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* Departments of Surgery and
Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA and
Dipartimento di Patologia animale, Universita degli Studi di Milano, Italy
1Correspondence: Beth Israel Deaconess Medical Center, Harvard Medical School, 99 Brookline Ave., Boston, MA 02215 USA. E-mail: hwang3{at}bidmc.harvard.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: islet transplantation • allograft survival
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. Immunosuppressive reagents used so far have many toxic side effects (3
4
5)
. Our goal is to minimize immune rejection by inducing the expression of HO-1, an enzyme that degrades heme, or administering the products of heme degradation to the donors, islets during their preparation and/or the recipients. We have shown that treatment of only the donor (HO-1 induction or administration of carbon monoxide or biliverdin or bilirubin), incubating islets in a CO-saturated solution during preparation or for longer periods of time, or treating only the recipient, frequently led to long-term survival (>100 days) and tolerance in B6AF1 recipients of DBA/2 islets (6
, 7)
. The mechanisms of such protection are not fully understood except that we know donor treatment suppresses inflammation in the islets after transplantation and presumably thus the protection of the islet allograft (the "donor" effect). In this study, we evaluated whether similar treatments prolong survival of islets in a stronger immunogenetic combination, i.e., BALB/c to C57BL/6. In addition, experiments were included to understand the mechanisms of any protection noted in addition to the donor effect.
CD4+CD25+ Tregs are a subset of T cells derived from the thymus that play a critical role in inducing and maintaining immunological tolerance to both self and foreign antigens by suppressing the aggressive T cell response (8
, 9)
. Several studies have demonstrated that CD4+CD25+ T cells are capable of prolonging allograft survival (10
11
12
13
14
15
16)
. Suppressive Treg cells express high level of CD25 (17)
, cytotoxic T-lymphocyte antigen 4 (CTLA-4) (18
, 19)
, glucocorticoid-induced tumor necrosis factor receptor (GITR) (20
, 21)
. The unique marker that characterizes the Tregs is the transcription factor Foxp3 (22
, 23)
. Foxp3, which encodes the transcription factor scurfin, is indispensable for the development and function of CD4+CD25+ Tregs (24)
. The mechanisms responsible for Treg cell-mediated inhibition of the immune response are still not clear. Cytokines such as IL-4, IL-10, and TGF-ß may mediate the suppressive effect of Tregs (25
26
27
28)
.
Studies show that HO-1 is engaged in Foxp3-mediated immune suppression (24
, 29)
. However, whether expression of HO-1 is essential for the development, maintenance, and function of Tregs is still controversial (30)
. Our own findings demonstrated that the tolerogenic effect of HO-1 plus DST in a cardiac transplantation model was dependent on CD4+CD25+ Tregs (31)
. In this study, we tested whether treatment of the donor, islets, and the recipient by induction of HO-1 or administration of the products of heme degradation can protect BALB/c islet grafts in C57BL/6 recipients from immune rejection by promoting Treg cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Islet isolation and transplantation
Pancreas was perfused with collagenase V at 0.8 mg/ml and digested for 14 min at 37°C. Islets were separated using density gradient centrifugation. Islet purity was assessed by dithizone (Sigma-Aldrich, St. Louis, MO, USA) staining after isolation. An algorithm was used for the calculation of the 150-µm diameter islet equivalent (IEQ) number (32
, 33)
. Islet-cell viability was assessed using fluorescence staining with acridine orange and propidium iodide (Sigma) (34)
. Recipients were rendered diabetic using streptozotocin (STZ, 225 mg/kg, i.p.; Sigma). Five days after STZ administration, mice with two consecutive blood glucose levels exceeding 350 mg/dl were used as recipients. Islets (500–550 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) following transplantation. Animals with a blood glucose <200 mg/dl were considered normoglycemic. Grafts were deemed rejected when two consecutive glucose levels were >300 mg/dl after a period of primary graft function.
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 (BALB/c) were transplanted under the other kidney capsule with no further treatments. If those second transplanted islets also survived >100 days, the recipients were considered tolerant. Antigen-specific tolerance was assessed by transplanting islets from a third-party strain (DBA/1) that does not share either class I or class II antigens with the original donor.
