Transgenic overexpression of human Bcl-2 in islet ß cells inhibits apoptosis but does not prevent autoimmune destruction
Janette Allison,
Helen Thomas,
Dianne Beck,
Jamie L. Brady,
Andrew M. Lew,
Andrew Elefanty,
Hiro Kosaka1,
Thomas W. Kay,
David C. S. Huang and
Andreas Strasser
The Walter and Eliza Hall Institute for Medical Research, Post Office, Royal Melbourne Hospital, Victoria 3050, Australia
1 Department of Dermatology, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan
Correspondence to:
J. Allison
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Abstract
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Insulin-dependent diabetes mellitus results when > 90% of the insulin-producing ß cells in the pancreatic islets are killed as a result of autoimmune attack by T cells. During the progression to diabetes, islet ß cells die as a result of different insults from the immune system. Agents such as perforin and granzymes, CD95 ligand and tumor necrosis factor-
, or cytokines and free-radicals have all been shown to cause ß cell apoptosis. The anti-apoptotic protein, Bcl-2, might protect against some of these stimuli. We have therefore generated transgenic mice expressing human Bcl-2 in their islet ß cells. Although Bcl-2 was able to prevent apoptosis induced by cytotoxic agents against ß cells in vitro, Bcl-2 alone could not prevent or ameliorate cytotoxic or autoimmune ß cell damage in vivo.
Keywords: ß cell death, insulin-dependent diabetes mellitus, non-obese diabetic mouse
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Introduction
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Mouse models of autoimmune or type I diabetes have provided valuable tools to study the initiation and progression of diabetes which are almost impossible to monitor in humans. The way in which the T cells kill mouse islets is complex and at least four immune-mediated, cell death mechanisms have been proposed: (i) granule exocytosis and perforin (1), (ii) the CD95 (Fas/APO-1) receptorCD95 ligand (CD95L) interaction (24), (iii) tumor necrosis factor (TNF) receptor type I (TNFRI)TNF-
interaction (5), and (iv) various pro-inflammatory cytokines inducing free-radical production and other effectors (6,7). These mechanisms all result in ß cell death by inducing apoptosis (8).
The distinct stimuli that cause apoptosis converge on a common cell death effector machinery which is driven by the cysteine proteases (caspases). These are present in all cells as zymogens which are activated to the mature form when the cell receives a death signal (9). Evidence also exists for caspase-independent pathways to apoptosis in certain cell types (10), although these appear to be an exception. Inhibitors of apoptosis have been identified and foremost amongst these is Bcl-2 (11,12). The mode of action of Bcl-2 has not been determined with certainty, but current models indicate that it blocks caspase activation by inhibiting the action of adaptor proteins such as Apaf1 (13). In addition, Bcl-2 is thought to prevent the release of cytochrome c from mitochondria, thus averting caspase 9 activation (14,15).
In hematopoietic cells, Bcl-2 protects against a range of stress stimuli including growth factor withdrawal, anoxia, cytotoxic drugs and irradiation, but does not prevent apoptosis signaled by cell death receptors such as CD95 and TNFRI (16). In certain cell types, expression of Bcl-2 has been shown to antagonize CD95 or TNFRI-mediated death (17,18). In these situations, cell death is believed to be mediated by caspase 8-induced activation of the pro-apoptotic Bcl-2 family member Bid (19,20), although the importance of this pathway has been questioned (21). In vitro studies have shown that Bcl-2 overexpression can protect a transformed ß cell line against IL-1ß (22), and renders primary ß cells resistant to the combined effects of IL-1ß, TNF-
and IFN-
(23). These cytokines probably mediate ß cell damage by inducing the formation of free-radicals, although TNF-
may also promote apoptosis through the TNF receptor death pathway. Evidence exists that anti-apoptotic proteins of the Bcl-2 family might also function in ß cells in vivo. Bcl-xL overexpression in ß cells was shown to accelerate SV40 large T antigen-induced tumorigenesis by inhibiting the apoptosis accompanying the transformation by this protein (24).
