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Concise Review |
a Division of Immunogenetics, Department of Pediatrics, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania, USA;
b Mirus Corporation, Madison, Wisconsin, USA
Key Words. Autoimmune diabetes • Allogeneic chimerism • NOD mouse
Tatiana D. Zorina, M.D., Ph.D., Division of Immunogenetics, Department of Pediatrics, University of Pittsburgh, School of Medicine, 3460 Fifth Avenue, Rangos Research Center, Pittsburgh, Pennsylvania, USA 15213. Telephone: 412-692-5238; Fax: 412-692-5809; e-mail: tatiana{at}pitt.edu
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ABSTRACT |
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INTRODUCTION |
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To substitute parenteral insulin administration, transplantation of ß cells and adult, fetal, or expanded islets of Langerhans ex vivo lately has become one of the highly regarded approaches for therapy of T1D [1–6]. However, despite successful advancements in current clinical protocols for islet transplantation [7, 8], the high sensitivity of ß cells to immunosuppressive conditioning [9, 10], limited life span of transplanted allogeneic islets, and the even more prominent shortage of donors for pancreata remain major limitations to the broad clinical adaptation of these therapies.
To promote engraftment, bone marrow transplantation (BMT) from autoimmunity-free allogeneic donors in combination with donor BM major histocompatibility complex (MHC) class I-matched islets has been attempted in experimental [11, 12] and clinical [13, 14] settings. Moreover, the potential benefit of hematopoietic chimerism in the treatment of diabetes was found not to be restricted to its ability to provide tolerance in islet transplantation. In numerous reports, it has been demonstrated that transplantation of allogeneic BM from autoimmunity-free donors can arrest and abolish the autoimmune diabetogenic process in T1D prior to the clinical onset of the disease [15–20]. However, to date, chimerism per se (even disregarding the general problem of side effects associated with transplantation of immunocompetent tissue) cannot be considered as a therapeutic approach for T1D since, by the time of disease diagnosis, the number of remaining ß cells is no longer adequate to sustain euglycemia.
Based on the accumulating data providing strong evidence that insulin-secreting tissue has a potential for reparative processes [21–28], we hypothesized that in T1D the eventual ß cell destruction could be regarded as a result of the defeat of the reparative processes in the endocrine pancreas in their struggle with autoimmune aggression. Accordingly, the hypothesis has been made that supporting endogenous ß cell recovery by restraining the autoimmune assault could become a condition adequate for the unhindered regeneration and/or recovery of the endogenous insulin-secreting tissue and become an alternative way to normalize glucose metabolism in T1D.
Generated data have proven the possibility of restoring autologous insulin regulation even when therapy is initiated after the clinical onset of the disease. This indicates that hemopoietic chimerism can be adapted as a condition adequate not only to arrest the autoimmune destruction of ß cells, but also to allow their functional recovery. To sustain euglycemia in the NOD mice after the clinical onset of overt diabetes until recovery of the endogenous insulin secretion occurred, in addition to the induction of allogeneic chimerism, donor BM MHC class I-matched islet transplantation was utilized. If similar patterns of ß cell recovery were shown in humans in the clinical protocol islet, transplantation would not be required since glycemia during the time required for the restoration of the autologous pancreas could be controlled by parenteral insulin administration.
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MATERIALS AND METHODS |
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Preparation of Allogeneic Chimerae
A basic protocol was adapted [29] and applied with slight modifications. Briefly, NOD recipient mice were irradiated with 700 cGy or 950 cGy of total body irradiation (TBI) and were reconstituted within 5 hours via i.v. injection of T-cell-depleted MHC-mismatched BM cells. For T-cell depletion, a rabbit-anti-mouse brain polyclonal serum (InterCell Technologies, Inc.; Somerville, NJ; http://www.intercell.com) and guinea pig complement (GIBCO; Grand Island, NY; http://www.lifetech.com) were used.
Islet of Langerhans Transplantation
Islets were harvested from euthanized B10 or B6-GFP mice and grafted into NOD mice previously transplanted with B10- or B6-GFP-derived BM, respectively. Pancreatic islet isolation was performed using a modified collagenase digestion procedure [30, 31]. Briefly, 3 ml of cold Hanks’ balanced salt solution containing 1.75 mg/ml collagenase (Sigma; St. Louis, MO; http://www.sigmaaldrich.com) were injected into the pancreatic duct. After pancreatectomy, the islets were purified from the digested pancreatic tissue by density gradient cell separation (Ficoll, 1.108, 1.096, 1.069, and 1.037 g/ml; Sigma). Approximately 300 islets were transplanted beneath the renal capsules of recipients under anesthesia induced by i.p. administration of 0.015–0.017 ml of 2.5% avertin per gram of body weight. Islet graft function was monitored by testing blood and urine glucose levels and by histological detection of insulitis and insulin-positive cells upon graft extirpation.
