motif), which shows a good correlation and better sensitivity compared with an electrophoretic mobility shift assay (EMSA) [18]. The TransAm NF-kB ELISA kit consists of 96-well microtiter plates precoated with an oligonucleotide containing the appropriate NF-kB binding consensus sequence. The active form of NF-kB binding to the target oligonucleotide was detected by incubation with primary antibody specific for the activated form of p65, visualized by anti-IgG horseradish peroxidase conjugate and developing solution, and quantified at 450 nm with a reference wavelength of 655 nm. The results were expressed after subtraction of background binding obtained by incubation with a mutated consensus oligonucleotide which has no effect on NF-kB binding.
were washed three times in phosphate buffer solution, and further incubated with a biotinylated secondary antibody for 30 min at room temperature. Antigen-antibody complexes were detected by the avidin-biotin-peroxidase method. For negative controls, the primary antibody was omitted, and unspecific rabbit IgG was used at the same concentration as the primary antibody. Finally, the slides were counterstained with hematoxylin and then examined by light microscopy.
Evaluation of Leukocyte Count, Hepatic, and Renal Functions The white blood cell counts of the blood samples were analyzed by an automatic Coulter counter (model Z1; Beckman Coulter Electronics, Fullerton, CA). Plasma levels of alanine aminotransferase (ALT) and creatinine were measured by a standard colorimetric method using a Synchron LX20 spectrophotometer (Beckman Coulter) and manufacturer-supplied reagents.
Statistical Analysis Comparison of means was performed using the Student’s two-tailed t-test. One-way analysis of variance (ANOVA) was used to compare skin graft survival among groups. The Bonferroni correction for multiple hypothesis testing was performed for post-hoc comparisons in pairs of groups. Bar graphs with error bars represent mean 6 standard error of the mean. A P value < 0.05 was considered to be statistically significant.
RESULTS Effects of Platonin on Graft Survival
For assessment of graft survival, we observed and recorded the area of viable graft of each recipient for consecutive 28 d. All allografts in the control group were rejected within 7 d with a median graft survival of 6 d after transplantation. Treatment with platonin
Immunohistochemistry Sections 5 mm thick were cut from representative tissue blocks, deparaffinized by rinsing with xylene, and rehydrated in distilled water through graded alcohol. Antigen retrieval was enhanced by autoclaving slides in sodium citrate buffer (pH 6.0) for 30 min. Endogenous peroxidase activity was quenched by 30 min of incubation in a 0.3% hydrogen peroxide-methanol buffer. The slides were then incubated overnight at 4 C in humid chambers with primary rabbit polyclonal anti-iNOS antibody (ab15323; Abcam, Cambridge, MA). The slides
FIG. 1. The effect of platonin on the survival of skin grafts. BALB/c mice, transplanted with skin grafts from C57BL/6 donors, were treated daily with platonin 1.0, 10, or 100 mg/kg starting on the day of transplantation. A control group was grafted with no additional treatment. At least 15 mice were tested in each group. There were significant differences in graft survival on d 1, 3, 5, and 7 among all groups (P < 0.001, one-way ANOVA) and between individual groups at all time points (Bonferroni post-hoc testing, P < 0.01).
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significantly prolonged skin graft survival (Fig. 1) in a dose-related manner, with a median survival of 8 d at a daily dose of 1.0 mg/kg, 14 d at 10 mg/kg, and 20 d at 100 mg/kg. The percentage of viable grafts at every time point after transplantation significantly differed among the various doses (one-way ANOVA, P < 0.001). Any two groups differed significantly throughout the experiment as well (Bonferroni posttest, P < 0.01 for all comparisons). A representative set of photographs showing dose- and time-dependent effect of platonin on the integrity of skin allografts is shown in Fig. 2A.
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neutrophils, with nuclear fragmentation, apoptosis, and vascular thrombosis. No eosinophils were observed. Epidermal changes included vacuolization, spongiosis, hyperkeratosis, and follicular plugging, eventually proceeding to extensive necrosis. By d 7, allografts in the control group had complete epidermal necrosis and necrotizing vasculitis. Platonin treatment clearly reduced the inflammatory reaction with better integrity of the epidermis and less marked inflammatory cell infiltrates (Fig. 2B). The microscopic changes correlated well with macroscopic observations. Changes in Body Weight and Leukocyte Count
Histologic Analysis
Histologic examination of the graft tissues showed increasingly severe vasculitis, epidermal degeneration, and dermal inflammation over time. The degree of perivascular inflammation and number of vessels involved increased along with progression of rejection. As rejection worsened, the infiltrate changed from predominantly mononuclear cells to polymorphonuclear
In the second set of experiments, the control group and platonin-treated recipients had statistically similar weights at various times after transplantation (Fig. 3A). The white blood cell count decreased after skin transplantation and then rose gradually in both groups. The leukocyte count after d 5 was slightly lower in platonin-treated mice without statistical significance (Fig. 3B).