Real-time RT-PCR analysis
Total RNA was extracted using Qiagen RNA kit (Qiagen, Charworth, CA, USA). DNase treatment was performed according to the manufacturer’s suggestion to avoid the contamination by genomic DNA. 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 (6)
.
CoPP, bilirubin, and CO treatments
Cobalt protoporphyrin-IX (CoPP, Frontier Scientific, Logan, UT) was used to induce HO-1 expression in the donor. CoPP was dissolved in 0.1 M sodium hydroxide and the pH was adjusted to 7.4 with hydrochloride acid. Treatment involved a single dose of CoPP at 20 mg/kg given to the donor at 24 h before harvesting the islets. CO exposure to the donor was performed in a chamber containing 250 parts per million (ppm) CO for 2 h before islet harvest and to the recipient for 1 h per day from day –1 until day 13 following transplantation. Freshly isolated islets were cultured in a CO chamber that contains 1% CO and 5% CO2 at 37°C for 3 h before transplantation. Islets cultured in 5% CO2 without CO were transplanted at the same time and used as a control. Bilirubin at 20 µmol/kg was given to the donor at 1 h before islet isolation or to the recipient at 20, 50, or 100 µmol/kg twice per day from day –1 until day 13. "Saturation" of the medium was achieved by bubbling 1% CO into the medium for 10 min at room temperature before the islets were placed into the medium.
HO-1 activity assay
HO-1 activity was measured by bilirubin generation as described (35)
. At 24 h after CoPP injection, pancreata were collected from the mice. Tissues were homogenized in MgCl2 (2 mM) phosphate buffer (100 mM, pH 7.4) and sonicated on ice before centrifugation at 4000 rpm for 10 min at 4°C. Protein concentration in the supernatant was measured. Bilirubin was extracted from 1 mg of protein with chloroform, and its concentration was determined spectrophotometrically by the difference in absorption between 464 and 530 nm with an absorption coefficient of 40 mM/cm.
Mixed lymphocyte reaction
Spleen cells were harvested from BALB/c and C3H/HeOuJ mice. After depletion of T cells using anti-CD4/CD8 beads (Miltenyi Biotec, Auburn, CA, USA), cells were seeded in round-bottomed 96-well plates at 8 x 104/well and treated with mitomycin C (Sigma, 50 µg/ml) for 30 min to be used as stimulators. Naive and tolerant CD4+ cells from spleens of C57BL/6 control and tolerant mice were obtained by FACS-sorting and used as responders (8x104) in mixed lymphocyte reaction (MLR). Cells were pulsed with [3H] methylthymidine (0.5 µCi/well; NEN) and harvested 4 days after. Incorporated radioactivity of triplicate wells was measured using a liquid scintillation counter.
Immunohistochemistry
Islet grafts, including a portion of the kidney, were harvested at 3 and 7 days post transplantation and snap frozen in liquid nitrogen for immunohistological staining. Anti-mouse Foxp3 antibody (eBioscience, San Diego, CA, USA) was used to detect the expression of Foxp3. Secondary antibody was FITC labeled anti-mouse antibody (Vector Laboratories, Burlingame, CA, USA). Costaining with an anti-insulin antibody on the same section was performed to localize the transplanted grafts. DAPI staining was used to identify the cell nuclei. Six random areas of interest (400x400 pixel area) were selected from each tissue section; total numbers of cells, as well as Foxp3-positive cells, were counted and the percentage of Foxp3-positive cells were calculated.
CD4+CD25+ cells depletion and FACS analysis
CD4+CD25+ cells depletion was carried out in the recipient by using an anti-CD25 antibody given at day –6 and –1 before islet transplantation (250 µg/mouse i.p.; Bioexpress, West Lebanon, NH, USA). Depletion of CD4+CD25+ cells was confirmed by FACS analysis at day 1 of transplantation using the anti-CD25-PE antibody. Briefly, single cell suppressions (10x106/ml) were prepared from splenocytes and lymph nodes of mice treated with the anti-CD25 antibody or not treated. Ten million cells were stained with the anti-CD25-PE antibody (BD Biosciences, San Jose, CA, USA) for 15 min at room temperature. Depletion of CD4+CD25+ cells was analyzed in FACSort with Cellquest software (BD Biosciences).