Urine and Blood Glucose Measurements
Urine was tested for glucose weekly with Keto-Diastix® (Baxter Healthcare; Elkhart, IN; http://www.baxter.com). A positive test for glucosuria was considered symptomatic for the onset of diabetes. A pen type glucometer (Precision QID; Medisense; Bedford, MA; http://www.medisense.com) was used to measure blood glucose. Mice were considered diabetic when a level of 300 mg/dl or above was obtained for three consecutive daily readings.
Characterization of Chimerae by Flow Cytometry
The level of chimerism assessed by flow cytometry was expressed as a percentage of peripheral blood leukocytes bearing donor or host MHC class I molecules (H-2Kb for B10 and B6-GFP or H-2Kd for NOD) after staining with directly labeled monoclonal antibodies (mAbs). To confirm the multilineage hematopoietic engraftment, two-color staining was carried out using anti-donor MHC class I and lineage-specific mAbs including myeloid cells (GR-1), T cells (
ßTCR), B cells (CD19), and dendritic cells (CD11c). Flow analysis was performed on a Becton Dickinson dual laser FACSCalibur (San Jose, CA; http://www.bd.com). All mAbs were purchased from BD PharMingen (San Diego, CA; http://www.bdbiosciences.com/pharmingen).
Islet Graft Removal
The islet-graft-bearing kidneys were surgically removed following the same anesthesia protocol as for islet transplantation, or excised from euthanized animals, routinely processed, and paraffin embedded.
Immunohistochemical Protocols
Intraperitoneally, 5-bromo-2'-deoxyuridine (BrDU; Sigma) was injected (50 mg/kg of body weight). One hour later, pancreata and graft-bearing kidneys were collected, routinely processed, and paraffin embedded or snap frozen in liquid nitrogen. Five-micron paraffin sections were stained with mouse anti-BrDU mAb (Amersham Biotech; Amersham, UK; http://www.apbiotech.com) and treated with biotinylated horse anti-mouse IgG (Vector Laboratories, Inc.; Burlingame, CA; http://www.vectorlabs.com). To visualize the secondary Ab, the ABC immunoperoxidase system (VECTASTAIN; Vector Laboratories) was used with chromogen 3-amino-9-ethylcarbazole (AEC; ScyTek Laboratories, Inc.; Logan, UT; http://www.scytek.com) for coloration. Two-color staining (for BrDU and insulin) was also performed to discriminate the proliferating ß cells from infiltrating mononuclear hematopoietic cells, and counterstained with hematoxylin. Insulin-positive cells were revealed with donkey anti-mouse anti-insulin alkaline phosphatase-labeled mAb (BioGenex Laboratory; San Ramon, CA; http://www.biogenex.com). Five-micron frozen sections were stained with rabbit-anti-Glut-2 Ab (Santa Cruz Biotechnology; Santa Cruz, CA; http://www.scbt.com) and treated with biotinylated goat anti-rabbit (ScyTek) secondary Ab. In some experiments, to reveal insulin-positive cell sections, paraffin-embedded specimens were stained with guinea pig anti-insulin Ab (one-color staining; Zymed Labs; South San Francisco, CA; http://www.zymed.com) and biotinylated goat anti-guinea pig secondary Ab that was visualized with streptavidin/horseradish peroxidase (both purchased from ScyTek). Additional sections were stained with either rabbit anti-somatostatin Ab (Dako Corporation; Carpinteria, CA; http://www.dako.dk), rabbit anti-glucagon Ab (Zymed Labs), or rabbit anti-pancreatic polypeptide Ab (Lab Vision; Fremont, CA; http://www.labvision.com) and treated with biotinylated goat anti-rabbit secondary Ab (ScyTek). To visualize biotinylated secondary Ab, AEC chromogen/substrate solution (ScyTek) was applied.
