Bilirubin Can Induce Tolerance to Islet Allografts
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《内分泌学杂志》
Department of Surgery, Beth Israel Deaconess Medical Center (H.W., S.S.L., J.D., E.C., B.Y.C., F.H.B.) and Joslin Diabetes Center (V.T.), Harvard Medical School, Boston, Massachusetts 02215
Department of Experimental Medicine and Biotechnology (C.D.), University of Milano-Bicocca, 20052 Monza-Milano, Italy
Abstract
Induction of heme oxygenase-1 (HO-1) expression in recipients of allogeneic islets can lead to long-term survival (>100 d) of those islets. We tested whether administration of bilirubin would substitute for the beneficial effects of HO-1 expression in islet transplantation. Administering bilirubin to the recipient (B6AF1) or incubating islets in a bilirubin-containing solution ex vivo led to long-term survival of allogeneic islets in a significant percentage of cases. In addition, administering bilirubin to only the donor frequently led to long-term survival of DBA/2 islets in B6AF1 recipients and significantly prolonged graft survival of BALB/c islets in C57BL/6 recipients. Donor treatment with bilirubin up-regulated mRNA expression of protective genes such as HO-1 and bcl-2 and suppressed proinflammatory and proapoptotic genes including monocyte chemoattractant protein-1 and caspase-3 and -8 in the islet grafts before transplantation. Furthermore, treatment of only the donor suppressed the expression of proinflammatory cytokines including TNF-, inducible nitric oxide synthase, monocyte chemoattractant protein-1, and other proapoptotic and proinflammatory genes normally seen in the islets after transplantation. Donor treatment also reduced the number of macrophages that infiltrated the islet grafts in the recipients. Preincubation of TC3 cells with bilirubin also protected the cells from lipid peroxidation. Our data suggests that the potent antioxidant and antiinflammatory actions of bilirubin may contribute to islet survival.
Introduction
TRANSPLANTATION OF ISLETS is an important approach to the treatment of diabetes. Although recent changes in immunosuppression and other facets of the transplant have improved results, there are still problems besetting the procedure (1). Transplanted islets are frequently rejected, likely in part because the islets frequently undergo apoptosis after transplantation (2), which reduces the effective number of transplanted islets. The need for more islets for each transplant leads to a shortage of islets. Immunosuppression of the recipients, which can be toxic, has been the sole approach to impede rejection (3).
Data from others and ourselves have shown that the expression of heme oxygenase-1 (HO-1) in islets can provide salutary effects (4, 5). HO-1 expression appears to protect islets from apoptosis and rejection (6).
HO-1 degrades heme into three products: carbon monoxide (CO), biliverdin, and free Fe2+ (7). Biliverdin is rapidly converted to bilirubin by biliverdin reductase, and the Fe2+ stimulates an iron pump that removes free iron from the cells as well as leading to the up-regulation of ferritin, an iron-binding protein. Each of these products has protective (antiapoptotic and antiinflammatory) properties, and one or more of them likely accounts for the protection afforded by HO-1 in any given situation (8, 9, 10).
This is true for islet transplantation. Administration of exogenous CO is, like HO-1, salutary for islet transplantation (11). In the present study, we tested whether bilirubin would prove to have similar salutary effects as CO and HO-1 in islet transplantation. There are reasons why such information may be valuable. In an occasional model we have found that biliverdin has salutary effects, whereas CO, at least at the doses and dosing schedule tested, did not. This raises the question whether biliverdin/bilirubin can in certain situations be more effective than CO. Furthermore, although the two substances can achieve the same disease-benefiting effects in a given condition, they may do so by different mechanisms, raising the possibility that their actions may be additive or synergistic. The results of our studies with bilirubin are reported here.
Materials and Methods
Animals
Male DBA/2, B6AF1, BALB/c, C57BL/6, and DBA/1 mice 6–8 wk of age (The Jackson Laboratory, Bar Harbor, ME) were used in the experiments. Mice were kept for 2 wk at four mice/cage and fed normal laboratory chow before using for the experiment. The animal experimentation protocol was approved by the Animal Care and Use Committees of the Beth Israel Deaconess Medical Center (Boston, MA).
Islet isolation and transplantation
Islets were isolated as described by Pileggi et al. (4). Islet purity was assessed by dithizone (Sigma-Aldrich, St. Louis, MO) staining after isolation. An algorithm was used for the calculation of the 150-μm diameter islet equivalent (IEQ) number (12, 13). Islet-cell viability was assessed using fluorescence staining with acridine orange (Sigma-Aldrich) and propidium iodide (Sigma-Aldrich) (14). Our isolation protocol usually yields 90–95% of viable cells before transplantation. Freshly harvested islets were transplanted into the recipient immediately after isolation. Recipients were rendered diabetic using streptozotocin (STZ; 225 mg/kg, ip, Sigma-Aldrich). Five days after STZ administration, mice with two consecutive blood glucose levels exceeding 350 mg/dl were used as recipients. Islets (450–500 IEQ) were transplanted under the kidney capsule of the recipients. Blood glucose levels of the recipients were measured twice weekly with a glucometer (Roche, Basel, Switzerland) after islet transplantation. Animals with a blood glucose less than 200 mg/dl were considered normoglycemic. Grafts were deemed rejected when two consecutive glucose levels were more than 300 mg/dl after a period of primary graft function.
Administration of bilirubin
Bilirubin (Sigma-Aldrich) was dissolved in a 0.2 N sodium hydroxide, subsequently adjusted to a pH of 7.4 with 1 N hydrochloride acid, diluted with 1x PBS to a final volume, and stored at –80 C until use. Light exposure was limited as much as possible. Bilirubin was either given ip to the donor (one dose) 1 h before islet harvest or to the recipient from d –1 until 13 d after transplantation (either one or two doses per day). No further treatment was given after the 14 d of injection. Control animals were given vehicle only.
