1 Division of Immunogenetics, Department of Pediatrics, University of Pittsburgh School of Medicine, Rangos Research Center, Childrens Hospital of Pittsburgh, Pittsburgh, Pennsylvania
2 Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania
3 Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
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ABSTRACT |
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Islet transplantation for young patients with type 1 diabetes is not currently considered as an option to replace the traditional therapy of daily insulin injections. The risks associated with chronic diabetes (managed with insulin therapy) are certainly less damaging for the patient than the risks associated with the chronic use of the immunosuppressive drugs necessary to prevent transplant rejection. Even with the success of the nonsteroidal antirejection drug regimen of the Edmonton protocol (1), islet transplantation is still considered only for adult patients who have a history of complications such as end stage renal failure; those who already have or are receiving another transplanted organ; or those who, despite strict adherence to insulin therapy, cannot adequately regulate their blood glucose levels (2).
If a way can be found to circumvent the need for a chronic immunosuppressive treatment while preserving donor islet function, then the relatively low risk and generally uncomplicated surgical procedure required to transplant allogeneic islets could become the therapy of choice for young patients with newly diagnosed diabetes. Islet replacement may also help curtail the development of long-term complications associated with the disease (3).
Tissue-specific gene therapy may provide a way to protect islet grafts from the host environment without systemic immunosuppression. Genes may be transferred ex vivo to the donor islets before transplantation to provide protection from the hosts immune response in the microenvironment surrounding the graft (4).
Antioxidant genes are not highly expressed in pancreatic islets compared with other tissues of the body (5,6). For this reason, islets may be particularly susceptible to cytotoxic superoxide radicals generated during the inflammatory immune response that promotes type 1 diabetes onset or occurs to some degree with any transplanted tissue. Isolated islets should be protected from oxidative stress during and after transplantation. This could lessen the early graft loss as a result of ischemia/reperfusion injury and nonspecific inflammation (7,8). MnSOD (or SOD2) is a nuclear encoded mitochondrial antioxidant enzyme that is expressed in response to the inflammatory cytokines interleukin (IL)-1ß (9,10), -interferon (IFN-
) (11,12), and tumor necrosis factor-
(TNF-
) (13). Previous studies have shown that overexpression of MnSOD protects insulinoma and other tumor cell types from IL-1ß damage in vitro (14,15).
This study was designed to determine whether protecting islets from oxidative stress can prolong islet function in vivo after isolation and transplantation in an autoimmune animal model without the added complication of allorejection. The MnSOD antioxidant enzyme was overexpressed in islets isolated from young, prediabetic NOD mouse donors. These MnSOD overexpressing or control islets were transplanted into a syngeneic but immunocompromised mouse model (NODscid). Before transplantation, NODscid mice were rendered diabetic with streptozotocin (STZ). After transplantation and return of euglycemia, donor islets were challenged with spleen cells from spontaneously diabetic NOD mice. These challenged grafts were then followed until hyperglycemia returned. Our results show that the mice that received islet transplants overexpressing MnSOD were euglycemic an average of 50% longer than those that received either untreated islets or islets carrying the LacZ marker gene.
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RESEARCH DESIGN AND METHODS |
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Islet isolation and culture.
Mouse islets were isolated from the exocrine pancreas by collagenase digestion under sterile conditions. The procedure follows the classical previously published method (16) with slight modifications. After separation on a Ficoll gradient, the islets were further purified by hand picking to eliminate any remaining exocrine tissue. Whole islets were maintained in culture medium consisting of RPMI 1640 medium supplemented with 10% FCS, 20 mmol/l HEPES, 1% L-glutamine, 1% penicillin/streptomycin, and 50 µmol/l ß-mercaptoethanol (2-ME) at 37°C in an atmosphere of 95% air, 5% CO2. All culture reagents were purchased from Life Technologies (Grand Island, NY). Collagenase Type V, Ficoll type 400, and 2-ME were purchased from Sigma (St. Louis, MO).
Adenoviral vectors.