Statistical analyses
Kaplan-Meier survival curves were performed using the Statview software, and the statistical differences were assessed by the Log-rank test. Values of P < 0.05 were considered significant. Survival data are expressed as mean survival time ± SD (MST ±SD). Differences between cytokine expressions were compared for statistical significance by the Mann-Whitney U test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B). Treatment of only the donor with induction of HO-1 with 20 mg/kg CoPP given 24 h before islet isolation significantly prolonged islet graft survival as compared to the control group. The mean graft survival time was 40.6 ± 10.4 days (Fig. 1B
). A single injection of CoPP at 20 mg/kg has been shown to efficiently induce HO-1 expression in the pancreatic islets for up to 6 days (6)
. HO-1 activity was also significantly increased in pancreata from CoPP-treated mice compared to those from controls (Fig. 1A
). Similar results were obtained when bilirubin was administered to the islet donor 1 h before islet isolation at a dose of 20 µmol/kg. Islets grafts survived for 31.33 ± 16.58 days (n=6, P=0.04 vs. control, Fig. 1B
). Administering CO at 250 ppm to the donor for 2 h before islet isolation also prolonged islet graft survival, although the difference between the CO-treated group and the control was not significant (P=0.059 vs. control, n=9). Nonetheless, one graft from CO-treated donors survived indefinitely in the recipient (Fig. 1B
).
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Our previous study showed that culturing islets in a "CO-saturated" medium prolonged islet allograft survival in a DBA/2 to B6AF1 combination (6)
. To test whether there would be an effect in a stronger immunogenetic combination, we isolated BALB/c islet in the CO-saturated solution and cultured those islets in 1% CO, 5% CO2 for 3 h before transplanting them into C57BL/6 recipients rendered diabetic by STZ. The mean survival time of those grafts was 31.4 ± 13.89 days, which is significantly longer than the control (n=5, P=0.034 vs. control, Fig. 2
).
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We then tested whether combining treatment of CO exposure and bilirubin administration to the recipient would promote islet allograft survival. Recipients were exposed to 250 ppm CO for 1 h every day, from day –1 until day 13 following transplantation, at the same time bilirubin was given twice per day at different concentrations (20 µmol/kg, 50 µmol/kg, and 100 µmol/kg). Administering 250 ppm CO plus 100 µmol/kg bilirubin to recipients led to graft rejecting in 24.5 ± 5.45 days (n=6), which is not significantly different than the control (data not shown). Treatment with CO at 250 ppm plus bilirubin at 50 µmol/kg led to one graft surviving long-term and the other four surviving up to 37.5 ± 17.23 days (n=5, data not shown). The best results were obtained when the recipient was treated with bilirubin at 20 µmol/kg, while exposing to 250 ppm CO. In 7 animals treated, 3 survived >100 days, two grafts were rejected at 22 and 27 days, while the other 2 animals died at 31 and 41 days with normoglycemia (n=7, Fig. 2
).
We tested whether better results could be obtained when both donor and recipient were treated than with treatment of only one or the other. Exposing the donor to 250 ppm CO for 2 h before islet harvesting, plus to the recipient for 1 h every day from day –1 until day 13 did not prolong islet graft survival: grafts were rejected in 26 ± 5.03 days (n=4, P=0.3 vs. control). However, islet grafts survived significantly longer when donors were exposed to 250 ppm CO for 2 h before islet isolation, and recipients received bilirubin at 20 µmol/kg from day –1 until day 13: two of 6 islet grafts survived >100 days, while the others were rejected in 32.75 ± 19.18 days (n=6, P=0.01 vs. control, Fig. 2
).
The best results were obtained when the donor, the islets ex vivo, and the recipient were all treated. By combining the donor, islets, and the recipient treatment, we developed the following protocol (which is referred to as the combined protocol in the text). First, to induce HO-1 expression in the donor at day –1 before islet isolation with 20 mg/kg CoPP, plus exposing the donor to 250 ppm CO for 2 h before harvesting the islets. Second, islets were isolated in the "CO-saturated" solution and cultured in 1% CO and 5% CO2 at 37°C for 3 h. Third, recipients were exposed to 250 ppm CO for 1 h every day, and in addition received 20 µmol/kg bilirubin twice per day from day –1 until day 13. This treatment led to prolonged islet survival in 5/5 animals: 3 animals survived >100 days, while the other two were rejected at 40 and 82 days, which is still significantly longer than the controls (Fig. 2)
.