Tolerance test
The kidneys under which the initial islets were transplanted were removed from a number of animals that had islets surviving long term, after which islets syngeneic with the original donor (DBA/2) were transplanted under the other kidney with no further treatments. If those second transplanted islets also survived more than 100 d, the recipients were considered tolerant. Antigen-specific tolerance was assessed by transplanting islets of a third party donor (DBA/1) that did not share either class I or II antigens with the original donor.
Real-time RT-PCR analysis
Real-time RT-PCR was performed to quantify the amount of target gene in each sample at mRNA level using the ABI PRISM 7700 Sequence Detection Systems as described (11). Total cellular RNA was extracted using a QIAGEN RNA kit (QIAGEN, Valencia, CA). DNAse, DNase I, RNase-free treatment was performed according to the manufacturer’s suggestion (QIAGEN) to exclude the contamination by genomic DNA during real-time RT-PCR. Samples were transcribed to cDNA in the presence of murine leukemia virus transcriptase (Applied Biosystems, Foster City, CA). RT reactions were set up in a total volume of 25 μl by using 2x Taqman Universal PCR Master Mix (Applied Biosystems). Each cDNA sample was analyzed in duplicate in the presence of 1x commercially available assay-on-demand primers together with 6-carboxyfluorescein-labeled fluorogenic probes (Applied Biosystems) specially designed for each target gene. Glyceraldehyde-3-phosphate dehydrogenase expression of each sample was performed in parallel by using the predeveloped primer and VIC-labeled fluorogenic probe to standardize the amount of sample cDNA added to the reaction. Freshly isolated islet cDNA was analyzed each time and used as a calibrator. The PCR ran for 2 min at 50 C, 10 min at 95 C, and for 40 cycles composed of an incubation at 95 C for 15 sec plus one at 60 C for 1 min. Relative quantification of all target genes was analyzed based upon a comparative cycle cross threshold method. The amount of target genes, normalized to glyceraldehyde-3-phosphate dehydrogenase and relative to the calibrator, was calculated based on the manufacturer’s instructions (http://www.appliedbiosystems.com/support/apptech/#rt_PCR). Expression of the following genes was analyzed in freshly isolated islets and islet grafts after transplantation: proinflammatory cytokines and chemokines including TNF-, inducible nitric oxide (NO) synthase (iNOS), monocyte chemoattractant protein-1 (MCP-1), macrophage migration inhibitory factor (MIF), IFN--inducible protein-10 (CXCL10), and CD40; death receptors including Fas (CD95) and TNF-related apoptosis-inducing ligand (TRAIL-R); caspases including caspase-3, -8, -9, and -12; and Bcl-2 family genes Bax, Bid, and Bad.
Cell culture
Cells of the insulinoma line TC3 (DSMZ, Braunschweig, Germany) were cultured in DMEM (Life Technologies, Grand Island, NY) containing 10% fetal bovine serum, 2 mmol/liter L-glutamine, 100 U/ml penicillin G, and 100 U/ml streptomycin.
Fenton reaction
TC3 cells were preincubated with bilirubin at different concentrations (1, 10, and 100 μmol/liter) for 1 h. H2O2 (100 μmol/liter) and FeSO4 (100 μmol/liter) were added to the cell culture after bilirubin incubation. The concentrations of lipid peroxides (i.e. malondialdehyde and 4-hydroxyalkenals) in the supernatant were measured 2 h later using the Lipid Peroxidation Assay kit according to the manufacturer’s recommendation (Calbiochem, San Diego, CA). Cells were harvested at 2 and 6 h after the Fenton reaction. Expression of genes including TNF-, iNOS, MCP-1, IL-6, and caspase-3 and -8 were quantified at the mRNA level by real-time RT-PCR.
Immunohistochemistry
Islet grafts including a portion of the kidney were harvested at 1 and 3 d posttransplantation and snap frozen in liquid nitrogen for immunohistological staining. Sections of 5 μm were stained with the macrophage marker F4/80 (Serotec, Oxford, UK) to detect the expression of infiltrated macrophages inside the islet graft. Secondary antibody was biotinylated antirat antibody (Vector Laboratories, Burlingame, CA). Avidin-biotin complex-horseradish peroxidase (DakoCytomation California Inc., Carpinteria, CA) and diaminobenzidine substrates were used to develop the color. Counterstaining with hematoxylin and eosin was performed after dehydration, and slides were covered with Cytoseal 280 mounting medium for observation. Sections from three individual animals within the same treatment group were studied for F4/80 expression. Six random areas of interest (400- x 400-pixel area) were selected from each tissue section, and the total number of F4/80-positive cells was enumerated. The percentage of macrophages in the total number of cells (blue nuclei) was calculated.
Statistical analyses
Kaplan-Meier survival curves were performed by using the Statview software, and the statistical differences were assessed by the log rank test. P < 0.05 was considered significant. Survival data are expressed as mean survival time ± SD. Differences between cytokine expressions were compared for statistical significance by the Mann-Whitney U test.
Results
Administration of bilirubin can induce long-term survival and tolerance to islet allografts in the DBA/2 to B6AF1 combination
DBA/2 (H-2d) islets transplanted into B6AF1 (H-2b,k/d) recipients, with no treatments of either, were rejected in 24.9 ± 5.3 d when 450–500 IEQs were transplanted (n = 11, Fig. 1). Islet grafts in every bilirubin-treated group survived significantly longer than the controls. Administering bilirubin at 8.5 μmol/kg to the islet donor 1 h before islet transplantation led to a significant percentage (41.7%, n = 12) of long-term surviving islets (P = 0.01 vs. control). In groups in which only the recipient was treated, we compared giving one dose of bilirubin at 17 μmol/kg to the recipient or giving two doses (every 12 h at 8.5 μmol/kg) per day from d –1 until d 13. The single dose led to 50% long-term survival (n = 6), whereas two doses led to 72.7% (n = 11) of islet grafts surviving long-term (P = 0.19 vs. one dose, Fig. 1). We also cultured isolated islets in 100 μmol/liter bilirubin for 1 h after isolation and before transplantation. In this treatment group, 40% of DBA/2 islets (n = 5) survived long term in the B6AF1 recipients (P = 0.0004 vs. control).