An E1- and E3-deleted, first-generation recombinant adenovirus (replication-deficient) was used as a vector in these experiments. Human MnSOD cDNA was cloned and sequenced previously (17), and its function was described by Wong et al. (18). The transgene was excised from the pRK5-MnSOD plasmid by digestion with EcoRI and PvuI (19). The resulting 700-bp fragment was then cloned into pAD.CMV Link that contains the CMV immediate early promotor/enhancer and an SV40 poly A site. Recombinant adenovirus was generated by co-transfection of NheI cut pAD plasmid with ClaI cut Ad5.sub360 (E1/E3-deleted) viral DNA (19) to insert the MnSOD cassette into the E1 region of the adenovirus. The adenovectors encoding the LacZ and GFP marker genes have been described previously (20).
Gene transfer to islets.
Islets were incubated with the Ad-MnSOD, Ad-LacZ, or Ad-GFP vectors in serum-free medium at a multiplicity of infection of 100 at room temperature for 1 h with agitation every 15 min. A multiplicity of infection of 100 was chosen based on dose-response experiments showing an infection rate of 7090% of the surface area of the islets without cytotoxic effects (21,22). After infection, islets were placed back in complete medium and incubated for 23 days before further analysis to allow for transgene expression.
Cell preparation and fluorescence-activated cell sorter analysis.
Two days after Ad-GFP infection, islets were dissociated to single cells with enzyme-free dissociation buffer (Gibco-BRL), washed, and placed in fluorescence-activated cell sorter (FACS) medium (Hanks balanced salt solution with 0.1% sodium azide and 0.5% BSA). They were then incubated with a Glut-2 mAb (Chromaprobe) for 20 min and then washed and fixed with 1% paraformaldehyde. Flow cytometry was done on a FACSCalibur flow cytometer (Becton Dickinson). Analysis was performed on groups of 200 islets isolated and infected on three separate occasions.
Antibody staining of MnSOD.
Whole islets infected with Ad-MnSOD were fixed in a 2% paraformaldehyde and permeabilized with 0.1% Triton-X solution. After blocking with goat serum, the primary rabbit polyclonal IgG MnSOD antibody (Upstate Biotechnology, Lake Placid, NY) was added for 2 h. A fluorophore-labeled secondary antibody, Alexa Fluor 488 goat anti-rabbit IgG (Molecular Probes, Eugene, OR), was added for a 1-h incubation protected from light. Hoechst 33342 (Molecular Probes) was used as a counter stain and applied for 20 min.
Multiphoton microscopy.
Islets were imaged using a multiphoton laser scanning confocal microscope comprising a titanium-sapphire ultrafast tunable laser system (Coherent Mira Model 900-F), Olympus Fluoview confocal scanning electronics, and an Olympus IX70 inverted microscope with custom-built power attenuation and external PMT detection systems. The three-dimensional multiphoton data stack acquisition used two-photon excitation at 800 nm (Hoechst 33342) and 870 nm (Alexa Fluor 488), with fluorescence emission detected respectively using D450/50 mol/l and HG510/50 steep bandpass emission filters (Chroma, Brattleboro, VT).
RT-PCR.
