Islet allotransplantation represents a possible therapy for the treatment of type 1 diabetes (T1D) (1). However, the acute rejection of the foreign islet tissue remains a major obstacle. Proinflammatory cytokines have been demonstrated to play a major role in the failure of islet allografts as well as the pathogenesis of autoimmune diabetes. In particular, expression of the cytokine IFN-γ has been shown to be an important mediator of β-cell death in T1D (2, 3) and islet allograft rejection (4, 5). In animal models of T1D, islet infiltrating lymphocytes produce IFN-γ during insulitis and the administration of anti-IFN-γ antibodies attenuates the development of disease (2, 6). Similarly, during islet allograft rejection, IFN-γ production by islet infiltrating T cells is sufficient to upregulate major histocompatibility complex (MHC) class I antigen expression on islet cells, rendering them more effective targets for CD8 T cell mediated attack, in the absence of FasL or perforin/granzyme pathways (5, 7–9). Hence, the blockade of IFN-γ signaling is important not only for inhibiting islet allograft rejection, but also in preventing autoimmune β-cell destruction (10).
Suppressor of cytokine signaling-1 (SOCS-1) is identified as a negative regulator of the JAK-STAT signaling pathway for cytokines. It was previously demonstrated that after stimulation with IFN-γ, SOCS-1 and SOCS-2 mRNA and protein are expressed in primary islets and NIT-1 insulinoma cells (11). Whereas islets of SOCS-1 deficient mice (which were also deficient in IFN-γ) are hypersensitive to IFN-γ and TNF via increased expression of iNOS, nitric oxide, and upregulation of class I MHC (11), overexpression of SOCS-1 can suppress responses to IFN-γ. It has been demonstrated that overexpression of SOCS-1 in transfected NIT-1 cells inhibits IFN-γ signaling in β-cells including MHC class 1 upregulation and protects β-cells from the cytotoxic effects of the IFN-γ. Overexpression of SOCS-1 has also been demonstrated to inhibit cytokines of the IL-6 cytokine family, type 1 interferons, IL-4 and TNF-α (12). We previously reported that SOCS-1-Tg NOD mice (mice overexpress SOCS-1 under the human insulin promoter) have a markedly reduced incidence of diabetes (13).
Taken together, the above findings suggest that IFN-γ is not only associated with regulation of islet allograft rejection, but also with autoimmune β-cell destruction during diabetes. Blocking this signaling pathway by over-expression of SOCS-1 may enhance islet allograft survival. In the present report, we examined SOCS-1 transgenic (SOCS-1-Tg) islet allograft survival in MHC-mismatched and spontaneously diabetic NOD mice and examined the differences in rejection kinetics between alloreactive and autoimmune responses. Our work demonstrates that alteration in cytokine signaling has divergent effects in these two situations, with the autoreactive barrier being harder to overcome than that presented by MHC mismatched grafts.
MATERIALS AND METHODS
C57BL/6J (non-Tg: H-2b; 8 weeks) and SOCS-1-Tg mice on the B6 background (14; 8 weeks) were pancreatic islet donors. The genotype of knockout and wildtype mouse strains was confirmed by polymerase chain reaction (PCR) analysis of tail DNA (14). BALB/c (H-2d; 8–12 weeks) and aged clinically-diabetic female NOD/Shi transplant recipients were obtained from the rodent-breeding colony at The Scripps Research Institute (TSRI; La Jolla, CA); NOD/Ltj mice were purchased from the Jackson Laboratories (Bar Harbor, ME). Mice were bred and maintained in a specific pathogen-free environment at TSRI, and experiments conducted in accordance with institutional guidelines for animal care and use.
Preparation and Transplantation of Islet Tissue
Pancreatic islets were isolated from SOCS-1-Tg and non-Tg donors by collagenase P digestion (4 mg/ml; Boehringer Mannheim, GmBH, Germany). Handpicked islets were cultured in RPMI 1640 medium supplemented with 10% FCS, 2 mM l-glutamine and 0.2% (v/v), penicillin/streptomycin and 10% CO2, 90% air for at least 6 days prior to transplantation. Islets were embedded in plasma clots prepared from recipient mouse strain plasma to facilitate transplantation. Approximately 500 islets were transplanted beneath the kidney capsule of BALB/c or NOD recipient mice anesthetized with avertin.
