DcR3/TR6 Effectively Prevents Islet Primary Nonfunction After Transplantation

The mouse counterpart of human TR6 might not exist, because a BLAST search of the mouse genome using the human TR6 cDNA sequence as a query failed to yield significant homologues. We therefore used human TR6 to study the role of TR6 in mouse PNF. Full-length TR6 fused with human IgG Fc (TR6-Fc) was prepared for this purpose, as described in our previous publication (10). The purified TR6-Fc protein was analyzed with 10% SDS-PAGE under reducing or nonreducing conditions, showing that the protein is a disulfide-linked dimer under nonreducing conditions (Fig. 1). Light-scattering analysis also confirmed that it behaves as a disulfide-linked dimer in solution (data not shown). NH₂-terminal sequencing revealed that the mature secreted TR6-Fc had the predicted sequence of VAETP (single-letter code of amino acids), starting at amino acid 30. The estimated purity of the protein preparation was >95% according to SDS-PAGE.

Murine L929 cells (1 × 10⁶) with or without murine FasL transfection were incubated with 1 μg of Fas-Fc, 10 ng of TR6-Fc, or 1 μg of phycoethrin-conjugated anti-murine FasL (clone Kay-10; BD PharMingen, San Diego, CA) in 100 μl of buffer for 20 min at room temperature. In certain samples, unlabeled soluble TR6 without Fc was added (1 μg/sample) as a competitor at this step. The cells were washed twice. For detection of the human-Fc fusion proteins, cells were reacted with goat F(ab)₂ anti-human IgG (Southern Biotechnology, Birmingham, AL) for 15 min at room temperature. After washing, the cells were resuspended in 0.5 μg/ml propidium iodide, and live cells were gated and analyzed by flow cytometry.

Two milliliters of digestion solution (Hanks’ balanced salt solution [HBSS] containing 20 mmol/l HEPES and 2 mg/ml collagenase IV; Worthington Biochemical, Lakewood, NJ) were injected into the common bile duct of BALB/c, C57BL/6, or lpr/lpr mice (20–24 g) after the distal end of the duct was ligated. The distended pancreas was isolated and put into a 15-ml tube containing an additional 0.5 ml of digestion solution. The pancreas was digested at 37°C for exactly 38 min, and the digestion process was stopped by the addition of 10 ml of cold HBSS containing 20 mmol/l HEPES. The islet suspension was filtered through No. 7880 cheesecloth gauze (Tyco Healthcare, Mansfield, MA) and centrifuged at 500g for 1–2 min. The pellet was washed with cold HBSS once at 500g for 1–2 min, and the supernatant was removed completely. The pellet was then resuspended in 3 ml of 25% Ficoll, and 2-ml layers of 23, 20, and 11% Ficoll were added sequentially. The Ficoll gradient was centrifuged at 700g for 5 min. Most of the islets were in the interface between the 20 and 23% Ficoll layers and were handpicked with Pasteur pipettes. The islets were then washed twice with cold HBSS. The islets were cultured overnight in RPMI 1640 containing 10% FCS on wells precoated with 0.5% BSA in PBS, and then used for transplantation or in vitro study.

Diabetes was induced in recipient C57BL/6 or gld/gld mice by intraperitoneal injection of streptozotocin in PBS (Sigma, Oakville, ON, Canada) at 220 mg/kg body wt. The diabetes status was defined as nonfasting blood glucose measuring over 20 mmol/l for two consecutive days. Approximately 400 islets from C57BL/6 or from BALB/c mice were injected intraperitoneally into the diabetic C57BL/6 mice. TR6-Fc was given to the recipients intraperitoneally twice a day at 15 mg · kg⁻¹ · day⁻¹, starting right after islet transplantation. PNF was defined as blood glucose remaining above 18 mmol/l 48 h after islet transplantation. For lpr/lpr (in C57BL/6 background) to gld/gld (also in C57BL/6 background) islet transplantation, the gld/gld recipients with streptozotocin-induced diabetes received a transplant of ∼250–270 lpr/lpr islets, and recipient blood glucose was monitored for 14 days; for the controls of this experiment, C57BL/6 to C57BL/6 islet transplantation was conducted under similar conditions.