The primary islet grafts were removed by nephrectomy from recipients that carried long-term surviving grafts based on treatment with bilirubin. Blood glucose levels of those recipients increased sharply to more than 350 mg/dl within a few hours, suggesting that euglycemia was maintained by the transplanted grafts. Tolerance was demonstrated in three of three recipients; second islet grafts from the same donor strain (DBA/2) placed under the capsule of the remaining kidney survived long term without further treatment. One experiment failed due to the death of a recipient animal at 29 d after transplantation of the second graft; that animal was euglycemic at that time. Grafts from a third party donor (DBA/1, H-2q) were rejected in 14 ± 2.08 d (n = 3, Table 1).
Administration of bilirubin to BALB/c donors prolongs islets graft survival in C57BL/6 recipients
The protective effect of administering bilirubin to islet donors was confirmed in a donor-recipient combination differing by a stronger immunogenetic disparity: islets from BALB/c (H-2d) mice transplanted to C57BL/6 (H-2b) mice. A very significant prolongation of islet graft survival was observed when bilirubin was given to the donor (8.5 μmol/kg, 1 h before islet isolation, n = 6) compared with the control, which received vehicle only (n = 6, P = 0.0074 vs. control, Fig. 2).
Bilirubin treatment to the donor up-regulates expression of protective genes and suppresses proapoptotic genes at the mRNA level in the islets before transplantation
To better understand the protective effect of treating only the donor with bilirubin, we analyzed the expression levels of the protective genes, HO-1 and bcl-2, the proapoptotic genes, caspase-3 and -8, and the proinflammatory genes, IL-6, MCP-1, iNOS, and TNF-, at the mRNA level using real-time RT-PCR. We studied freshly isolated islets from either control or bilirubin-treated donors (DBA/2). Bilirubin was given at 8.5 μmol/kg 1 h before islet isolation. The islet isolation procedure took approximately 4 h to complete. Therefore, the mRNA expression levels detected represent the mRNA level 5 h after bilirubin injection and immediately before the islets would be transplanted. As shown in Fig. 3, the HO-1 and bcl-2 genes were expressed significantly more strongly in islets from bilirubin-treated donors compared with islets from controls. On the contrary, expression of proapoptotic and proinflammatory genes including caspase-3 and -8 and MCP-1 were significantly suppressed in the islets from bilirubin-treated donors compared with islets from controls. No detectable expression of iNOS, TNF-, MIF, and IL-6 was observed in islets isolated from either bilirubin-treated or control animals.
Bilirubin treatment to the donor suppresses proinflammatory gene expression in the islet graft at various times after transplantation
We tested whether bilirubin treatment to the donor could suppress the inflammatory response seen in islets after transplantation to allogeneic recipients without any treatment. We analyzed the expression of proinflammatory, proapoptotic, and death receptor genes that are induced after transplantation in the untreated condition using real-time RT-PCR. These included TNF-, iNOS, MCP-1, Fas, TRAIL-R, caspases, and others. The proinflammatory cytokine products of these genes are among the leading contributors that play a role in the destruction of -cells after transplantation. Expression levels of TNF-, iNOS, MCP-1, Fas, TRAIL-R, caspase-3, -8, and -9, BID, and CXCL10 in the bilirubin-treated groups were very significantly lower than in control groups 1 d posttransplantation (Fig. 4). Significant differences were also observed at some later time points for some genes. No significant difference was observed for expression of genes such as MIF, Bax, Bad, and CD40 between bilirubin-treated and control grafts at various days posttransplantation.
Bilirubin suppresses macrophage infiltration into the graft after allogeneic transplantation
Infiltrating macrophages that secrete proinflammatory cytokines, such as TNF-, IL-1, and IFN-, contribute to the destruction of transplanted islets. Our question was whether bilirubin pretreatment to the donor would reduce the number of macrophages infiltrating into the islet grafts after transplantation. Islet grafts at 1 and 3 d posttransplantation were stained with the macrophage marker, F4/80. Immunohistological staining showed that fewer macrophages infiltrated into the islet grafts from bilirubin-treated donors compared with grafts from nontreated donors at the same time points (Fig. 5). One day after transplantation, 25.83 ± 9.89% of cells (in 554 cells counted) stained positive for F4/80 in grafts from nontreated donor when compared with grafts from bilirubin-treated donor (3.13 ± 3.74% of positive cells in 527 cells counted, P = 0.005 vs. control). Similarly, 3 d after transplantation, 29.18 ± 14.54% of cells (in 504 cells counted) in the graft from control donor stained positive for macrophage when compared with graft from bilirubin-treated donor (8.31 ± 3.66% of positive cells in 518 cells counted, P = 0.001 vs. control). This is consistent with the reduced proinflammatory cytokine levels detected after bilirubin treatment.
In vitro culture of TC-3 cells with bilirubin can protect the cells from lipid peroxidation induced by hydroxyl radicals
To test the hypothesis that the antioxidant property of bilirubin may contribute to islet graft survival after transplantation, we investigated whether bilirubin could protect cells of the insulinoma line TC3 from lipid peroxidation, a well-established mechanism of cellular injury induced by hydroxyl radicals generated by H2O2 and FeSO4. Lipid peroxidation leads to the production of lipid peroxides (such as malondialdehyde and 4-hydroxyalkenals) and their byproducts, and ultimately the loss of membrane function and integrity. Cells precultured in 100 μmol/liter bilirubin for 1 h had significantly less lipid peroxidation compared with the nontreated controls, suggesting that bilirubin can scavenge free radicals in the cell culture medium, therefore protecting cells from injury (Fig. 6A). In addition, lower levels of MCP-1 (Fig. 6B) and caspase-8 (data not shown) were detected in the bilirubin-treated cells than in control cells 6 h after the Fenton reaction.