Immunofluorescence
Pancreata were fixed in 2% paraformaldehyde, washed in phosphate-buffered saline and frozen in Optimal Cutting Temperature (OCT) compound (Sakura, Finetek USA; Torrance, CA; http://www.sakuraus.com). Sections were blocked with donkey serum and incubated with anti-insulin Ab (DAKO), followed by indocarbocyanine (Jackson ImmunoResearch Laboratories, Inc.; West Grove, PA; http://www.jacksonimmuno.com) and a Hoechst 33342 nuclear stain (Molecular Probes; Eugene, OR; http://www.probes.com). Sections were kept in the dark until fluorescent microscopic examination. Images were acquired using Olympus Magnafire image analysis software.
Evaluation of the Autoimmune Damage to Pancreatic Islets
Morphometric assessment of islets was performed in the paraffin sections of pancreata based on the evaluation of Index N, as earlier reported [20]. Briefly, Index N was estimated as a ratio of the insulitis score and the parameter "A" (N = insulitis score/A), where parameter A quantifies the fraction of unaffected endocrine pancreas: A = area of pathology-free islets/entire pancreas. This new Index N was introduced because in chimeric animals, insulitis is eventually cleared, and alone this characteristic is not adequate for assessment of islet condition. Insulitis was characterized according to a scoring system with grades from 0 to 6. To estimate the value of parameter A, each pancreas was evaluated using 3–6 hematoxylin/eosin (H&E)-stained sections obtained 50 µm apart. The slides were examined using an optical grid with 314 divisions per field. At a magnification of 200x, each division represented 1 mm2 of tissue. The area occupied by islets that were free of insulitis and apoptosis was summed over 20 fields and averaged over three replicate assessments. In a few specimens, serial sections of pancreata were stained with H&E and with anti-insulin mAb to verify our judgment of ß cell mass as "normal" in H&E preparations. The t-test for independent samples was used to determine statistical significance of our comparisons. A curve of the age-related kinetics of islet damage in unmanipulated NOD mice was used as a control. To confirm the analysis based on Index N, pancreata were also tested by an enzyme-linked immunosorbent assay (ELISA; ALPCO; Windham, NH; http://www.alpco.com) for the direct measurement of insulin content, as described elsewhere [32].
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RESULTS |
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). Animals were monitored for weight loss, skin lesions, diarrhea, and hyperglycemia as the clinical manifestations of graft-versus-host disease (GVHD) and diabetes. None of these animals displayed clinical disease features, which was confirmed by histological assessments of the livers and pancreata (for GVHD and insulitis, respectively) harvested upon the completion of experiments. Absence of the features of GVHD was expected since it was intentionally precluded by the transplantation of T cell-depleted BM in all experiments.
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) or nonlethal (Fig. 2B) doses of TBI, an arrest of the destructive processes and total normalization of Index N were observed. The difference between Index N in the control (N = 0.09 ± 0.004) and all three chimeric groups (transplanted with allogeneic BM at 8, 10, and 12 weeks of age; N = 0.01 ± 0.003) was shown to be statistically significant (p < 0.001) using one-way analysis of variance. Once normalized, Index N remained at a plateau level up to 14 weeks (length of observation), confirming that normalization of the structure and function of the insulin-secreting tissue in the endogenous pancreata of chimeric NOD mice was stable and long lasting. Based on these data, a hypothesis was made that, as indicated by normalized Index N, the insulin-producing tissue of the endogenous pancreas had undergone a reparative process.
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). Age-matched unmanipulated female NOD mice (data not shown) and mice that underwent the BMT procedure but did not develop a detectable chimerism, developed a high degree of insulitis and showed a significant decrease in the unaffected ß cell mass (Index N > 0.09), resulting in the onset of diabetes. These mice were compared with chimeric animals divided into groups based on achieved levels of chimerism: <1%; 1%–3%; 9%–15%, and 45%–55%. As described elsewhere [20], there was no direct correlation between doses of the transplanted BM and the resulting levels of chimerism. However, once established, donor chimerism ranging from 1%–55% in the recipient peripheral blood was shown to be equally sufficient to abolish the autoimmune damage of insulin-secreting tissue in all chimeric animals. The difference in islet destruction between chimeric (Index N = 0.0054 ± 0.00315) and nonchimeric animals (N = 0.09 ± 0.0066) was statistically significant (p < 0.001).
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To detect proliferating cells, animals were injected with BrDU prior to euthanization. To recognize dividing insulin-secreting cells, double staining with anti-insulin and anti-BrDU mAbs was performed. In accordance with earlier reports [25–27], we found proliferating ß and duct cells. The photomicrographs representative of all samples studied showed proliferating (i.e., BrDU positive) ß cells (Fig. 4A) and duct cells (Fig. 4B) in the pancreata of B10 mice. Double staining revealed a regenerative capacity of ß cells in prediabetic NOD mice undergoing different stages of insulitis as well (Fig. 4C and D). In all randomly chosen BM/islet recipients (n = 9), cells double positive for insulin and BrDU were also identified in the islet allografts (Fig. 4E and F).