Islet viability was assessed by ethidium bromide and acridine orange (EB/AO) staining. Islets were stained with 0.04 μg/ml ethidium bromide and 4 μg/ml acridine orange in PBS for 15 min at room temperature. After being washed once with PBS, they were examined under a fluorescent microscope. Islets from triplicate wells were counted for AO-positive ones (defined as having >50% EB/AO-positive cells in the islet mass) among total islets. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) was used to detect islet apoptosis with apoTACS kits (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. Apoptotic islets were quantified by one-way blinded examination of TUNEL-labeled islets, with the examiner unaware of the islet treatment. Islets whose mass was >50% TUNEL-positive were considered apoptotic.

After 48 h of culture in complete RPMI 1640 medium in the presence of various reagents, the islets were transferred to 12-well plates at a density of 10 islets/well. The islets were gently washed twice with 1 ml of Kreb’s buffer (135 mmol/l NaCl, 3.6 mmol/l KCl, 5 mmol/l NaH₂PO₄, 0.5 mmol/l MgCl₂, 1.5 mmol/l CaCl₂, 2 mmol/l NaHCO₃, 10 mmol/l HEPES [pH 7.4], 0.07% BSA) and then incubated in Kreb’s buffer containing 2.8 mmol/l glucose for 5 min at 37°C. A total of 200 μl of supernatant was removed for determination of basal insulin levels. The islets were cultured for an additional 40 min, and all of the supernatants were harvested. The islets were then cultured in Kreb’s buffer containing 16.7 mmol/l glucose for 45 min at 37°C, and the supernatants were harvested. The harvested supernatants were assayed for insulin by enzyme-linked immunosorbent assay. The basal insulin levels were deducted from the 2.8 and 16.7 mmol/l glucose-stimulated levels in final data presentation.

As we were to use human TR6 in a mouse model, it is essential to establish whether human TR6 could associate with mouse FasL, which might be involved in PNF. As shown in Fig. 2, both Fas-Fc and anti-mouse FasL monoclonal antibodies (mAb) bound to the mouse FasL-transfected L929 cells (solid lines) but not to untransfected wild-type cells (shaded areas). TR6-Fc presented similar binding to transfected L929 cells as Fas-Fc and anti-FasL mAb. Binding by Fas-Fc, anti-FasL mAb, and TR6-Fc to the transfected cells could be inhibited by soluble TR6 at 1 μg/ml, demonstrating the binding specificity. This result proved for the first time that human TR6 could associate with mouse FasL and established a molecular basis for inhibition of mouse Fas-mediated apoptosis by human TR6-Fc.

PNF, one of the major problems occurring during islet transplantation, can be easily demonstrated in animal models. When transplanted with a lower-than-optimal number of BALB/c islets (∼400), 26.5% diabetic C57BL/6 (H-2^b) recipient mice failed to achieve a blood glucose level <18 mmol/l with in 48 h after transplantation (Fig. 3), indicative of PNF. This was not due to allograft rejection responses, in which cytotoxic T-cells need 4–5 days to complete processes of activation, proliferation, and differentiation; our test confirmed that within 48 h of mixed lymphocyte culture, cytotoxic lymphocyte activity was not detectable (data not shown). In support of this notion, the control isogeneic islet transplantation from C57BL/6 donors to C57BL/6 diabetic recipients with 400 islets per recipient had a similar incidence (25%) of PNF 48 h after transplantation. When TR6-Fc was administered at 15 mg · kg⁻¹ · day⁻¹ intraperitoneally twice a day to the allograft recipients, PNF was completely prevented. This result showed that TR6 could prevent islet PNF in vivo.