Blood Glucose Determinations
Venous blood glucose (BG) concentrations were measured in nonfasting mice using a Glucometer elite (Bayer, PA) to determine graft rejection in NOD autoimmune diabetic mice. Animals were considered diabetic if they had a nonfasting BG value >250 mg/dl for at least 2 consecutive measurements.
Histology and Immunohistochemistry
Paraffin sections of 10% formalin fixed organs or frozen sections of organs fixed in liquid N2 and embedded in OCT compound (Tissue-Tek, Torrance, CA) were prepared, cut in 5-μm thick sections, and stained with a primary antibodies against insulin (DAKO, Carpinteria, CA), CD4, CD8, IL-4 and IFN-γ (BD Pharmingen La Jolla, CA). Primary antibodies were detected with a biotinylated secondary antibody (anti-guinea pig IgG or anti-rat IgG) in conjunction with Vectastatin ABC peroxidase kit (Vector Laboratories, Carpenteria, CA) and chromogen diaminobenzidine (Sigma, St. Louis, MO). Slides were counterstained in Mayer’s Hematoxylin.
Flow Cytometric Analysis
Single cell suspensions from the peripheral lymph node cells (PLN) were prepared, followed by red blood cell (RBC) lysis with ammonium chloride lysis buffer. Cells (5× 105 per sample) were incubated with the appropriate amounts of mAbs (1 μg per million cells) and stained for the cell surface markers CD4 and CD8 obtained from BD Pharmingen (La Jolla, CA). Cells were then washed in PBS, fixated, and permealized before intracellular cytokine staining for IFN-γ and IL-4 (BD Pharmingen).
Culture medium or rIFN-γ (1000U/ml; BD Pharmingen) treated (24 hr) islets were dispersed into single cells using Accutase (Innovative Cell Technologies, San Diego, CA) and stained with the monoclonal antibodies to hemagglutinin, H-2Db and H-2Kd (BD Pharmingen). FACS analysis was performed on a FACScalibur and samples acquired were analyzed by FlowJo (Becton Dickinson). Islets were identified by staining for hemagglutinin and their high flavin adenine dinucleotide autofluorescence detectable on the FL1 channel (at 488 nm islet emission is 510–550 nm (15)). Isotype controls stained with anti-IgG2a mAb, anti-CD49f, and splenocytes were negative controls for staining.
Results are expressed as means ± standard error of mean (SEM). Statistical analysis was performed using Student’s t test (single comparisons) and the nonparametric Mann-Whitney U test (two independent groups).
SOCS-1 Expression Delays Islet Allograft Rejection
We tested whether islet expression of SOCS-1 in pancreatic islets protected them from conventional T1-type CD8 T cell mediated islet allograft rejection. Islets were isolated from transgenic SOCS-1 and control non-Tg mice on the C57BL6/J (H-2b) background, and transplanted into the kidney of completely MHC-mismatched BALB/c (H-2d) mice. Control non-Tg islet allografts were completely rejected by 14 days posttransplant, with no visible evidence of intact islet tissue and intense inflammatory cell infiltrates (Fig. 1A; n=3). In comparison, immunohistochemical staining for insulin confirmed the presence of donor SOCS1-Tg islets for up to 22 days posttransplant (Fig. 1B; n=3). Furthermore, histological analysis of SOCS-1-Tg islet allografts showed that entry of disruptive cellular infiltrates was hindered, and instead inflammatory infiltrates were mainly observed in the periphery of the islets in the graft. This data suggested that overexpression of SOCS-1 in pancreatic islets may be protective against early islet allograft rejection.
Induction of Class I Expression on SOCS-1-Tg Islets
SOCS-1 inhibits cytokine responses, and recent work has demonstrated that its expression can inhibit pathogenicity. To determine whether this inhibition was due to the inability to upregulate expression class I MHC alloantigens, we examined the capacity for islets to upregulate class I MHC in response to in vitro stimulation with rIFNγ (1000 U/ml) for 24 hr. The islets were dispersed into single cells and class I MHC expression was measured by flow cytometry using anti-H-2b/d monoclonal antibodies. For this experiment, we used pancreatic β-cells from SOCS-1.NOD mice crossed with NOD.HA (NOD mice with hemagglutinin under the control of the insulin promoter mice) to specifically identify β-cells via staining for hemagglutinin. Beta-cells were also identified and analyzed quantitatively based upon their autofluorescence due to high accumulation of flavin adenine dinucleotide compared to other islet cells (15).