Enforced expression of Bcl-2 may therefore protect islet ß cells against many of the effects of autoimmune attack including free radical-induced damage or killing mediated by death receptors. To test this, we have generated transgenic mice expressing high levels of Bcl-2 in their ß cells. Islets of these animals were exposed to a number of in vitro and in vivo stimuli that lead to apoptotic death of ß cells to determine the protective effects of Bcl-2. Our data show that Bcl-2 can protect ß cells against certain apoptotic stimuli but not against autoimmunity in three mouse models of insulin-dependent diabetes mellitus (IDDM).
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Methods
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Mice
Mice were bred at the Walter and Eliza Hall Institute's specific pathogen-free facility at Kew, Victoria. Strains used were non-obese diabetic (NOD)/Lt Jax (NOD) and the bm1 mutation of C57BL/6 (bm1). Transgenic mice were generated by microinjecting fertilized eggs of bm1 mice (which in our facility provide larger numbers of fertilized eggs than C57BL/6 mice) with a construct containing the rat insulin promoter (RIP), position 695 to position +8 (25), linked to the EcoRITaqI fragment of the human Bcl-2 cDNA (26) and the blunt-ended SmaIEcoRI 3' sequences containing the polyadenylation signals of the human growth hormone gene (27). The construct (RIP-Bcl-2) was built in pIC20H and the vector sequences were removed prior to microinjection. Two transgenic lines were obtained which expressed human Bcl-2 in the islet ß cells (lines 407-3 and 405-5). Line 407-3 was backcrossed to the NOD/Lt background for six generations. Offspring were screened for the transgene by Southern blotting using sequences from the RIP as a hybridization probe. The C57BL/6.RIP-mOVA transgenic mice express a membrane-bound form of ovalbumin (OVA) in the ß cells (28). The OT-I TCR transgenic mice, on a rag-1/ background, produce H-2Kb class I-restricted CD8+ T cells specific for OVA (28). The bm1.RIP-B7-1 transgenic mice express the murine B7-1 co-stimulator in their ß cells (29) and have normal islets. The bm1.RIP-IL-2 transgenic mice express a single copy of murine IL-2 in their islet ß cells which results in an inflammatory response into the islets but does not lead to diabetes in bm1 animals (30).
Transfer of OT-I T cells
bm1.RIP-Bcl-2 hemizygous mice were crossed to B6.RIP-mOVA hemizygous mice and the four types of offspring were genotyped by Southern blots using probes to RIP and OVA. Mice aged 1016 weeks old, were irradiated (500 rad from a 60Co source) and injected in the tail vein with 3x106 B6.rag-deficient OT-I T cells. Urinary glucose was monitored daily to determine onset of diabetes at which time mice were killed and pancreata taken for histology.
Preparation of islets
Preparation of islets was as described (3133). Briefly, the mouse bile duct was cannulated and injected with medium containing collagenase to distend the pancreas. After digestion at 37°C islets were separated from exocrine tissue by density-gradient centrifugation with BSA (First Link, Brierly Hill, UK) and then hand picked.
Cell death assays
Whole islets (100 per sample) were isolated and cultured overnight in CMRL-1066/10% FCS medium in untreated Petri dishes. Titrated concentrations of staurosporine were added and the islets cultured for 36 h. At the end of the incubation protocol, the culture medium containing non-attached cells was transferred to a polypropylene tube. Islets were washed with PBS, dispersed in trypsin, washed and pooled with the culture medium. After centrifugation the cells were resuspended in 150 µl of hypotonic buffer containing 50 µg/ml of propidium iodide (Sigma, St Louis, MO), 0.1% sodium citrate and 0.1% Triton X-100 as described (34). The percentage of apoptotic nuclei was determined by flow cytometry on a FACScan analyzer using the FL3 channel. Apoptotic nuclei have an apparent DNA content of <2C.