We next explored mechanisms with which TR6 protected islets from damage. Islets were cultured in complete medium for 24 h after isolation and then treated with different apoptosis-inducing factors. After an additional 48-h culture, they were stained with EB/AO for viability assessment. As shown in Fig. 4A, FasL (SuperFasL; Q-Biogene, Carlsbad, CA) at 60 ng/ml or IFN-γ at 1,000 units/ml alone had no apparent effect on viability of the islets after 48 h. However, when these two factors were combined, they significantly decreased islet viability. This was apparent as yellow staining on the islets was increased. TR6-Fc at 10 μg/ml protected the islets from death caused by the combination of FasL and IFN-γ, as the yellow staining in TR6-Fc–treated islets was reduced. Semiquantitative assessment confirmed the impression above. As shown in Fig. 4B, islets that were treated with FasL or IFN-γ alone had no statistical difference from islets that were cultured in medium in the EB/AO staining (89.6, 82.8, and 89.9% viable, respectively). When FasL and IFN-γ were combined, the viability of the islets decreased to 58.4%, and this was statistically different from all of the above-mentioned three treatments (P < 0.01 in all three comparisons, i.e., medium versus FasL/IFN-γ, FasL versus FasL/IFN-γ, and IFN-γ versus FasL/IFN-γ). When TR6-Fc was added to the culture, it significantly reversed the FasL plus IFN-γ–induced islet death with the viability raised to 82.8% (P < 0.01, FasL/IFN-γ versus FasL/IFN-γ/TR6-Fc). This showed that TR6 had a protective effect against islet death induced by a combination of FasL and IFN-γ.

The TUNEL assay was performed after islet culture to assess whether islet death was due to apoptosis and whether TR6 could prevent the apoptosis. IFN-γ (1,000 units/ml) plus FasL (60 ng/ml)-treated islets showed significantly increased apoptosis as seen in the photos and bar graph of Fig. 5 (P < 0.01, control versus IFN-γ/FasL). TR6-Fc (10 μg/ml) significantly protected the islets from IFN-γ plus FasL-induced apoptosis (P < 0.01, IFN-γ/FasL versus IFN-γ/FasL/TR6-Fc). The results of this section disclosed that a possible mechanism of the protective effect of TR6 on PNF was inhibition of islet apoptosis.

We next assessed β-cell function using islets treated with FasL and IFN-γ in the presence or absence of TR6-Fc. As shown in Fig. 6, as with the EB/AO staining, a combination of FasL and IFN-γ caused significant compromise in insulin release upon stimulation with 16.7 mmol/l glucose (P < 0.01, control versus FasL/IFN-γ), and such compromise could be prevented in the presence of TR6-Fc (P < 0.01, FasL/IFN-γ versus FasL/IFN-γ/TR6-Fc). The result suggests that the morphologic damage on islets seen in EB/AO staining and in TUNEL assays occurred on β-cells. It is to be noted that we intentionally used a condition under which the damage caused by FasL and IFN-γ was moderate and was sensitive to the rescue effect of TR6. The compromise in insulin release and the rescue effect of TR6 was comparable in scale to the islet viability and apoptosis as shown in the previous section.