After IFN-γ treatment, class I H-2Kd expression was only upregulated 1.2-fold in control NOD.HA islets compared to 1.9-fold in SOCS-1-Tg.NOD.HA islets (Fig. 2, E–H). In the case of H-2Db expression, we did not observe upregulation in SOCS-1-Tg.NOD.HA, although this haplotype was upregulated 2.2-fold in NOD.HA (Fig. 2, A–D). These results are consistent with a recent report by Chong et al. (16), which also demonstrated that H-2Db expression was not upregulated in RIP-SOCS-1.NOD in response to 100U/ml IFNg for 4 hr, although H-2Kd expression was not examined. Our data clearly demonstrates that SOCS-1 islets can respond to IFN-γ and upregulate specific subtypes of MHC class I molecules. The weak response to IFN-γ stimulation in the NOD may be due defects in class I MHC antigen processing (17).
SOCS-1-Tg Islets are Rejected in Spontaneously Diabetic NOD Mice
We previously reported that mice expressing SOCS-1 in their pancreatic beta cells on the NOD background have a markedly reduced incidence of diabetes (13). We therefore performed studies to assess whether the SOCS-1-Tg islets on the C57BL6/J background could reverse autoimmune diabetes in NOD mice that had already succumbed to clinical disease. Approximately 500 SOCS1-Tg or non-Tg islets were transplanted beneath the kidney capsule of diabetic (>250 mg/dL) NOD mice. Although, both transgenic (n = 4) and control (n = 3) islet allografts did show evidence of function 1 week after transplant (Fig. 3) and were able to temporarily reverse diabetes, the blood glucose quickly rose above 250 mg/dL after 2 weeks and the grafts were dramatically destroyed on histological examination of the graft site (Fig. 1, C and D). Histology revealed a rapid intense mononuclear infiltrate in both control and SOCS-1-Tg islet allografts that entirely destroyed islet tissue suggesting that diabetes recurred in these mice following engraftment.
The Kinetics of SOCS-1-Tg Islets Allograft Rejection Does Not Differ from Control Non-Tg Islets in Autoimmune Diabetic NOD Mice
On reversion of hyperglycemia, we examined frozen sections of NOD islet allografts for the presence of CD4+ and CD8 + T cells, and for the T1-cytokine IFN-γ and T2-cytokine IL-4. Additionally, intracellular cytokine staining for IFN-γ and IL-4 was performed on peripheral lymph node cells of recipient NOD mice, which received non-Tg and SOCS-1-Tg islet allografts. Interestingly, both immunohistochemistry and intracytokine staining revealed that rejection of both non-Tg and SOCS-1-Tg islets is predominately T2-mediated, as evidenced from the increased IL-4 production and the less prominent T1-type IFN-γ-mediated immune response normally associated with rejection of allogeneic islets (Table 1); IFN-γ production by allogeneic CD8+ T cells has been demonstrated previously (5). However, there were no significant differences in the expression of IL-4 when gated on CD4+ and CD8+ T cells in non-Tg islet allografts and SOCS-1-Tg islet allografts. Furthermore, less than 0.5% of CD4+ and CD8+ T cells taken from NOD hosts that received either non-Tg (n = 3) or SOCS-1-Tg (n = 4) islets expressed IFN-γ. Immuno-histochemical staining further revealed that the islet allografts contained both a mixture of both CD4+ and CD8+ T cells (data not shown). These results suggest that the transplanted islets are destroyed via a distinct population diabetogenic T cells.