Immunofluorescent staining and flow cytometric analysis
Human Bcl-2 protein was identified in islet ß cells by an intracellular staining protocol as described (35) with a mAb to human Bcl-2, clone Bcl-2-100 (36) and this detected with goat anti-mouse IgG conjugated to FITC (Silenus, Melbourne, Australia). WEHI 164 fibrosarcoma cells, transfected with a human Bcl-2 expression construct (37), were used as a positive control for Bcl-2 staining. Cells were analyzed on a FACScan (Becton and Dickinson, Mountain View, CA) using Lysys II software. Forward scatter versus side scatter was set on unstained ß cells or WEHI 164 cells by eye. FL1 baseline was set on unstained cells by eye.
Immunohistochemistry
Immunohistochemistry was done as previously described (38) using Bouin's solution-fixed paraffin-embedded tissue or acetone-fixed frozen tissue sections. Human Bcl-2 protein was identified with the mAb clone Bcl-2-100 (36) detected with anti-mouse IgG conjugated to horseradish peroxidase (Dako, Carpinteria, CA). Color development was done using diaminobenzidine. To detect apoptotic cells, dual-color immunofluorescence staining for insulin and TUNEL was done on 4% paraformaldehyde-fixed paraffin sections as described (39).
In vivo streptozotocin (STZ) treatment
STZ (Sigma) was dissolved in 0.01 M sodium citrate at 20 mg/ml and immediately injected i.p. into mice. For multiple low-dose STZ treatments, the drug was injected into male mice at 40 mg/kg, daily for 5 days. Mice were monitored daily for diabetes by urinary glucose analysis. For high-dose STZ treatments, the drug was injected once into male mice at 140 mg/kg body wt. Mice were killed after 3, 5 and 7 h, and pancreata taken for fixation in Bouin's solution and 4% PFA. Slides were stained for haematoxylin & eosin and for Gomori's aldehyde fuchsin (GAF). Dying islet cells were identified in the haematoxylin & eosin-stained slides by their pyknotic appearance. In the GAF-stained slides, ß cells could be identified by blue staining, and dying ß cells by their intense red staining nuclei and rounded cell shape.
Monitoring of diabetes
Urine glucose levels were monitored daily or weekly depending on the nature of the experiment, with two consecutive readings of >60 mM glucose taken to indicate the onset of diabetes. Blood glucose readings were taken at this time and the mice were killed.
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Results
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Production of transgenic mice expressing human Bcl-2 in islet ß cells
Two RIP-Bcl-2 transgenic lines (407-3 and 405-5) were derived on a bm1 genetic background. The 407-3 transgenic line was subsequently backcrossed onto the NOD genetic background. Transgene-encoded human Bcl-2 protein levels in ß cells were determined by flow cytometry using an intracellular staining method (Fig. 1
, left panels) and by immunohistochemistry (Fig. 1
, right panels). The amount of Bcl-2 protein expressed in the ß cells was comparable to that described for the WEHI 164/Bcl-2 cell line in which Bcl-2 conferred resistance to a range of death stimuli (40).

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Fig. 1. Expression of transgenic human Bcl-2 protein in islet ß cells of RIP-Bcl-2 transgenic mice. (Left panel) Transgene-encoded human Bcl-2 expression was detected in RIP-Bcl-2 ß cells of 8-week-old mice by immunofluorescent staining and flow cytometric analysis from two transgenic lines on a bm1 background: (A) bm1.407-3 line, (B) bm1.405-5 line and (C) non-transgenic littermate cells. The negative peak in the transgenic samples represents non-ß islet cells and other contaminating cells. (D) WEHI 164 parental cells and WEHI 164 Bcl-2 transfectants were used as controls for the antibody staining. (Right panel) Bcl-2 and insulin protein were detected by immunohistochemical staining in a bm1.407-3 transgenic (tg) islet (aged 6 weeks). The same expression levels and staining patterns were seen in 407-3 transgenic mice on the NOD genetic background.