Discussion
The preservation of islet cell viability after transplantation is a major concern. In most systems used to date, there is significant death of the islet cells by apoptosis (2). The danger hypothesis (15, 16) suggests that the aggressiveness of an immune response will be greater in the presence of inflammation than in its absence; thus, by suppressing inflammation in the islets, one may be able to achieve better survival because there is less of a rejection response (17).
Expression of HO-1 in a transplanted organ can be critical to the survival of that graft after transplantation (4). One example of this came from studies of mouse to rat transplantation in which the recipient is treated with the immunosuppressive agent, cyclosporine A, plus cobra venom factor to inhibit complement, leading to indefinite survival of the mouse heart graft. However, if the graft is from a mouse deficient in HO-1 (HO-1–/– mouse), the graft is rejected as though there is no immunosuppression. Because the wild-type donor and the HO-1–/– donor differ only by this one gene, it is compelling evidence that HO-1, in at least some transplant situations, is essential for survival (18). Which byproduct(s) of HO-1 degradation of heme is responsible for this effect is still not clear. However, when the action of HO-1 was blocked with tin protoporphyrin, in analogy with the HO-1–/– mouse donor, giving CO to the donor and to the recipient compensated for the action of HO-1 and allowed long-term survival (19).
As shown above, the administration of bilirubin results in significant benefits in terms of survival of islets in allogeneic recipients, in many cases resulting in long-term survival and antigen-specific tolerance. This is true when the donor, the islets, or the recipient were treated with bilirubin. Unlike HO-1 induction or the administration of CO, little is known about the mechanisms underlying the beneficial effects of administering biliverdin or bilirubin except that both these molecules exert potent antioxidant effects (20, 21). The power of the antioxidant effect may be extremely potent given the recent observation that in addition to the conversion of biliverdin to bilirubin by biliverdin reductase, the bilirubin can be oxidized and reconverted to biliverdin, thus setting up a cycle that would amplify the antioxidant effects (22). Although biliverdin or bilirubin may well have actions beyond their antioxidant ones, we suggest that the antioxidant effects may themselves contribute greatly to the results presented here.
In our other studies with biliverdin/bilirubin, we have made some progress in determining the mechanisms by which bilirubin acts. Transplantation of syngeneic small intestines in rats leads to a potent proinflammatory response including the up-regulation of IL-6, IL-1, and other proinflammatory molecules. Treatment of the donor and recipient with biliverdin significantly reduced expression of these proinflammatory molecules and improved organ survival. The antioxidant properties of biliverdin may account for these effects (23).
-Cells are unusually vulnerable to damage by reactive oxygen species (24, 25, 26). Reactive oxygen species generated after transplantation can induce proinflammatory cytokine expression and destroy -cells within the islets. The two commonly used chemicals to induce diabetes, alloxan and STZ, induce O2– and iNOS with the consequent production of NO, which results in the destruction of the -cells (27, 28, 29). We have shown above that bilirubin can scavenge free radicals in the insulinoma cell culture medium, thus protecting cells from lipid peroxidation. Our results provide the basis for a potential approach initiated by biliverdin/bilirubin that would protect the islets. There are three aspects of this action. Firstly, bilirubin treatment of the donor leads to a lesser inflammatory response in the islets after transplantation to the recipient as compared with islets from untreated donors. Inflammation, as discussed earlier, heightens the allo-aggressive immune response; thus, suppression of inflammation should lead to a lesser rejection response. Secondly, biliverdin treatment of the donor leads to suppression of iNOS and IL-1 (30, 31). There is strong evidence that both NO, the product of iNOS action, and IL-1 can damage islets. Thus, the diminution of NO and IL-1 should have salutary effects. Lastly, biliverdin/bilirubin may also be able to protect islets by up-regulating protective genes such as HO-1 and bcl-2. The up-regulation by biliverdin/bilirubin of HO-1 would lead to an amplification cycle because more biliverdin is produced by the induced HO-1. In addition to the protective effect of HO-1, up-regulation of bcl-2 in -cells can protect the cells from caspase-mediated cell death, thus contributing to the survival of transplanted islets (32, 33, 34). This is consistent with the suppressed level of caspases in freshly isolated islets and islet grafts at various days after transplantation.
It would seem that administration of bilirubin might be useful clinically for islet transplantation. Bilirubin is a natural product of the body that appears to have little if any toxicity (except in the immediate neonatal period) at the levels we induce by our treatments. Basal serum bilirubin levels were 9.45 ± 0.14 μmol/liter in the untreated mice. Serum bilirubin level peaked to a maximal level of 24.79 ± 1.9 μmol/liter after 15 min of a single injection of bilirubin (8.5 μmol/kg). The levels gradually decreased to 14.55 ± 1.2 μmol/liter at 2 h after injection and went back to normal (9.21 ± 0.21 μmol/liter) at 4 h after injection. Bilirubin in general appears to be protective. Several studies of normal human populations have shown that those individuals who have high normal or just above normal levels of bilirubin have less atherosclerosis-type disease than those with low normal levels of bilirubin (35, 36). Furthermore, individuals with Gilbert syndrome, who constitutively have significantly elevated levels of bilirubin, also enjoy such benefits (37).
We conclude that bilirubin has salutary effects on islet survival based on the suppression of inflammation that leads to a decreased immune response, the up-regulation of protective genes such as HO-1 and bcl-2 production, as well as the direct curbing of the proliferation and functional maturation of T cells (38).
Footnotes
This work was supported by the Riva Foundation (Grant 01-006 to F.H.B.).
First Published Online October 27, 2005
Abbreviations: CO, Carbon monoxide; HO-1, heme oxygenase-1; IEQ, islet equivalent; iNOS, inducible nitric oxide synthase; MCP-1, monocyte chemoattractant protein-1; MIF, macrophage migration inhibitory factor; NO, nitric oxide; STZ, streptozotocin.