To test whether recipients carrying long-term surviving grafts were antigen specifically tolerant to the transplanted grafts, we transplanted a second graft either syngeneic with the first graft (BALB/c) or a third party graft (DBA/1) after removing the initial islets. As evident in Table 1
, 2nd grafts from the same strain survived >100 days without any further treatment (n=3), while grafts from the third party were rejected in 19.3 ± 1.9 days (n=3).
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A high level of Foxp3 and TGF-ß expression was detected in grafts surviving long term in the recipient
Expression of Foxp3, IL-10, and TGF-ß in islet grafts, as well as in spleens of recipients carrying long-term surviving grafts was measured by real-time RT-PCR. Islets and spleens from nontreated C57BL/6 mice were used as controls. GAPDH expression was measured and used to normalize the amount of total RNA in each sample. In recipients carrying long-term surviving grafts there was a significantly higher expression of Foxp3 in islets and spleens (Fig. 3
A), a much higher level of TGF-ß (detected in the islet grafts but not in the spleens, Fig. 3B
), and high level of IL-10 in the spleens but not in the islets (Fig. 3C
).
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The combined protocol renders T cells in a donor-specific tolerant state in treated animals
To determine whether T cells from mice treated with the combined protocol are hyporesponsive to donor antigens, we performed MLR in vitro. CD4+ T cells from naive and tolerant C57BL/6 mice were isolated by FACS-sorting and stimulated with mitomycin C-treated, T cell-depleted, BALB/c or C3H/HeOuJ splenocytes. CD4+ cells from tolerant animals responded poorly to donor (BALB/c) splenocytes (P<0.05), but similarly as compared to naive CD4+, T cells to the third party (C3H/HeOuJ) splenocytes (P>0.05, Fig. 4
A). IL-2 mRNA measured by RT-PCR in cells from the MLR showed a similar trend (Fig. 4B
). Thus, our combined protocol renders T cells in a donor-specific tolerant state in treated animals.
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The combined protocol promotes migration of recipient-derived Foxp3+ Treg cells to the site of the transplanted islets
To understand whether the combined treatment protocol induces islet long-term survival and tolerance by promoting Treg cells, we transplanted BALB/c islets into C57BL/6 mice treated with the combined protocol and analyzed the presence of Foxp3+ cells at 3 and 7 days following transplantation. Expression of Foxp3 in grafts from control mice (Fig. 5
A), and mice treated with the combined protocol (Fig. 5C
) was detected with an anti-Foxp3 antibody. Islet grafts were also stained with an anti-insulin antibody. Overlapping images indicate that 8.38 ± 2.31% of 568 cells counted (identified by nuclear staining with DAPI) stained positive for Foxp3 in grafts from treated animals (n=3, Fig. 5D
) as compared to 4.28 ± 1.07% positive cells in grafts from control animals (523 cells in total, n=3, P=0.015 vs. control, Fig. 5B
) at 7 days following transplantation.
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These data suggest that our treatment protocol promotes the migration or de novo generation of recipient-derived Treg cells to the site of transplanted grafts. The presence of Treg cells may inhibit the activity of allo-aggressive T cells and protect transplanted grafts from immune rejection following transplantation.
CD4+CD25+cells are essential for the protective effect of the combined treatment protocol
To test whether CD4+CD25+ cells are essential for islet graft survival following transplantation using the combined protocol, we transplanted BALB/c islets to recipients predepleted of CD4+CD25+ cells. Depletion of CD4+CD25+ cells by giving recipients anti-CD25 antibody was confirmed by FACS analysis on day 1 post-transplantation: 0.43 ± 0.14% of CD4+CD25+ cells remained in the spleen of animals treated with the anti-CD25 antibody compared to 4.79% ± 0.26 in spleens of nontreated animals (Fig. 6
A). BALB/c islets were rejected in 20.4 ± 2.3 days after being transplanted to CD25-depleted C57BL/6 recipients (n=5). Grafts treated with the combined protocol were rejected in 23.8 ± 2.5 days (n=5, P=0.06 vs. control) in those recipients, i.e., no prolongation of graft survival was observed (Fig. 6B
), suggesting that CD4+CD25+ Tregs are essential for the long-term survival and tolerance induction using the combined treatment protocol.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Protocols that have attempted to achieve successful transplantation have relied primarily on treatment of the recipient with immunosuppressive therapy (4)
. We have taken a somewhat different approach and have shown that treatment of only the donor with either cobalt protoporphyrin to induce HO-1 or administration of carbon monoxide or bilirubin leads to prolonged survival in the untreated recipient. Further, those recipients carrying long-term surviving islet allografts became antigen specifically tolerant in that a second transplant from a donor syngeneic with the original donor survived indefinitely without further treatment, whereas third-party islets were promptly rejected.