To confirm the contribution of FasL and Fas to PNF in vivo, we conducted islet transplantation from lpr/lpr mice to diabetic gld/gld recipients. The former strain has mutation in FasL and the latter, in Fas. As a consequence, the FasL and Fas interaction is not functional in this in vivo model. As the lpr/lpr and gld/gld mice used both were in the C57BL/6 background, this model represents isogeneic islet transplantation with no allograft rejection involved. A marginal dose of 250–270 islets was used per recipient. The recipient mice had blood glucose levels between 20 and 30 mmol/l before islet transplantation (day 0). After islet transplantation, blood glucose was monitored daily. At such an islet dosage, normoglycemia was restored only in ∼20% of the control C57BL/6-to-C57BL/6 (B6 to B6) group around day 13 posttransplantation. Therefore, this regimen was sensitive for the detection of the putative contribution of FasL and Fas to in vivo islet damage after transplantation. As shown Fig. 7A, starting from day 6 posttransplantation, the lpr/lpr-to-gld/gld group had significantly lower blood glucose than the B6-to-B6 group (P < 0.05 at all points, starting from day 6; ANOVA). Figure 7B depicted the percentage of mice returned to normoglycemia (defined as ≤12 mmol/l) after the isogeneic islet transplantation. In the B6-to-B6 group, only 20% of the mice had normoglycemia until day 13 posttransplantation. However, in the lpr/lpr-to-gld/gld group, 33% of the mice achieved normoglycemia on day 5 (with some fluctuation between day 5 and day 10), and 44% of the mice achieved that on day 13. The difference between the two groups was highly significant (P < 0.001, two-way repeated measurement of ANOVA). These data indicate that with nonfunctional FasL and Fas in the donors and recipients, respectively, the efficiency of islet transplantation was improved. This suggests that Fas-mediated apoptosis contributes to PNF.

In this study, we reported that a soluble death decoy DcR3/TR6 could prevent islet PNF, and this protective effect was, at least in part, due to interference of TR6 with Fas-mediated islet apoptosis. The mechanisms of PNF have not been clearly delineated, but multiple factors, such as inflammatory cytokines, free radicals, macrophages, and withdrawal of trophic support from tissues surrounding the islets, are likely involved. The effective prevention of PNF by TR6 suggested that Fas-mediated islet apoptosis was an essential mechanism for PNF, because TR6 is known to bind to FasL and prevent FasL-triggered apoptosis. Indeed, Fas is reported being expressed on murine islets (18), which undergo apoptosis when infected ex vivo with FasL-expression adenovirus vector, resulting in PNF when transplanted into isogeneic or allogeneic diabetic recipients (19). Using lpr/lpr mice as donors and syngeneic gld/gld mice as recipients, we demonstrated that without effective FasL and Fas interaction, the efficacy of islet transplantation was significantly improved. This convincingly confirms that after transplantation, in the absence of allograft rejection, there is significant loss of islet function (i.e., PNF) as a result of Fas-mediated apoptosis.

Soluble FasL alone at the concentration used (60 ng/ml) or at a higher concentration (1 μg/ml, data not shown) did not cause significant islet death in our in vitro experiments, even though the FasL used was in a form capable of inducing apoptosis of many cell lines at a concentration of 10–100 ng/ml in solution. In vivo, leukocytes of donor origin or leukocytes attracted to islets by inflammatory cytokines might be a source of membrane-bound FasL, which might give a more potent signal through Fas. Alternatively, soluble FasL needed cofactors to effectively induce islet apoptosis.

In many in vitro apoptosis models, cells need to be sensitized or conditioned by measures such as protein synthesis inhibition, using cycloheximide, in addition to FasL, for apoptosis induction (20,21). In our model, when FasL was applied in combination with IFN-γ, significant islet damage was observed, and the TUNEL assay revealed that it was due to apoptosis. What is the physiologic relevance of IFN-γ in PNF, which is defined as a reaction unrelated to classical allograft rejection? Although a definitive cell source of IFN-γ production during PNF remains to be elucidated, IFN-γ mRNA can indeed be detected in freshly isolated islets (22). Also, it is known that memory T-cells under the influence of inflammatory cytokines can nonspecifically produce IFN-γ without T-cell receptor ligation (23). It is possible that some nonspecific memory T-cells of donor or recipient origin in the islets produce some IFN-γ, which sensitizes the islets. Another potential source of IFN-γ is NK cells. In the clinical islet transplantation, IFN-γ can also be produced by alloantigen-specific host T-cells within hours of alloantigen stimulation. Therefore, IFN-γ might be available in vivo locally during islet transplantation and serves as a conditioner for Fas-mediated islet apoptosis. We have tested only the combination of IFN-γ and FasL in our in vitro experiments. In vivo, other cytokines, in addition to IFN-γ, might also participate in the conditioning process for Fas-mediated apoptosis.