Accepted for publication October 17, 2005.
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Department of Experimental Medicine and Biotechnology (C.D.), University of Milano-Bicocca, 20052 Monza-Milano, Italy
Abstract
Induction of heme oxygenase-1 (HO-1) expression in recipients of allogeneic islets can lead to long-term survival (>100 d) of those islets. We tested whether administration of bilirubin would substitute for the beneficial effects of HO-1 expression in islet transplantation. Administering bilirubin to the recipient (B6AF1) or incubating islets in a bilirubin-containing solution ex vivo led to long-term survival of allogeneic islets in a significant percentage of cases. In addition, administering bilirubin to only the donor frequently led to long-term survival of DBA/2 islets in B6AF1 recipients and significantly prolonged graft survival of BALB/c islets in C57BL/6 recipients. Donor treatment with bilirubin up-regulated mRNA expression of protective genes such as HO-1 and bcl-2 and suppressed proinflammatory and proapoptotic genes including monocyte chemoattractant protein-1 and caspase-3 and -8 in the islet grafts before transplantation. Furthermore, treatment of only the donor suppressed the expression of proinflammatory cytokines including TNF-, inducible nitric oxide synthase, monocyte chemoattractant protein-1, and other proapoptotic and proinflammatory genes normally seen in the islets after transplantation. Donor treatment also reduced the number of macrophages that infiltrated the islet grafts in the recipients. Preincubation of TC3 cells with bilirubin also protected the cells from lipid peroxidation. Our data suggests that the potent antioxidant and antiinflammatory actions of bilirubin may contribute to islet survival.
Introduction
TRANSPLANTATION OF ISLETS is an important approach to the treatment of diabetes. Although recent changes in immunosuppression and other facets of the transplant have improved results, there are still problems besetting the procedure (1). Transplanted islets are frequently rejected, likely in part because the islets frequently undergo apoptosis after transplantation (2), which reduces the effective number of transplanted islets. The need for more islets for each transplant leads to a shortage of islets. Immunosuppression of the recipients, which can be toxic, has been the sole approach to impede rejection (3).
Data from others and ourselves have shown that the expression of heme oxygenase-1 (HO-1) in islets can provide salutary effects (4, 5). HO-1 expression appears to protect islets from apoptosis and rejection (6).
HO-1 degrades heme into three products: carbon monoxide (CO), biliverdin, and free Fe2+ (7). Biliverdin is rapidly converted to bilirubin by biliverdin reductase, and the Fe2+ stimulates an iron pump that removes free iron from the cells as well as leading to the up-regulation of ferritin, an iron-binding protein. Each of these products has protective (antiapoptotic and antiinflammatory) properties, and one or more of them likely accounts for the protection afforded by HO-1 in any given situation (8, 9, 10).
This is true for islet transplantation. Administration of exogenous CO is, like HO-1, salutary for islet transplantation (11). In the present study, we tested whether bilirubin would prove to have similar salutary effects as CO and HO-1 in islet transplantation. There are reasons why such information may be valuable. In an occasional model we have found that biliverdin has salutary effects, whereas CO, at least at the doses and dosing schedule tested, did not. This raises the question whether biliverdin/bilirubin can in certain situations be more effective than CO. Furthermore, although the two substances can achieve the same disease-benefiting effects in a given condition, they may do so by different mechanisms, raising the possibility that their actions may be additive or synergistic. The results of our studies with bilirubin are reported here.
Materials and Methods
Animals
Male DBA/2, B6AF1, BALB/c, C57BL/6, and DBA/1 mice 6–8 wk of age (The Jackson Laboratory, Bar Harbor, ME) were used in the experiments. Mice were kept for 2 wk at four mice/cage and fed normal laboratory chow before using for the experiment. The animal experimentation protocol was approved by the Animal Care and Use Committees of the Beth Israel Deaconess Medical Center (Boston, MA).
Islet isolation and transplantation
Islets were isolated as described by Pileggi et al. (4). Islet purity was assessed by dithizone (Sigma-Aldrich, St. Louis, MO) staining after isolation. An algorithm was used for the calculation of the 150-μm diameter islet equivalent (IEQ) number (12, 13). Islet-cell viability was assessed using fluorescence staining with acridine orange (Sigma-Aldrich) and propidium iodide (Sigma-Aldrich) (14). Our isolation protocol usually yields 90–95% of viable cells before transplantation. Freshly harvested islets were transplanted into the recipient immediately after isolation. Recipients were rendered diabetic using streptozotocin (STZ; 225 mg/kg, ip, Sigma-Aldrich). Five days after STZ administration, mice with two consecutive blood glucose levels exceeding 350 mg/dl were used as recipients. Islets (450–500 IEQ) were transplanted under the kidney capsule of the recipients. Blood glucose levels of the recipients were measured twice weekly with a glucometer (Roche, Basel, Switzerland) after islet transplantation. Animals with a blood glucose less than 200 mg/dl were considered normoglycemic. Grafts were deemed rejected when two consecutive glucose levels were more than 300 mg/dl after a period of primary graft function.
Administration of bilirubin
Bilirubin (Sigma-Aldrich) was dissolved in a 0.2 N sodium hydroxide, subsequently adjusted to a pH of 7.4 with 1 N hydrochloride acid, diluted with 1x PBS to a final volume, and stored at –80 C until use. Light exposure was limited as much as possible. Bilirubin was either given ip to the donor (one dose) 1 h before islet harvest or to the recipient from d –1 until 13 d after transplantation (either one or two doses per day). No further treatment was given after the 14 d of injection. Control animals were given vehicle only.