FIG. 2. Comparison of gross and microscopic appearance of skin grafts between the platonin-treated and control mice. (A) Representative photographs of mouse skin allografts. Skin grafts were evaluated by visual and tactile inspection until necrosis. The necrotic graft was nonvascularized and had dried up or dropped off. (B) Representative hematoxylin and eosin stained allografts harvested on d 1, 3, 5, and 7 after grafting (original magnification, 3100). The extent of perivascular infiltration, epidermal degeneration, and dermal inflammation correlated with gross changes. (Color version of figure is available online.)
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Platonin thus suppressed the rise of proinflammatory cytokines after skin transplantation without changing the overall Th1/Th2 cytokine balance. In Vivo NF-kB DNA-Binding Activity
In direct allorecognition, rejection of organ allografts is a process initiated with the migration of donor passenger antigen-presenting cells to recipient lymphoid tissue. To determine if the regulation of proinflammatory cytokines was accompanied by inhibition of the activity of an upstream transcription factor NF-kB, we analyzed its DNA binding activity in the nuclear extracts from draining lymph nodes of grafted skin in recipients after transplantation. As shown in Fig. 5, platonin treatment significantly reduced binding of the p65 subunit to its target DNA sequence in draining lymph nodes. Assessment of iNOS Expression
FIG. 3. Body weight (A) and leukocyte count (B) of platonintreated mice compared with controls after skin transplantation. There were no significant differences in either variable between the two groups (n ¼ 6 in each group).
It is known that platonin suppresses iNOS expression in LPS-stimulated macrophages and peripheral blood mononuclear cells [12, 19]. To examine the in vivo effect of platonin on iNOS expression in our skin graft model, we harvested draining lymph nodes for protein expression of iNOS. As a result, the expression pattern of iNOS in draining lymph nodes shows a distribution from medullary sinus to paracortex from d 1 to 3 (Fig. 6). Treatment with platonin clearly reduced the intensity and distribution of iNOS signals in draining lymph nodes in a manner resembling the inhibition of NF-kB activity.
Serum Cytokine Levels
Evaluation of Hepatotoxicity and Nephrotoxicity
Serum levels of IL-2, IL-4, IL-5, and IL-12p70 were low and remained stable after transplantation, with no significant difference between the platonin-treated and control groups (Fig. 4). IL-10 and IFN-g levels were gradually up-regulated after grafting and, to a slightly lower but not statistically significant extent, in platonin-treated mice. In the control group, IL-6 levels were markedly elevated after transplantation. They dropped at d 5, although not to baseline. IL-6 levels in the control group again rose at d 7, the same time as rejection was apparent (Fig. 4D). A comparable rise was not seen in any of the platonin-treated mice, suggesting that platonin profoundly inhibits the production of IL-6. Levels of TNF-a demonstrated a pattern similar to IL-6 over time in the control group (Fig. 4 G), rising very evidently along with gross and histologic rejection. Serum TNF-a levels of the mice treated with platonin were lower than those in the control group, but the trend did not reach statistical significance except at d 7 (P < 0.1 at d 1, 3, and 5; P < 0.05 at d 7).
After skin transplantation, plasma ALT levels in both control and platonin-treated mice increased slowly (Fig. 7A), reaching approximately twice baseline levels at d 7. There were no significant differences between the control and platonin-treated mice in either ALT or creatinine levels (Fig. 7B). DISCUSSION
The present study demonstrates that treatment with platonin prolongs survival of skin allografts in major histocompatibility complex-incompatible mice. Concurrently, platonin suppresses production of the proinflammatory cytokines IL-6 and TNF-a, selectively inhibits NF-kB activation, and reduces iNOS expression in draining lymph nodes. No significant toxicity to bone marrow, liver, or kidney is noted. This is the first in vivo animal study to show the therapeutic potential of platonin in the field of transplantation. Recent cases of hand and face transplantation have renewed interest in studies on skin allograft rejection.