These studies just discussed were primarily done in donor-recipient combinations with a relatively weak immunogenetic disparity. In this study, we extended our results to a strong disparity: BALB/c to C57BL/6. The frequency with which islets survived long term after treatment of only the donor, only the recipient or only the islets during the isolation procedure was less than in the weaker combination. However, as in the earlier studies, each of the three treatments alone confirmed our earlier findings that a significant percentage of islets do survive long-term with any one of these three treatments. Also as previously found by other groups and us, the combination of the three treatments was more effective than any one treatment alone (6
, 37)
.
Our main goal in the present studies was to obtain further information about the underlying mechanisms accounting for the long-term survival of the islets and the induction of tolerance. We thus performed a series of studies in which we evaluated the potential role of T regulatory cells in the tolerance induced by increased HO-1 expression plus CO and bilirubin administration. We found that there was higher expression of Foxp3 in islets and spleens (Fig. 3A
), a much higher level of TGF-ß (detected in the islet grafts but not in the spleens, Fig. 3B
), and high levels of IL-10 in the spleen but not in the islets (Fig. 3C
). These findings were accompanied by a reduced antigen-specific response of T cells of tolerant recipients against antigens of the donor strain but not antigens of a third-party strain.
There are studies that relate Tregs, Foxp3, and HO-1. HO-1 is involved with Foxp3 mediated immune suppression (12
, 29)
. We have shown that the ability of HO-1 plus donor specific transfusions (DST) to lead to tolerance is dependent on CD4+CD25+ Tregs (31)
. Further, in the tolerance-inducing protocol using anti-CD40L antibodies plus DST, tolerance induction, which is dependent on Tregs, is strictly dependent on HO-1 expression: hmox1–/– recipients do not develop tolerance under conditions that lead to tolerance in wild-type recipients. HO-1 induced in nonobese diabetic (NOD) mice enhances ß cell survival and moderates the diabetic state in those mice by decreasing infiltration of CD11c+ dendritic cells (38)
. In the present investigation, we studied whether the combined treatment (induction of HO-1 or administration of CO or bilirubin) of the donor, the islets and the recipient would protect BALB/c islet grafts in C57BL/6 recipients from immune rejection and lead to tolerance by promoting Tregs. Our data show clearly that the combined protocol led to the migration or de novo generation of Tregs, thereby presumably preventing rejection of those islets.
Last, to evaluate whether the Treg was responsible for the long-term survival induced by the combined protocol, we effectively predepleted CD4+CD25+ Treg cells from the recipient mice prior to transplantation. In the absence of the Treg, the same protocol that led to long-term survival failed significantly to prolong survival of the transplanted islets.
These studies extend the work of others and us (7
, 39)
. We show that the long-term survival and tolerance can be achieved in a strong immunogenetic combination. Further, the basis of long-term survival and tolerance gained by treatments with induced HO-1 or products of heme degradation are based on the preferential generation of Tregs. Not only do we see the markers of Tregs associated with these treatments, but we also trace the accumulation of Tregs in the islets after transplantation.
It would seem to us that a similar protocol used in humans might provide at least a supplementary treatment to try to achieve successful islet transplantation. Several of the components of the protocol used here are beginning to be used in humans.
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ACKNOWLEDGMENTS |
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Received for publication March 5, 2007. Accepted for publication May 3, 2007.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, n=5), administering bilirubin (
, n=6) significantly prolonged islet allograft survival. Exposing donors to CO also prolonged islet allograft survival (
, n=9).
, n=7), or CO to donors and bilirubin (20 µmol/kg) to recipients (•, n=6), significantly prolonged islet allograft survival and frequently led to long-term survival of islet allograft. Treating the donor, islets, and the recipient using the combined protocol led to most grafts surviving long term in the recipients (