Tolerance test
The kidneys under which the initial islets were transplanted were removed from a number of animals that had islets surviving long term, after which islets syngeneic with the original donor (DBA/2) were transplanted under the other kidney with no further treatments. If those second transplanted islets also survived more than 100 d, the recipients were considered tolerant. Antigen-specific tolerance was assessed by transplanting islets of a third party donor (DBA/1) that did not share either class I or II antigens with the original donor.
Real-time RT-PCR analysis
Real-time RT-PCR was performed to quantify the amount of target gene in each sample at mRNA level using the ABI PRISM 7700 Sequence Detection Systems as described (11). Total cellular RNA was extracted using a QIAGEN RNA kit (QIAGEN, Valencia, CA). DNAse, DNase I, RNase-free treatment was performed according to the manufacturer’s suggestion (QIAGEN) to exclude the contamination by genomic DNA during real-time RT-PCR. Samples were transcribed to cDNA in the presence of murine leukemia virus transcriptase (Applied Biosystems, Foster City, CA). RT reactions were set up in a total volume of 25 μl by using 2x Taqman Universal PCR Master Mix (Applied Biosystems). Each cDNA sample was analyzed in duplicate in the presence of 1x commercially available assay-on-demand primers together with 6-carboxyfluorescein-labeled fluorogenic probes (Applied Biosystems) specially designed for each target gene. Glyceraldehyde-3-phosphate dehydrogenase expression of each sample was performed in parallel by using the predeveloped primer and VIC-labeled fluorogenic probe to standardize the amount of sample cDNA added to the reaction. Freshly isolated islet cDNA was analyzed each time and used as a calibrator. The PCR ran for 2 min at 50 C, 10 min at 95 C, and for 40 cycles composed of an incubation at 95 C for 15 sec plus one at 60 C for 1 min. Relative quantification of all target genes was analyzed based upon a comparative cycle cross threshold method. The amount of target genes, normalized to glyceraldehyde-3-phosphate dehydrogenase and relative to the calibrator, was calculated based on the manufacturer’s instructions (http://www.appliedbiosystems.com/support/apptech/#rt_PCR). Expression of the following genes was analyzed in freshly isolated islets and islet grafts after transplantation: proinflammatory cytokines and chemokines including TNF-, inducible nitric oxide (NO) synthase (iNOS), monocyte chemoattractant protein-1 (MCP-1), macrophage migration inhibitory factor (MIF), IFN--inducible protein-10 (CXCL10), and CD40; death receptors including Fas (CD95) and TNF-related apoptosis-inducing ligand (TRAIL-R); caspases including caspase-3, -8, -9, and -12; and Bcl-2 family genes Bax, Bid, and Bad.
Cell culture
Cells of the insulinoma line TC3 (DSMZ, Braunschweig, Germany) were cultured in DMEM (Life Technologies, Grand Island, NY) containing 10% fetal bovine serum, 2 mmol/liter L-glutamine, 100 U/ml penicillin G, and 100 U/ml streptomycin.
Fenton reaction
TC3 cells were preincubated with bilirubin at different concentrations (1, 10, and 100 μmol/liter) for 1 h. H2O2 (100 μmol/liter) and FeSO4 (100 μmol/liter) were added to the cell culture after bilirubin incubation. The concentrations of lipid peroxides (i.e. malondialdehyde and 4-hydroxyalkenals) in the supernatant were measured 2 h later using the Lipid Peroxidation Assay kit according to the manufacturer’s recommendation (Calbiochem, San Diego, CA). Cells were harvested at 2 and 6 h after the Fenton reaction. Expression of genes including TNF-, iNOS, MCP-1, IL-6, and caspase-3 and -8 were quantified at the mRNA level by real-time RT-PCR.
Immunohistochemistry
Islet grafts including a portion of the kidney were harvested at 1 and 3 d posttransplantation and snap frozen in liquid nitrogen for immunohistological staining. Sections of 5 μm were stained with the macrophage marker F4/80 (Serotec, Oxford, UK) to detect the expression of infiltrated macrophages inside the islet graft. Secondary antibody was biotinylated antirat antibody (Vector Laboratories, Burlingame, CA). Avidin-biotin complex-horseradish peroxidase (DakoCytomation California Inc., Carpinteria, CA) and diaminobenzidine substrates were used to develop the color. Counterstaining with hematoxylin and eosin was performed after dehydration, and slides were covered with Cytoseal 280 mounting medium for observation. Sections from three individual animals within the same treatment group were studied for F4/80 expression. Six random areas of interest (400- x 400-pixel area) were selected from each tissue section, and the total number of F4/80-positive cells was enumerated. The percentage of macrophages in the total number of cells (blue nuclei) was calculated.
Statistical analyses
Kaplan-Meier survival curves were performed by using the Statview software, and the statistical differences were assessed by the log rank test. P < 0.05 was considered significant. Survival data are expressed as mean survival time ± SD. Differences between cytokine expressions were compared for statistical significance by the Mann-Whitney U test.
Results
Administration of bilirubin can induce long-term survival and tolerance to islet allografts in the DBA/2 to B6AF1 combination
DBA/2 (H-2d) islets transplanted into B6AF1 (H-2b,k/d) recipients, with no treatments of either, were rejected in 24.9 ± 5.3 d when 450–500 IEQs were transplanted (n = 11, Fig. 1). Islet grafts in every bilirubin-treated group survived significantly longer than the controls. Administering bilirubin at 8.5 μmol/kg to the islet donor 1 h before islet transplantation led to a significant percentage (41.7%, n = 12) of long-term surviving islets (P = 0.01 vs. control). In groups in which only the recipient was treated, we compared giving one dose of bilirubin at 17 μmol/kg to the recipient or giving two doses (every 12 h at 8.5 μmol/kg) per day from d –1 until d 13. The single dose led to 50% long-term survival (n = 6), whereas two doses led to 72.7% (n = 11) of islet grafts surviving long-term (P = 0.19 vs. one dose, Fig. 1). We also cultured isolated islets in 100 μmol/liter bilirubin for 1 h after isolation and before transplantation. In this treatment group, 40% of DBA/2 islets (n = 5) survived long term in the B6AF1 recipients (P = 0.0004 vs. control).