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FIG. 4. Serum cytokine levels of platonin-treated mice compared with those of controls. The levels of IL-2 (A), IL-4 (B), IL-5 (C), IL-6 (D), IL10 (E), IL-12p70 (F), TNF-a (G), and IFN-g (H) (pg/mL) were determined by cytometric bead array on d 1, 3, 5, and 7 after skin transplantation. *P < 0.05; **P < 0.005 versus respective control (n ¼ 6 in each group).
Acute rejection of skin grafts is an antigen-specific T cell-dependent response by which activated T cells specifically destroy major histocompatibility complexincompatible cells [20]. Skin transplantation is often referred to as a ‘‘strong’’ model of rejection, as skin allo-
FIG. 5. NF-kB activity in draining lymph nodes after skin transplantation. NF-kB activity was determined by measuring binding of the NF-kB subunit p65 to its NF-kB consensus binding sequence with an enzyme-linked immunosorbent assay-based technique. *P < 0.05; **P < 0.01 versus respective control (n ¼ 5 in each group).
grafts are highly susceptible to destruction [21]. Once a drug is effective against skin graft rejection, it is likely to be active, probably more so, in other rodent models of transplantation [20]. Thus, testing in skin transplantation may be more predictive of the clinical action of drugs. Our in vivo study clearly demonstrated, both grossly and histologically, the beneficial effect of platonin in limiting skin graft rejection in mice. The importance of cytokines has already been studied extensively in transplant immunology [22]. Viewed somewhat simplistically, Th1-type immunogenic cytokines are involved in cellular immunity and allograft rejection, whereas Th2-type regulatory, tolerogenic cytokines are involved in humoral immunity and graft acceptance [23]. The polarization of the immune response towards either Th1 or Th2 has been described in various animal transplantation models [24]. In the present study, however, platonin treatment did not significantly influence the levels of Th1 (IL-2, IL-12p70, and IFN-g) and Th2 (IL-4, IL-5, and IL-10) cytokines, implying that platonin’s suppression of rejection may not be related to a shift in the balance of Th1 and Th2 cytokines.
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FIG. 6. Representative photographs of iNOS expression in tissues from platonin-treated and control mice undergoing skin transplantation. After 1 and 3 d, draining lymph nodes were dissected and fixed in 4% formalin. Paraffin-embedded tissue sections were immunohistochemically stained with anti-iNOS antibody. Compared with controls, decreased iNOS expression was visualized by less intensive brown staining in the platonin-treated group (original magnification, 340). (Color version of figure is available online.)
TNF-a is a proinflammatory cytokine with various immunologic functions affecting the activation of antigen-presenting and T cells, and altering the production of other cytokines. TNF-a plays an important role in early graft injury [25], and serum TNF-a levels are increased in liver, kidney, and pancreas transplant rejection [26]. Preliminary studies of anti-TNF-a monoclonal antibody in renal transplantation have yielded encouraging results [27]. Our previous studies demonstrated that platonin suppressed TNF-a production in rats before or after onset of heatstroke [28, 29]. In this study, although TNF-a production in platonin-treated mice was significantly lower than in controls only on d 7 after transplantation, the levels did not appear to rise in the treated animals as they did in controls. Differences in TNF- a levels could not be solely responsible for platonin’s effects. Regardless, these results suggest a plausible partial explanation for the prolonged skin graft survival we demonstrated in the platonin-treated mice. IL-6 is another pleiotropic cytokine of great importance in the induction of acute phase reactions and immune responses [30]. Consistent with our findings, He and coworkers [31] found that serum levels of IL-6 increased on d 1 following cardiac transplantation, indicating that initial IL-6 up-regulation did not require an adaptive immune response. They found that IL-6 levels decreased on d 3 and 5 but rose again on d 7 in mice with allogeneic transplants, suggesting that a second phase of IL-6 production was induced during the rejection process. Interestingly, Liang et al. [32] found that IL-6-deficient cardiac grafts transplanted into
allogeneic wild-type recipients had significantly longer survival than in controls, but allogeneic grafts transplanted into IL-6-deficient recipients did not survive better than in controls. The evidence to date for the role of IL-6 thus suggests it has a complex relationship with other factors in the rejection process. We found that the serum IL-6 levels were lower in the platonin-treated group, mice whose skin grafts lasted much longer than those of the controls. Part of platonin’s effect in prolonging graft survival, then, may involve suppression of IL-6. With respect to molecular mechanism, selective NFkB inhibition may be responsible for the extended survival. NF-kB functions as a master switch in a variety of processes including reperfusion injury and graft rejection [7, 33]. However, NF-kB is also involved in normal cellular physiology, and global NF-kB inhibition might result in serious side effects, such as hepatotoxicity and opportunistic infections [34]. It is therefore of interest that there are no significant changes in Th1 cytokines in our study. As aforementioned, skin allografts are so immunogenic that interventions that can successfully prevent rejection of heart or pancreatic islets may fail to elicit acceptance of skin transplants. In this regard, Zhou and colleagues [35] have shown that inhibition of T-cell-intrinsic NF-kB activation attenuates cardiac but not skin allograft rejection. Generally, after skin transplantation, Langerhans cells (a type of epidermal DC) migrate out of the donor skin into the lymph node of the recipient, where they can activate T cells. Allogeneic Langerhans cells have been shown to allow NF-kB-impaired T cells to reach an activation
CHENG ET AL.: PLATONIN PROLONGS SKIN GRAFT SURVIVAL
FIG. 7. Plasma alanine aminotransferase (ALT) (A) and creatinine (B) of levels platonin-treated mice compared with those of controls after skin transplantation. There were no significant differences in either variable between the two groups (n ¼ 6 in each group).