For RT-PCR, total RNA was extracted from whole islets using TRIzol reagent according to the manufacturers directions (Life Technologies). First-strand cDNA was synthesized from total RNA by RT reaction using the SuperScript Preamplification System (Life Technologies), and the reaction was run on a Perkin-Elmer GeneAmp PCR System 9600 thermocycler (Perkin-Elmer). Oligonucleotide primers were synthesized in house using an Applied Biosystems synthesizer, model 394-08. MnSOD primers are 25 mer each in length covering nucleotides 128152 for the upper primer and 388412 for the lower primer with sequence as follows: 5' primer-GCTGGCTCCGGCTTTGGGGTATCTG; 3' primer-GCTGAGGTTTGTCCAGAAAATGCTA. These primers amplify a section of the MnSOD gene that is specific for the human sequence and yields a PCR product of 284 bp. Primer sequences were obtained from Epperly et al. (19). As an internal control for islet RNA, the murine ß-actin gene was amplified with the primer sequences 5' primer-ATCCGTAAAGACCTCTATGC (nucleotides 945964) and 3' primer-AACGCAGCTCAGTAACAGTC (nucleotides 1,2121,231), yielding a 287 bp PCR product (23). The same PCR conditions were used for both MnSOD and ß-actin amplifications: one cycle of 94° for 3 min; 35 cycles of 94° for 30 s, 59° for 50 s, 72° for 90 s; and one cycle of 72° for 5 min. Each 25-µl reaction mixture contained 2.5 µl of 10x buffer, 1.5 µl of 25 mmol/l MgCl2, 0.5 µl of 10 mmol/l dNTPs, 0.5 µl of 10 pmol upper primer, 0.5 µl of 10 pmol lower primer, 0.25 µl Taq polymerase, 1 µl of cDNA, and 18.25 µl of distilled H2O. Amplification products were visualized by electrophoresis on a 1% agarose gel. Fragment size was confirmed using a 100-bp DNA ladder (Boehringer Mannheim, Mannheim, Germany).
Assessment of islet cell viability.
The assay that we chose to determine cellular damage of whole islets was based on a method by Liu et al. (24) in which viability was determined by a semiquantitative test using propidium iodide (PI), a vital dye that can permeate only cells with damaged membranes. The percentage of dead cells in the islet is then approximated by visual enumeration. PI was added to whole islets for 45 min before examination under a fluorescent microscope with a rhodamine (TRITC) filter. The number of dead or damaged (PI-positive) cells was counted by two independent observers blinded to islet group identity. In some instances, Hoechst 33342 was added before PI for 20 min as a counterstain. Because an average-size murine islet (150 µm in diameter) consists of
1,000 cells, an islet was considered nonviable when ≥500 cells (>50%) stained positive for PI. When an islet had <500 cells staining positive for PI, it was considered viable. An average of 35 islets were counted for each islet group in each independent set of experiments. Each set of experiments was performed in triplicate.
Glucose stimulation assay.
Whole islets were cultured in first low (3 mmol/l) and then high (20 mmol/l) concentrations of glucose in Krebs-Ringers Buffer (KRB). KRB consisted of 115 mmol/l NaCl, 24 mmol/l NaHCO3, 5 mmol/l KCl, 2.5 mmol/l CaCl2, 1 mmol/l MgCl2, 25 mmol/l HEPES, 0.1% BSA, and either 3 or 20 mmol/l glucose. The static incubation assay was performed in a 96-well flat-bottomed culture plate with 23 islets/well and 36 duplicate wells for each islet group. After a 30-min preincubation in 3 mmol/l KRB, supernatant from each well was collected after each 30-min incubation, and the concentration was measured using a mouse insulin enzyme-linked immunosorbent assay (ALPCO Diagnostics). This kit has a measurable range of 2507,500 ng/ml. ß-cell function was measured as the fold increase in insulin secretion when going from a low to high glucose concentration (21).
Cytotoxic exposure in vitro.
Islets from uninfected, Ad-LacZinfected, and Ad-MnSOD-infected groups (3040 islets per group) were exposed to three different agents in vitro: oxygen free radicals, nitric oxide (NO), and inflammatory cytokines. Each agent was tested in triplicate on independently infected islet groups for a total of 100 islets of each type tested per agent. For the first set of experiments, alloxan was used as an oxygen free radical generator (25,26). Alloxan was dissolved in a cold 0.01% HCl solution and used immediately after preparation. Islets were exposed to 4 mmol/l alloxan for 5 min at room temperature. The islets were then washed, placed into fresh complete medium, and incubated at 37°C for an additional 2 h before being examined for cellular damage. Second, the three islet groups were exposed to a generator of NO radicals, S-nitroso-N-acetylpenicillamine (SNAP). SNAP produces NO and disulfides as it degrades. Similar compounds such as SIN-1 and SNP were not used because they release products other than NO that also mediate cell death. SNAP was dissolved in DMSO to stabilize the compound before it was added to islet cultures. Islets were incubated with medium containing 5 mmol/l SNAP for 18 h at 37°C and then examined. The third test exposed the islet groups to inflammatory cytokines that have been reported to initiate events in vivo that may lead to ß-cell death. (27) On the basis of previous studies, islets were cultured with 50 units/ml recombinant mouse IL-1ß (Genzyme, Cambridge, MA), 103 units/ml TNF-
, and 103 units/ml IFN-
(R & D, Minneapolis, MN) for 48 h to induce ß-cell damage before being examined (24,28).