The primary islet grafts were removed by nephrectomy from recipients that carried long-term surviving grafts based on treatment with bilirubin. Blood glucose levels of those recipients increased sharply to more than 350 mg/dl within a few hours, suggesting that euglycemia was maintained by the transplanted grafts. Tolerance was demonstrated in three of three recipients; second islet grafts from the same donor strain (DBA/2) placed under the capsule of the remaining kidney survived long term without further treatment. One experiment failed due to the death of a recipient animal at 29 d after transplantation of the second graft; that animal was euglycemic at that time. Grafts from a third party donor (DBA/1, H-2q) were rejected in 14 ± 2.08 d (n = 3, Table 1).
Administration of bilirubin to BALB/c donors prolongs islets graft survival in C57BL/6 recipients
The protective effect of administering bilirubin to islet donors was confirmed in a donor-recipient combination differing by a stronger immunogenetic disparity: islets from BALB/c (H-2d) mice transplanted to C57BL/6 (H-2b) mice. A very significant prolongation of islet graft survival was observed when bilirubin was given to the donor (8.5 μmol/kg, 1 h before islet isolation, n = 6) compared with the control, which received vehicle only (n = 6, P = 0.0074 vs. control, Fig. 2).
Bilirubin treatment to the donor up-regulates expression of protective genes and suppresses proapoptotic genes at the mRNA level in the islets before transplantation
To better understand the protective effect of treating only the donor with bilirubin, we analyzed the expression levels of the protective genes, HO-1 and bcl-2, the proapoptotic genes, caspase-3 and -8, and the proinflammatory genes, IL-6, MCP-1, iNOS, and TNF-, at the mRNA level using real-time RT-PCR. We studied freshly isolated islets from either control or bilirubin-treated donors (DBA/2). Bilirubin was given at 8.5 μmol/kg 1 h before islet isolation. The islet isolation procedure took approximately 4 h to complete. Therefore, the mRNA expression levels detected represent the mRNA level 5 h after bilirubin injection and immediately before the islets would be transplanted. As shown in Fig. 3, the HO-1 and bcl-2 genes were expressed significantly more strongly in islets from bilirubin-treated donors compared with islets from controls. On the contrary, expression of proapoptotic and proinflammatory genes including caspase-3 and -8 and MCP-1 were significantly suppressed in the islets from bilirubin-treated donors compared with islets from controls. No detectable expression of iNOS, TNF-, MIF, and IL-6 was observed in islets isolated from either bilirubin-treated or control animals.
Bilirubin treatment to the donor suppresses proinflammatory gene expression in the islet graft at various times after transplantation
We tested whether bilirubin treatment to the donor could suppress the inflammatory response seen in islets after transplantation to allogeneic recipients without any treatment. We analyzed the expression of proinflammatory, proapoptotic, and death receptor genes that are induced after transplantation in the untreated condition using real-time RT-PCR. These included TNF-, iNOS, MCP-1, Fas, TRAIL-R, caspases, and others. The proinflammatory cytokine products of these genes are among the leading contributors that play a role in the destruction of -cells after transplantation. Expression levels of TNF-, iNOS, MCP-1, Fas, TRAIL-R, caspase-3, -8, and -9, BID, and CXCL10 in the bilirubin-treated groups were very significantly lower than in control groups 1 d posttransplantation (Fig. 4). Significant differences were also observed at some later time points for some genes. No significant difference was observed for expression of genes such as MIF, Bax, Bad, and CD40 between bilirubin-treated and control grafts at various days posttransplantation.
Bilirubin suppresses macrophage infiltration into the graft after allogeneic transplantation
Infiltrating macrophages that secrete proinflammatory cytokines, such as TNF-, IL-1, and IFN-, contribute to the destruction of transplanted islets. Our question was whether bilirubin pretreatment to the donor would reduce the number of macrophages infiltrating into the islet grafts after transplantation. Islet grafts at 1 and 3 d posttransplantation were stained with the macrophage marker, F4/80. Immunohistological staining showed that fewer macrophages infiltrated into the islet grafts from bilirubin-treated donors compared with grafts from nontreated donors at the same time points (Fig. 5). One day after transplantation, 25.83 ± 9.89% of cells (in 554 cells counted) stained positive for F4/80 in grafts from nontreated donor when compared with grafts from bilirubin-treated donor (3.13 ± 3.74% of positive cells in 527 cells counted, P = 0.005 vs. control). Similarly, 3 d after transplantation, 29.18 ± 14.54% of cells (in 504 cells counted) in the graft from control donor stained positive for macrophage when compared with graft from bilirubin-treated donor (8.31 ± 3.66% of positive cells in 518 cells counted, P = 0.001 vs. control). This is consistent with the reduced proinflammatory cytokine levels detected after bilirubin treatment.
In vitro culture of TC-3 cells with bilirubin can protect the cells from lipid peroxidation induced by hydroxyl radicals
To test the hypothesis that the antioxidant property of bilirubin may contribute to islet graft survival after transplantation, we investigated whether bilirubin could protect cells of the insulinoma line TC3 from lipid peroxidation, a well-established mechanism of cellular injury induced by hydroxyl radicals generated by H2O2 and FeSO4. Lipid peroxidation leads to the production of lipid peroxides (such as malondialdehyde and 4-hydroxyalkenals) and their byproducts, and ultimately the loss of membrane function and integrity. Cells precultured in 100 μmol/liter bilirubin for 1 h had significantly less lipid peroxidation compared with the nontreated controls, suggesting that bilirubin can scavenge free radicals in the cell culture medium, therefore protecting cells from injury (Fig. 6A). In addition, lower levels of MCP-1 (Fig. 6B) and caspase-8 (data not shown) were detected in the bilirubin-treated cells than in control cells 6 h after the Fenton reaction.