threshold necessary for T-cell differentiation and allograft rejection [36]. As depicted in this study, selective NF-kB inhibition was observed in draining lymph nodes. We previously demonstrated that platonin modulates the differentiation, maturation, and functions of DC toward an intermediate stage of maturation [14]. Ex vivo treatment by incubating pretransplant grafts with platonin also delayed the rejection of fully allogeneic skin transplants in mice (preliminary unpublished observations). These results together suggest that selective NF-kB inhibition may involve the effect of platonin against rejection. NF-kB is required for maximal transcription of many cytokines, including TNF-a and IL-6 [37]. Classic activation of macrophages requires IFN-g and the engagement of receptors, which recognize exogenous molecules such as the toll-like receptors [38]. Exposure of macrophages to the ligands of these receptors activates the downstream cascades, particularly NF-kB,which rapidly induces the expression of inflammation-associated genes, resulting in the production of proinflammatory cytokines, Th1 type inflammation, and release of NO
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by virtue of iNOS activity [39, 40]. In line with the observations that platonin suppressed the production of the proinflammatory cytokines, platonin attenuates NO production in LPS-stimulated macrophages and peripheral blood mononuclear cells through inhibiting iNOS expression [12, 19]. In our study, platonin significantly suppressed iNOS expression in draining lymph nodes. Blockade of NO production by iNOS inhibitors has been shown to prolong skin xenograft survival by down-regulation of the expressions of proinflammatory cytokines [41]. On the other hand, it is reported that NO derived from iNOS has a dual role in cardiac transplantation: detrimental in acute rejection and beneficial in chronic rejection [42]. Causal relationships between platonin-induced iNOS inhibition and attenuated skin graft rejection deserve further investigation. Platonin in our study caused no obvious hepatic or renal toxicity. If further investigation confirms the apparent safety of this substance, this would add to the attractiveness of platonin as an immunosuppressive agent. Infection is a common adverse complication of immunosuppression, yet platonin has antibacterial activity, and has been used for treatment of periodontal disease [3]. An agent that simultaneously suppresses graft rejection and bacterial infection would be a tremendous addition to our current armamentarium. Platonin is a water-soluble agent suitable for intravenous injection [29], making it suitable for clinical trials without a need to do drug formulation. No significant effect on leukocyte counts by platonin further renders this agent falling in a new category of immunosuppressants possessing a better pharmacologic profile than that of mTOR inhibitors. Furthermore, platonin possesses free radical-scavenging activities, and acts as an antioxidant [4, 43]. Recent data suggest that concurrent treatment with antioxidants may attenuate the renal dysfunction associated with cyclosporine [44]. In summary, our data provide clear evidence that platonin effectively prolongs skin allograft survival in mice without major toxicity. This effect appears to involve suppression of the up-regulation of proinflammatory cytokines, selective inhibition of NF-kB activity, and reduced iNOS expression, but without changing the overall Th1/Th2 cytokine balance. Our results along with the other evidence available to date on platonin’s effects, suggest that this agent may have unique benefits in transplant medicine. Clearly, this promising agent is worthy of further investigation.
ACKNOWLEDGMENTS The authors thank Dr. Mary Jeanne Buttrey and Dr. Hsin-Chen Lee for their critical reading and helpful editorial comments on the manuscript. They also thank Hui-Ru Shieh and Chien-Ying Huang for technical assistance and help.
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