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Mice that received islet grafts became euglycemic within 24 hours following the transplantation procedure and remained so for the length of observation. In animals that received parenteral insulin administration, true euglycemia was not sustained. Although 1–3 units of humulin per day were injected s.c., the control of glycemia was limited to 300–350 mg/dl. After surgical removal of islet-graft-bearing kidneys performed at 17–26 weeks after islet transplantation, all mice remained euglycemic for up to 18 days following nephrectomy (length of observation). Two mice that received insulin injections instead of islet transplants no longer required exogenous insulin by 16 and 17 weeks after induction of chimerism, and remained euglycemic for 16 additional weeks (length of observation). Direct assessment of the insulin content in the endogenous islets was performed by histological evaluation of pancreata harvested from euthanized animals upon termination of the experiments. Insulin-positive ß cells in these pancreata collected from mice about 5 months after the clinical onset of the disease (including those that did not undergo islet transplantation) were found in quantities and morphologies similar to those of the normal mouse pancreas (Fig. 5).
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In contrast, in mice subjected to syngeneic T-cell-depleted BM but not islet transplantation or exogenous insulin administration (n = 5), the therapeutic effect was not achieved. These animals and those that received radiation only (n = 4) without BMT progressed to a diseased condition, and all animals were euthanized 10–14 days after either procedure (data not shown).
ß Cell Regeneration or Functional Recovery?
Beta cell destruction is an intrinsic feature of autoimmune diabetes. Immunohistological staining of pancreatic specimens from diabetic NOD mice 3 weeks after being rendered chimeric showed very few to no insulin-positive cells, suggesting that destruction of ß cells in our experimental setting occurred as well (Fig. 6, bottom panel, middle microphotograph). These data were supported by the direct ELISA measurement of the insulin content of pancreatic extracts that showed equally low (>1% of the normal values) insulin in chimeric (3 weeks after induction of chimerism) and unmanipulated diabetic NOD mice (Table 1). However, to rule out the possibility that, in our model, at the time when the treatment of the diabetic animals was initiated, ß cells were functionally silent rather than destroyed, an additional series of histological evaluations of the pancreata from such animals was performed. Five spontaneously diabetic NOD mice were conditioned with 700 cGy of TBI, transplanted with 40 x 106 T-cell-depleted, B10-derived BM cells, and the next day grafted with B10-derived islets. Three weeks later, endogenous pancreata were collected for immunohistological evaluation. Sections were obtained throughout the whole organ of each pancreatic specimen. Compared with normal islets (Fig. 7A), these islets (Fig. 7B) were significantly reduced in size and altered in shape. Immunostaining for insulin (Fig. 7D) and Glut-2 (specific ß cell marker; Fig. 7F) was negative in all specimens examined. Cells positive for glucagon, somatostatin, and pancreatic polypeptide were still present in these islets (Fig. 7H, 7J, and 7L, respectively), although their distribution was altered compared with normal islets (Fig. 7G, 7I, and 7K). Perhaps due to the absence of ß cells that normally compose the large, inner part of the islet, somatostatin, glucagons, and pancreatic polypeptide-producing cells, normally localized on the periphery of islet, formed small dense conglomerates.
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DISCUSSION |
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To quantitate reparative processes in the insulin-secreting tissue in the preclinical stage of the disease, revealed in mouse pancreata by us and others [15–19], we introduced a new morphometric parameter, Index N [20]. This new parameter was needed because insulitis was cleared in diabetic NOD mice after induction of chimerism and could no longer be used as an adequate parameter to characterize the endocrine pancreas. We have shown, using protocols with either lethal or nonlethal doses of TBI used for recipient conditioning, that BMT performed at 8, 10, or 12 weeks in prediabetic NOD mice led to normalization of insulin-producing tissue within 14 weeks after chimerae preparation.