Discussion
The preservation of islet cell viability after transplantation is a major concern. In most systems used to date, there is significant death of the islet cells by apoptosis (2). The danger hypothesis (15, 16) suggests that the aggressiveness of an immune response will be greater in the presence of inflammation than in its absence; thus, by suppressing inflammation in the islets, one may be able to achieve better survival because there is less of a rejection response (17).
Expression of HO-1 in a transplanted organ can be critical to the survival of that graft after transplantation (4). One example of this came from studies of mouse to rat transplantation in which the recipient is treated with the immunosuppressive agent, cyclosporine A, plus cobra venom factor to inhibit complement, leading to indefinite survival of the mouse heart graft. However, if the graft is from a mouse deficient in HO-1 (HO-1–/– mouse), the graft is rejected as though there is no immunosuppression. Because the wild-type donor and the HO-1–/– donor differ only by this one gene, it is compelling evidence that HO-1, in at least some transplant situations, is essential for survival (18). Which byproduct(s) of HO-1 degradation of heme is responsible for this effect is still not clear. However, when the action of HO-1 was blocked with tin protoporphyrin, in analogy with the HO-1–/– mouse donor, giving CO to the donor and to the recipient compensated for the action of HO-1 and allowed long-term survival (19).
As shown above, the administration of bilirubin results in significant benefits in terms of survival of islets in allogeneic recipients, in many cases resulting in long-term survival and antigen-specific tolerance. This is true when the donor, the islets, or the recipient were treated with bilirubin. Unlike HO-1 induction or the administration of CO, little is known about the mechanisms underlying the beneficial effects of administering biliverdin or bilirubin except that both these molecules exert potent antioxidant effects (20, 21). The power of the antioxidant effect may be extremely potent given the recent observation that in addition to the conversion of biliverdin to bilirubin by biliverdin reductase, the bilirubin can be oxidized and reconverted to biliverdin, thus setting up a cycle that would amplify the antioxidant effects (22). Although biliverdin or bilirubin may well have actions beyond their antioxidant ones, we suggest that the antioxidant effects may themselves contribute greatly to the results presented here.
In our other studies with biliverdin/bilirubin, we have made some progress in determining the mechanisms by which bilirubin acts. Transplantation of syngeneic small intestines in rats leads to a potent proinflammatory response including the up-regulation of IL-6, IL-1, and other proinflammatory molecules. Treatment of the donor and recipient with biliverdin significantly reduced expression of these proinflammatory molecules and improved organ survival. The antioxidant properties of biliverdin may account for these effects (23).
-Cells are unusually vulnerable to damage by reactive oxygen species (24, 25, 26). Reactive oxygen species generated after transplantation can induce proinflammatory cytokine expression and destroy -cells within the islets. The two commonly used chemicals to induce diabetes, alloxan and STZ, induce O2– and iNOS with the consequent production of NO, which results in the destruction of the -cells (27, 28, 29). We have shown above that bilirubin can scavenge free radicals in the insulinoma cell culture medium, thus protecting cells from lipid peroxidation. Our results provide the basis for a potential approach initiated by biliverdin/bilirubin that would protect the islets. There are three aspects of this action. Firstly, bilirubin treatment of the donor leads to a lesser inflammatory response in the islets after transplantation to the recipient as compared with islets from untreated donors. Inflammation, as discussed earlier, heightens the allo-aggressive immune response; thus, suppression of inflammation should lead to a lesser rejection response. Secondly, biliverdin treatment of the donor leads to suppression of iNOS and IL-1 (30, 31). There is strong evidence that both NO, the product of iNOS action, and IL-1 can damage islets. Thus, the diminution of NO and IL-1 should have salutary effects. Lastly, biliverdin/bilirubin may also be able to protect islets by up-regulating protective genes such as HO-1 and bcl-2. The up-regulation by biliverdin/bilirubin of HO-1 would lead to an amplification cycle because more biliverdin is produced by the induced HO-1. In addition to the protective effect of HO-1, up-regulation of bcl-2 in -cells can protect the cells from caspase-mediated cell death, thus contributing to the survival of transplanted islets (32, 33, 34). This is consistent with the suppressed level of caspases in freshly isolated islets and islet grafts at various days after transplantation.
It would seem that administration of bilirubin might be useful clinically for islet transplantation. Bilirubin is a natural product of the body that appears to have little if any toxicity (except in the immediate neonatal period) at the levels we induce by our treatments. Basal serum bilirubin levels were 9.45 ± 0.14 μmol/liter in the untreated mice. Serum bilirubin level peaked to a maximal level of 24.79 ± 1.9 μmol/liter after 15 min of a single injection of bilirubin (8.5 μmol/kg). The levels gradually decreased to 14.55 ± 1.2 μmol/liter at 2 h after injection and went back to normal (9.21 ± 0.21 μmol/liter) at 4 h after injection. Bilirubin in general appears to be protective. Several studies of normal human populations have shown that those individuals who have high normal or just above normal levels of bilirubin have less atherosclerosis-type disease than those with low normal levels of bilirubin (35, 36). Furthermore, individuals with Gilbert syndrome, who constitutively have significantly elevated levels of bilirubin, also enjoy such benefits (37).
We conclude that bilirubin has salutary effects on islet survival based on the suppression of inflammation that leads to a decreased immune response, the up-regulation of protective genes such as HO-1 and bcl-2 production, as well as the direct curbing of the proliferation and functional maturation of T cells (38).
Footnotes
This work was supported by the Riva Foundation (Grant 01-006 to F.H.B.).
First Published Online October 27, 2005
Abbreviations: CO, Carbon monoxide; HO-1, heme oxygenase-1; IEQ, islet equivalent; iNOS, inducible nitric oxide synthase; MCP-1, monocyte chemoattractant protein-1; MIF, macrophage migration inhibitory factor; NO, nitric oxide; STZ, streptozotocin.
Accepted for publication October 17, 2005.
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