Our findings indicate that overexpression of SOCS-1 is beneficial against T1-type CD8 T cell mediated allogeneic islet allograft destruction in normal mice, extending islet survival beyond 22 days in control mice compared to just 14 days in control mice. Whereas many diabetes models induce hyperglycemia chemically in transplant recipient mice using β-cell toxins such as streptozotocin or alloxan monohydrate, these study only allorecognition of the islet tissue and do not account for the effects that recurrence of autoimmunity has on the fate of the islet allografts. Transplantation of islets directly into spontaneously diabetic NOD mice is valuable for analysis of islet allotransplantation as a possible treatment of T1D. Here we demonstrate that SOCS-1 expression in islet allografts is a less effective strategy to prevent rejection in the NOD mouse model of autoimmune diabetes than in normal mice. We found that there was no difference in the tempo and kinetics of SOCS-1-Tg and non-Tg islet allograft destruction in the NOD mouse. Both SOCS-1-Tg islets and control islet allografts were rejected by a predominately T2-type cytokine (IL-4) immune mechanism and infiltrate of both CD4+ and CD8+ T cells at 2 weeks posttransplant. In NOD mice, transplanted syngeneic and allogeneic islets are rapidly destroyed by CD4+ and not CD8+ T cell dependent mechanisms (18).
Recent studies have suggested that over-expression of SOCS-1 in islets would be beneficial in protection also against autoimmune destruction. Flodstrom-Tullberg et al. (13) demonstrated that diabetogenic T cells transfer disease less efficiently to NOD mice, which overexpress SOCS-1 in their islets. Additionally, lymphocytes infiltrating the pancreas of SOCS-1-Tg mice had a reduced efficiency to transfer diabetes than lymphocytes from non-Tg mice (13).
Chong et al. also demonstrated that over-expression of SOCS-1 under the control of the rat insulin promoter in CD8+ TCR transgenic NOD 8.3 mice completely prevented progression of diabetes (16). They concluded that SOCS-1 protection was mediated by blocking β-cell responses to cytokine signaling, such as Fas upregulation and IFN-γ-induced class I upregulation, thereby inhibiting recognition of islets by islet reactive CD8+ T cells (16). However, the contribution of CD4+ T cells to autoimmune diabetes (19) was not recognized, and the fact that islet allograft rejection is predominately CD8 T cell mediated may account for the effects observed (5). Our observation that SOCS-1 could not completely inhibit islet allograft rejection and class I MHC upregulation may be accounted for by an incomplete inhibition of IFN-γ signaling by SOCS-1, cross talk between other secondary messengers, low expression of the transgene, or superfluous effects on other cytokine signaling (20). Chong et al. have similarly demonstrated that IFN-γ signaling is not fully regulated by SOCS-1 when NIT-1 insulinoma cells were transfected with SOCS-1 (11, 21). Overall, these studies indicate that SOCS-1-Tg β-cells are less sensitive to destruction by activated self-reactive immune cells.
Transfer experiments have shown that CD4+ and CD8+ T cells are essential for the development of insulitis in the NOD mouse (19, 22). The indefinite survival of syngeneic β2m-deficient islet transplants in NOD mice demonstrate that MHC class I molecules and CD8 T cells play a major role in autoimmune β-cell destruction and recurrent diabetes in transplanted islets (23). Moreover, whereas graft antigens such as class I MHC are targeted by alloreactive CD8 T cells, anti-islet autoantibodies such as glutamic acid decarboxylase (GAD) and insulin exist which can perpetuate islet destruction. Gagnerault et al. (24) demonstrated the importance of autoantibodies, demonstrating protection from insulitis and diabetes in NOD mice when they removed pancreatic lymph nodes at 3 weeks of age. This suggests that anti-islet response plays an important role in the development of T-cell mediated autoimmune diabetes, and may initiate or accelerate to islet allograft rejection in autoimmune diabetic mice.
The successful treatment of T1D patients with an allogeneic islet transplant requires two major obstacles to be overcome: the transplanted islets must withstand allogeneic rejection and the destruction by the host’s anti-islet immune response. Here we demonstrated that modulation of immunogenicity of islet allografts through overexpression of the SOCS-1 genes prolonged islet allograft rejection. SOCS-1 expression, however, could not protect from the recurrence of disease in spontaneously-diabetic NOD mice. In conclusion, SOCS-1 may be successfully combined with the expression of other genes altering β-cell vulnerability to autoimmune destruction in order to develop new strategies to induce tolerance in islet allograft recipients with T1D.
We would like to thank Daisy Dietz, Patrick Secrest, Mary Cleary, and Leah Varney for their technical assistance.
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