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When pancreata were taken from animals at different ages (E16, day 1, 3, 10 and 17, and week 6 and >4 months) and analyzed by immunohistology, high levels of Bcl-2 protein were detected in the transgenic islets at all timepoints (see, e.g. Fig. 1
, right panels). At each stage, the islets from transgenic mice and non-transgenic littermates were indistinguishable histologically and expressed similar levels of the islet hormones, insulin (ß cells), glucagon (
cells) and somatostatin (
cells) (Fig. 1
and data not shown). These results showed that RIP-Bcl-2 transgenic mice expressed high levels of Bcl-2 in islet ß cells, and that this had no obvious consequences for the development and function of the ß cells.
Bcl-2 expression in islet ß cells confers resistance against staurosporine
It has been established that murine and human islet ß cells can undergo apoptosis (39,4145). However, we have found that normal primary ß cells were relatively resistant to a number of apoptosis-inducing stimuli in vitro, such as
irradiation, anoxia and cytokines (unpublished results and 31,33). Staurosporine, a broad-spectrum protein kinase inhibitor, induces all cells to die by apoptosis within 2436 h and this death can be inhibited by Bcl-2 (46), providing a stringent assay for function of Bcl-2. To test if Bcl-2 can inhibit cell death in islet ß cells, whole islets from control and RIP-Bcl-2 transgenic mice were cultured in the presence of titrated concentrations of staurosporine. The percentage of apoptotic nuclei was determined by flow cytometry according to the procedure of Nicoletti et al. (34). Transgenic and nontransgenic ß cells were unaffected by staurosporine at 0.1 and 1 µM concentrations, but at 5 µM most of the non-transgenic ß cells underwent apoptosis (Fig. 2
). Expression of Bcl-2 inhibited cell death even at drug concentrations of 10 µM. It is of note that normal mouse ß cells were highly resistant to staurosporine-induced cell death. At 1 µM, most ß cells of non-transgenic mice were alive after 36 h. By comparison, these concentrations of staurosporine killed the majority of NIT cells (a cell line derived from an SV40 large T antigen-induced pancreatic ß cell tumor with a NOD genetic background) (data not shown).

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Fig. 2. Islet ß cells overexpressing Bcl-2 are protected against cell death induced by staurosporine. Islets isolated from 40-day-old NOD.407-3 transgenic mice (RIP-Bcl-2) or their littermates (non-tg) were treated in culture for 36 h with staurosporine at concentrations of 0, 0.1, 1, 5, 10 and 20 µM. The data for 0.15 µM represent the mean of two experiments, and for 10 and 20 µM the mean of three experiments with the SD given. Similar results were obtained with islets from bm1.407-3 transgenic mice.
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Bcl-2 expression in islet ß cells does not prevent the destructive effects of high dose STZ treatment
We showed above that Bcl-2 could protect islet ß cells from the effects of a toxic drug, staurosporine, in culture. To determine if Bcl-2 could protect islet ß cells from death stimuli in the whole animal, we tested the effect of the cytotoxic drug STZ in bm1.407-3 male mice. STZ carries a glucose moiety and is transported into the ß cell via the GLUT 2 receptor. The mechanism of its cytotoxicity is not known, but it methylates DNA directly and this results in DNA strand breaks. Recent evidence indicates that the DNA strand breaks lead to the over-activation of the DNA repair enzyme PARP and this to depletion of NAD (47). At high doses, STZ acts directly on the ß cell resulting in damage to the islets within a few hours. Three hours after injection of STZ, bm1.RIP-Bcl-2 transgenic (N = 3) and non-transgenic (N = 3) mice had islets that appeared intact when assessed by histology, but TUNEL staining labeled nuclei in most of the islet cells (Fig. 3
). It is likely that this labeling was due to DNA damage induced by cell death rather than that caused by STZ since the sensitivity of the TUNEL assay requires large numbers of DNA ends to give a signal. By 5 h, transgenic mice (N = 4) and non-transgenic littermates (N = 5) had islets showing many apoptotic-like ß cells (Fig. 4
). Pyknotic nuclei were evident and the cells had rounded up. Morphology is not an exact indicator of apoptosis, however, and a clear demonstration of apoptosis would require identification of the cleavage products of the caspase cascade. By 7 h, many of the ß cells had disappeared and only small islets remained. Counting the number of pyknotic ß cells in transgenic and nontransgenic islets showed that the expression of Bcl-2 did not reduce the cytotoxic effects of high-dose STZ (Table 1
).