In addition to FasL, TR6 could bind to LIGHT, which is a ligand of LTβR and is sufficient to trigger apoptosis via LTβR in some tumor cells (16). This has raised a possibility that TR6 might also inhibit islet apoptosis mediated by LTβR in vivo. Two recent articles showed that the administration of soluble LTβR-Ig or transgenic overexpression of LTβR-Ig effectively prevents or reverses insulinitis and diabetes in NOD mice (24,25); prevention of LTβR-mediated islet apoptosis by soluble LTβR-Ig might be implicated in these models. Therefore, the protective action of TR6 might involve multiple mechanisms.

It is worth pointing out that for the purpose of quantifying, we arbitrarily defined the PNF as failure of glucose decrease <18 mmol/l with 48 h after islet transplantation. Although TR6 treatment rescued all PNF under such a definition, ∼50% of the mice that received TR6 still had blood glucose >12 mmol/l at 48 h (data not shown). Thus, the result of this study should not be interpreted in such a way that the TR6 treatment is a cure for PNF. We believe that there are still islets not being able to function properly in vivo despite the treatment, because 1) TR6-Fc in its present form has only a very short half-life of 20 min (data not shown), and there is certainly room to improve its effect in this regard; 2) the 400 islets transplanted to a recipient are empirically determined to achieve euglycemia in ∼80% of the mice, and probably this number has already factored in nonfunctional islets; and 3) PNF is probably caused by multiple factors, not all of which could be corrected by TR6; indeed, TR6 was not able to protect islet function damaged by a combination of TNF-α and interleukin-1α (data not shown).

The Edmonton protocol has proved that islet transplantation is a successful treatment for diabetes. Rough calculations show that >60% of transplanted islets are nonfunctional. PNF and rejection are the chief reasons for such a low efficiency. Considering the organ donor shortage, to reduce the number of islets needed for successful transplantation becomes an essential task to significantly enhance our capability for treating more patients with diabetes. Many preclinical and clinical studies are under way to optimize immunosuppressant regimens in islet transplantation, but the problem of PNF remains to be addressed. In vivo, tumor cells may use TR6 to protect themselves from immune surveillance and gain survival advantages (9). By the same token, islets can benefit from the protective role of TR6. Our animal study proved that this is indeed the case. We envision that such an approach could be applied for clinical islet transplantation. Soluble TR6 can be added to buffers used for islet isolation, transportation, and injection. Alternatively, islets can be transfected with TR6 expression adenovirus vectors for long-lasting secretion of TR6 in situ. As discussed above, such treatments with TR6 might not totally prevent PNF, but they could probably reduce PNF significantly and bring down the number of islets needed for insulin independence after islet transplantation.

This work was supported by grants from the Canadian Institutes of Health Research (CIHR; MOP57697 and PPP57321); the CIHR/Canadian Blood Service Partnership Program; the Kidney Foundation of Canada; the Heart and Stroke Foundation of Quebec; the Roche Organ Transplantation Research Foundation, Switzerland (ROTRF 280039611); the Juvenile Diabetes Research Foundation (5-2001-540); and the J-Louis Levesque Foundation to J.W. This work was also supported in part by National Natural Science Foundation of China (nos. 30070724 and 30271273) and National Natural Science Foundation of Zhejiang, China (nos. 399124 and 300479) to Y.W. Y.W is a recipient of a Wyeth-Ayerst/CIHR fellowship, and R.R. was supported by a fellowship from Swiss National Science Foundation. J.W. is a National Researcher of Fonds de la recherche en santé du Québec (FRSQ).

We sincerely thank Ovid Da Silva and Robert Boileau of the Research Support Office, CHUM Research Center, for editorial assistance and statistical analysis, and Dr. Stephen Ullrich of Human Genome Sciences for reviewing the manuscript.