STZ treatment.
NODscid recipients were rendered diabetic with a single high dose (200 mg/kg) of STZ delivered intraperitoneally in a 0.9% saline solution (29). The animals were monitored by blood glucose readings from the tail vein until hyperglycemia was established. An animal was considered diabetic after two consecutive readings of 300 mg/dl or higher.
Islet transplantation and graft recovery.
A total of 400 islets were transplanted per mouse. Under sterile conditions, the recipient animal was anesthetized with a 2.5% solution of avertin, injected intraperitoneally at a dose of 0.0150.017 ml/g body wt (30). The kidney was externalized through a small incision in the flank. The islets were aspirated into PE50 tubing (Harvard Apparatus, Holliston, MA), then inserted into the space between the capsule and the kidney. The kidney was replaced in the abdominal cavity, and the incision was closed with small wound clips. Uninfected, Ad-LacZ-infected, or Ad-MnSOD-infected islets isolated from 3- to 4-week-old NOD mice were transplanted into a total of 40 STZ-treated NODscid animals. Of these animals, 29 were challenged with spleen cells from diabetic NOD mice, 10 were monitored for long-term function (≥90 days), and 1 was killed after 14 days for histological examination of the graft. For graft recovery, the graft-bearing kidney was removed. When hyperglycemia was evident in 25 days after graft removal, the animal was considered graft-dependent.
Autoimmune challenge in vivo.
Spleens were removed from spontaneously diabetic NOD female mice (blood glucose >300 mg/dl). A single-cell suspension was prepared by manual disruption of the spleen and passage through a 40-µm nylon mesh. The cells were washed, counted, and resuspended at a concentration of 66.7 x 106 cells/ml. NODscid recipients to be challenged received an intraperitoneal injection of 20 x 106 spleen cells (31).
Histologic analysis.
Paraffin sections (4 mm) were cut from graft-bearing kidneys of treated animals. Tissue sections were stained with either an insulin (diluted 1:150; DAKO, Carpinteria, CA) or an MnSOD (diluted 1:300; Upstate Biotechnology, Lake Placid, NY) primary antibody followed by biotinylated secondary antibody (30 min) and labeled streptavidin (30 min). Chromogen substrate 3,3'-diaminobenzidine (Sigma, St. Louis, MO) was added for 10 min for enzymatic stain development. Mayers hematoxylin was applied as a counterstain.
Statistical analysis.
A two-sample test of binomial proportions with a 95% CI was used to determine significant differences between groups for the in vitro results. A two-sample t test for independent samples with unequal variances was used to test for statistical significance of the mean time of graft function for the experimental animal group in comparison with the control animal groups.
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RESULTS |
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Detection of MnSOD transgene.
RT-PCR amplification was used to detect the presence of human MnSOD mRNA in the Ad-MnSOD-infected islets. The gel picture in Fig. 2 shows that MnSOD mRNA was detected in the three independently infected Ad-MnSOD islet preparations (lanes 13) but not in uninfected or Ad-LacZ-infected islet preparations (lanes 4 and 5, respectively). The primers used were specific for the human gene as they did not amplify MnSOD in murine kidney (lane 6), which is known to express abundant levels of endogenous MnSOD (5).
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Islet function after autoimmune challenge.
Of the 40 animals that received a transplant, 29 were challenged with T-cells from NOD mice and were followed until hyperglycemia was evident. In addition to these 29 animals, 22 animals that did not receive a transplant were challenged in parallel as controls for disease transfer. Table 3 summarizes the length of time that these islet-engrafted animals or animals that did not receive a transplant remained disease-free after the introduction of activated T-cells.