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Fig. 3. Islet ß cells overexpressing Bcl-2 are not protected from the in vivo effects of high-dose STZ treatment. Three hours after high-dose STZ treatment, pancreata were recovered and analyzed for insulin expression and TUNEL staining was performed to detect cleaved DNA. Both transgenic (tg) and non-transgenic (non-tg) islets have insulin-expressing cells with similar numbers of TUNEL-positive cells. Transgenic mice that received no STZ treatment (No STZ) had no TUNEL-positive cells in their islets.
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Fig. 4. Islet ß cells overexpressing Bcl-2 are not protected from the in vivo effects of high-dose STZ treatment. GAF staining of islets from (A) a RIP-Bcl-2 transgenic mouse and (B) a non-transgenic littermate 5 h after treatment with high-dose STZ. (C) Islets from a transgenic mouse that was injected with buffer only. Note the pyknotic nuclei and rounded cell shape in the treated samples.
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Bcl-2 expression in islet ß cells does not prevent the destructive effects of multiple low-dose STZ treatment
Multiple low-dose injections of STZ into male C57BL/6 (or bm1) mice cause diabetes which is associated with lymphocytic infiltration. The mechanism of this process is not clear but is believed to involve, at least in part, damage to ß cells mediated by T cells (48). Others have shown that ß cells undergo apoptosis, in vivo, in response to multiple low-dose STZ treatment (49). The bm1.407-3 transgenic males and littermate controls were injected with five daily injections of STZ (40 mg/kg). Both groups of animals developed diabetes at similar incidence (Table 2
). Unexpectedly, some animals from each group spontaneously recovered from their diabetes. Staining of pancreata from the recovered and non-recovered mice after 30 days, showed the presence of small disorganized islets with staining for insulin, glucagon or somatostatin (and Bcl-2 if islets were from transgenic mice) (data not shown). Infiltrating leukocytes were sparse. Two explanations may account for the recovery of some mice from STZ-induced diabetes. Islets may have regenerated enough insulinproducing cells to maintain normo-glycemia. Alternatively, infiltrating leukocytes producing cytokines may have inhibited insulin secretion and as these cells became more sparse, insulin secretion from islets remains recovered well enough to reverse hyper-glycemia.
Bcl-2 expression in islet ß cells does not antagonize cell death induced by CD8+ T cells
We also investigated the effect of Bcl-2 overexpression on cytotoxic T lymphocyte (CTL)-induced ß cell death. Transgenic mice that express a membrane-bound form of OVA (RIP-mOVA mice) can be made diabetic by transfer of a critical number (>3x106) of OVA-specific CD8+ T cells (OT-I T cells). Diabetes occurs by day 56 after transfer in 100% of mice (28). Double-transgenic RIP-mOVA/Bcl-2 mice that expressed mOVA and Bcl-2 in their islet ß cells and RIP-mOVA littermate controls were injected with 3x106 OT-I T cells. At 13.5 days after transfer, no CD8+ T cells were seen in the islets of recipients, but by day 5, large numbers of CD8+ OT-I T cells and macrophages were seen in the islets of both RIP-mOVA and RIP-mOVA/Bcl-2 mice (data not shown). Five of five RIP-mOVA/Bcl-2 double-transgenic mice tested became diabetic by day 6 after transfer of OT-I T cells, as did two of two RIP-mOVA control mice. Islets from both groups were destroyed, with only a few ß cells remaining and these were in the process of undergoing apoptosis (data not shown and 50). Littermate controls that did not express the OVA antigen (none of six non-transgenic; none of two RIP-Bcl-2 mice) had no leukocyte infiltrates or pathology. In conclusion, the expression of Bcl-2 in ß cells of RIP-mOVA mice did not protect ß cells against CTL attack and did not alter the kinetics with which diabetes developed.