Experiments designated to elucidate whether the same pattern of reparative processes in the endocrine pancreas could be elicited in overtly diabetic animals have demonstrated that about the same time span (14–17 weeks) was required to achieve insulin independence in animals that were rendered hematopoietic chimerae after onset of diabetes. Functional recovery of the endogenous pancreata in the antea-diabetic mice was confirmed by the persistence of euglycemia in the chimeric mice after removal of the islets grafted into these animals to control glycemia for the time required for the reparative process to occur. Subsequent histological examination of endogenous pancreata revealed patterns and distribution of the insulin-positive cells similar to those of normal pancreata. Preliminary data showed that islet transplantation was not an obligatory element of this therapy. Even though true euglycemia was not sustained in the chimeric animals supported by parenteral insulin administration through the time required for the restoration of pancreata destroyed by the diabetogenic processes, a full recovery of endogenous insulin supply was demonstrated to be similar to mice that were transplanted with donor BM MHC class I-matched islets, and, for the length of the experiment, they were truly euglycemic.
An assumption was made that the observed restoration of the insulin homeostasis in the antea-diabetic mice was due to the regenerative processes in the endocrine pancreas. The neogenesis of ß cells may not necessarily be the only process underlying the observed functional recovery of the endocrine pancreas after induction of chimerism. However, immunostaining for insulin and Glut-2 of the specimens from the diabetic, rendered chimeric, NOD mice (at the beginning of therapy, prior to the occurrence of reparative processes) showed very little to no positive staining, thus indicating a high probability for extensive physical loss of ß cells as opposed to solely failure of their function. Cells positive for glucagon, somatostatin, and pancreatic polypeptide were still present in these islets, indicating the selective destruction of ß cells. These data thus support our assumption that recovery of insulin independence in our chimeric model was the result of ß cell regeneration rather than their recovery from a nonfunctional state. Certainly, further study is needed to ultimately define the role of all mechanisms, such as recovery of preexisting salvaged ß cells, repopulation of a ß-cell subset due to regenerative potential of the ß-cell precursors (duct cells? stem cells?), and the ability of the mature ß cells to replicate, possibly contributing to the observed phenomenon of the endocrine pancreas restoration. According to data generated to date, ß cells double positive for insulin and GFP were not found, thus indicating that functional ß cells originated from the endogenous tissues rather than from transplanted BM. However, in light of the recent report that autologous BM harbors cells capable of differentiation into functionally competent ß cells [36], a possible contribution of donor-derived HSCs to islet recovery will be further explored in future experiments.
It is worthy to note that in NOD mice, unlike the autoimmunity-free strains, as we and others [20, 36] have demonstrated, a gradual replacement of endogenous hematopoietic tissue by donor-derived hematopoiesis during the recipient’s life span was observed. The observation that the level of allochimerism in NOD recipients significantly increases over time makes our finding that as low as 1% of chimerism is sufficient to abrogate diabetogenesis clinically relevant: induction of a low level of chimerism implies milder conditioning required for the allogeneic HSC engraftment, which is a vital benefit from a clinical perspective.
Clinical observations have shown that inhibiting autoimmunity by immunosuppressive therapies does not allow native cell regeneration in long-lasting diabetics, since otherwise diabetic kidney-alone recipients over time would become nondiabetic. This is fully consistent with the understanding of the basic mechanisms mediating immunosuppression; they suppress mitotic activity, and hence provide a negative effect on any cell proliferation, which is a substrate of the regenerative processes. This is one of many reasons why an approach for therapy of diabetes that does not include general immunosuppression needs to be found. Based on our findings, we can state now that the negative effects of immunosuppression in diabetic patients very likely go beyond the general disadvantages, such as compromised general immunity, increased risk for malignancies, and toxicity to ß cells and kidneys, but it also precludes recovery of the autologous insulin supply, even though some amelioration of the autoimmunity can be achieved. Thus, it is not only the waning of autoimmunity that is a major element of our therapy, but the way in which it was achieved.
A future direction of this study is to elucidate the immunomodulatory mechanisms allowing/mediating recovery of the autologous insulin secretion after the clinical onset of diabetes. The possible contributions of both central and peripheral tolerance will be addressed. The understanding of the machinery responsible for the observed phenomenon of the reversibility of diabetogenesis in our model will help to better understand the biology of the immunomodulatory processes that limit autoimmunity in allogeneic chimerae in general.
In summary, a potential for restoration of endogenous insulin regulation was shown in diabetic NOD mice. In light of this finding, a new challenge to cure diabetes solely by abolishment of autoimmunity arises. Future studies are needed to elucidate whether the same patterns of the reparative processes observed in the mouse model could be triggered in the human endocrine pancreas, and how long after disease onset the reparative process can be induced. Once these parameters are defined, BMT and other approaches to impede autoimmunity already described [37–41], or yet to be found, may become new therapies for T1D.
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ACKNOWLEDGMENT |
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REFERENCES |
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