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DISCUSSION |
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However, MnSOD conferred very little protection against the NO-generating compound SNAP. The mechanism for this may be associated with the formation of excess peroxynitrite (ONOO-). This highly cytotoxic anion, formed by superoxide and NO, takes place at a faster rate than the dismutation reaction (34). Two studies by MacMillan-Crow et al. (35,36) reported that peroxynitrite targets nitration of tyrosine residues (especially Tyr34) in human MnSOD, resulting in enzyme inactivation. They found 3-nitrotyrosine and increased but inactive MnSOD in the tubular epithelium of chronically rejecting human renal allografts. Enzyme inactivation allows increased levels of peroxynitrite that may function to promote apoptosis (37).
The above observations seem to coincide with the results of our cytokine experiments. The inflammatory cytokines TNF-, IFN-
, and IL-1ß mediate the generation of oxygen free radicals and NO (12,13,27,38). In this context, as NO levels increase, the dismutation activity of MnSOD is not fast enough to prevent some oxygen radicals from reacting with NO to form peroxynitrite that, in turn, deactivates MnSOD. Thus, the MnSOD antioxidant would be able to protect the islets only until peroxynitrite formation reached a level that affected a decrease in enzyme activity. Studies are now under way to determine whether this is the mechanism that eventually leads to graft failure of Ad-MnSOD-modified islets.
The results of the confocal microscopic study showed the adenoviral infection pattern of a typical islet. The peripheral cells of the islet are infected by the adenovirus construct, yet the antioxidant overexpression in the outer layers of cells seems to be sufficient to protect the entire islet, including the non-transgene-bearing cells, from exogenous oxygen radicals. Our in vitro findings after oxygen radical exposure support this interpretation.
Protection was also seen in vivo. The antioxidant-bearing transplanted islets were able to withstand the onslaught of factors involved in this model of islet destruction 50% longer (P = 0.001) than uninfected, Ad-LacZ-infected, or endogenous (nontransplanted) islets. Although it can be argued that the method of giving the transplanted islets a chance to engraft before introducing diabetogenic T-cells is artificial, this model is a valid, quantitative test of the potential protective properties of the transgene being studied. If this test had been completely successful (i.e., if the grafts did not eventually fail after autoimmune challenge), then more demanding tests that resemble natural disease onset and clinical transplantation situations more closely would have been attempted.
Because in our model, graft function eventually failed, it is reasonable to believe that overexpression of the MnSOD gene alone will not be enough to protect islets from destruction after transplantation into the diabetic host. However, the MnSOD oxygen radical scavenger has therapeutic value in extending the life of autoimmune challenged islet grafts. A combination of this antioxidant enzyme with another gene (or genes) acting on other pathways of cell death could potentially provide improved protection from the host immune response and disease recurrence. Although the actual mechanism for the destruction of transplanted pancreatic islets remains unknown, it is clear from this and other studies that NO and, consequentially, peroxynitrite generation must be controlled to provide greater graft protection.
When perfected, this type of virtual encapsulation, provided by transgenic overexpression in the outer cell layers, could act as a barrier to protect the interior (mostly ß) cells of the islet. This would provide the type of protection for transplanted islets that was theorized as being the most beneficial for young patients, i.e., islet protection without systemic immunosuppression therapy.
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ACKNOWLEDGMENTS |
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The MnSOD antibody used in this study was recommended and developed by Dr. Larry Oberley (University of Iowa). We thank Judy Burnham and Sean Alber for excellent technical assistance.
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FOOTNOTES |
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Received for publication 15 February 2002 and accepted in revised form 29 October 2002.
FACS, fluorescence-activated cell sorter; IFN-,
-interferon; IL, interleukin; KRB, Krebs-Ringers Buffer; MnSOD, manganese superoxide dismutase; NO, nitric oxide; PI, propidium iodide; SNAP, S-nitroso-N-acetylpenicillamine; STZ, streptozotocin; TNF-
, tumor necrosis factor-
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REFERENCES |
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