Bcl-2 expression in islet ß cells does not influence diabetes onset in the RIP-B7-1 transgenic mouse model of autoimmunity
Transgenic mice expressing B7-1 on their islet ß cells have been studied (29). With the exception of a low percentage of aged mice (>225 days) these animals do not develop autoimmunity spontaneously and have normal islets. If, however, inflammation is induced in the islets by co-expression of a transgene encoding the pro-inflammatory cytokine, IL-2, RIP-B7-1/IL-2 double-transgenic animals all develop diabetes within 4060 days. Bcl-2 expression in islet ß cells of these animals (RIP-B7-1/IL-2/Bcl-2 mice) did not influence the onset of diabetes (Table 3
).
Bcl-2 expression in islet ß cells has no influence on diabetes onset and incidence in NOD mice
The 407-3 RIP-Bcl-2 transgenic mice were backcrossed to the NOD genetic background and at generation 6 a cohort of female mice was monitored for spontaneous diabetes incidence. RIP-Bcl-2 transgenic NOD mice and littermate controls showed a similar incidence of diabetes (Fig. 5A
). At generation 8, transgenic mice (six of seven) and non-transgenic mice (four of five) were diabetic by 225 days (Fig. 5B
). Histological examination of islets failed to reveal any obvious differences between diabetic RIP-Bcl-2 transgenic mice and diabetic NOD controls. Both had destroyed islets with residual leukocyte infiltrates (not shown).

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Fig. 5. Incidence of diabetes in NOD.RIP-Bcl-2 mice. The incidence of diabetes in NOD.RIP-Bcl-2 (N = 22) and non-transgenic littermates (N = 25) was assessed after the 407-3 transgene was backcrossed to the NOD genetic background for 6 generations (A). RIP-Bcl-2 transgenic mice (N = 7) and non-transgenic littermates (N = 5) were also assessed at the eighth backcross (B).
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Discussion
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There were no adverse effects associated with the expression of high levels of human Bcl-2 protein in islet ß cells of transgenic mice. Similarly, overexpression of Bcl-xL, a Bcl-2 homologue, did not perturb development or function of islet ß cells (24). The functional capacity of transgenic Bcl-2 was demonstrated by its ability to protect ß cells, in vitro, from the apoptosis-inducing agent, staurosporine. Of interest was the observation that relatively high concentrations of staurosporine (5 µM) were needed to induce apoptosis in normal islet ß cells. Resistance of normal mouse ß cells to apoptotic stimulation was also found with
-irradiation where doses as high as 3000 rad failed to induce apoptosis in normal ß cells after 4 days (H. Thomas, unpublished data). Consistent with this observation, high doses of UV-B irradiation (300 rad) have been to used to deplete human or pig islets of antigen-presenting cells without affecting islet function (51). One explanation for this resistance of normal islets is that they may already express relatively high levels of cell survival proteins. So far, only limited information is available on the expression of such proteins. For example, in situ hybridization studies revealed low levels of Bcl-xL, no Bcl-2 and relatively high levels of the pro-apoptotic protein, Bax, in ß cells (24).
To test if Bcl-2 overexpression had any beneficial effects on ß cell survival in the whole animal, we treated mice with the drug STZ. At high doses, this induced cell death in ß cells within 3 h of administration. High-dose STZ is directly toxic to ß cells and it is thought to damage DNA by alkylation leading to induction of DNA strand breaks. Overexpression of Bcl-2 might be expected to prevent such a process because it has been shown to inhibit cell death resulting from DNA damage, after irradiation, in lymphoid cells (52). We did not observe any differences in the response of transgenic from non-transgenic mice to treatment with this drug. It is possible that subtle protective effects were present but were not detected in our assay. Alternatively, it is possible that DNA damage kills different cell types by different molecular mechanisms.
To determine whether overexpression of Bcl-2 in islet ß cells would protect against, or ameliorate the effects of autoimmunity, three mouse models of autoimmunity were analyzed: the OT-I transgenic T cell transfer model (28), the RIP-B7-1 transgenic mouse model (29,53) and the spontaneously diabetic NOD mouse model (54,55). In all of these we saw no beneficial effects of overexpressing Bcl-2 in islet ß cells. In the OT-I transgenic mouse model the effector CD8+ T cells kill ß cells directly by MHC-restricted CTL attack (56), but it is not yet known if this is perforin/granzyme or CD95L meditated, or both. Either way, the finding that Bcl-2 did not protect ß cells expressing OVA from CTL attack is consistent with data from other systems showing that Bcl-2 does not prevent killing by intact CTL in culture (57) nor influence killing of lymphocytes through CD95 (35,37). In the RIP-B7-1 model, both CD4+ and CD8+ T cells seem to be involved (58), and may use multiple mechanisms to kill ß cells including perforin/granzymes, CD95L and cytokines. Likewise spontaneous diabetes in the NOD mouse is dependent on both CD4+ and CD8+ T cells with perforin being particularly important (1). Bcl-2 alone would be unlikely to provide sufficient protection to prevent or slow down ß cell killing from all these insults. The possibility exists that the protection conferred by Bcl-2 is too subtle to be detected in our assays. For example, although NOD.RIP-Bcl-2 mice and their littermates at backcross 6 and 8 became diabetic at similar incidence, none of the NOD.RIP-Bcl-2 mice became diabetic before day 125, whereas some control littermates did (Fig. 5A and B
). Analysis of a larger cohort of mice (>50) at backcross 10 might demonstrate a statistically significant difference.
Because Bcl-2 alone did not protect ß cells against autoimmune attack, combinations of protective strategies may be required. CD8+ effector T cells form a major component of the autoimmune process in IDDM in the NOD mouse (1) and, as discussed above, the cytolytic mechanisms that CTL use are not influenced by Bcl-2. It is therefore necessary to study the protective effects of Bcl-2 in situations where CD8+ T cell effector mechanisms have been removed. For example, perforin-deficient NOD mice are protected from diabetes but their islets still have some pathology (1). Expression of Bcl-2 in islets of perforin-deficient NOD mice may ameliorate this pathology. Alternatively, expression of Bcl-2 in islets of NOD mice deficient in CD95 may prove beneficial since we have shown a minor role for CD95 in ß cell death in the NOD mice (4).
Overexpression of Bcl-2 in islet ß cells may prove useful in other situations. During transplantation into diabetic recipients, islets are exposed to unfavorable growth conditions and high glucose concentrations both of which can result in apoptotic cell death (59), and these may be preventable by overexpression of Bcl-2. Bcl-2 may also enhance the viability of islets during the isolation procedure. Encapsulation of islets in biomembranes prevents access by T cells but does not provide a favorable environment for islets to function and this may be another situation in which Bcl-2 could prove beneficial.
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Acknowledgments
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We thank Tanya Templeton for animal husbandry and collection of diabetes data, Merryn Ekberg for excellent technical assistance, and Steven Mihajlovic and Ellen Tsui for histology preparation. This work was supported by grants from the National Health Medical Research Council of Australia, the 1998 JDFI/NHMRC/JDF Australia consortium, the Dr Josef Steiner Cancer Research Fund and the Anti-Cancer council of Victoria. A. S. is a scholar of the Leukemia Society of America and a recipient of a Clinical Investigator Fellowship of the Cancer Research Institute.
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Abbreviations
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CD95L CD95 ligand |
CTL cytotoxic T lymphocyte |
GAF Gomori's aldehyde fuchsin |
NOD non-obese diabetic |
OVA ovalbumin |
RIP rat insulin promoter |
STZ streptozotocin |
TNF tumor necrosis factor |
TNFRI tumor necrosis factor receptor I |
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Notes
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Transmitting editor: M. Feldmann 
Received 27 May 1999,
accepted 14 September 1999.
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