Introduction

Type 1 diabetes (T1D) is an immune-mediated disease resulting from inflammatory cell and cytokine-mediated absolute destruction of the pancreatic insulin-producing beta (β) cells [1]. The cytokine products of T helper (Th) cells are crucial to the development of an effective immune response [2]. A current hypothesis is that Th1 cells and their cytokine products, such as interleukin(IL)-2, IFN-γ and TNF-β, activate macrophages and cytotoxic T cells to destroy β-cells, causing T1D, whereas Th2 cells and their cytokine products, such as IL-4 and IL-10, down-regulate Th1 cells and cytokines and thereby prevent T1D [3]. The balance between Th1 and Th2 cells appears to be vitally important. Hence, a shift of the immune system from Th1-like immunity to Th2-like immunity may represent an attractive and reasonable therapeutic strategy for T1D. Th2-like cytokine IL-10 has been one of the most extensively investigated and promising candidates for effective immune diversion for diabetes treatment [4].

IL-10 was first described as cytokine synthesis inhibitory factor (CSIF) [5], an activity produced by mouse Th2 cells that inhibited activation of and cytokine production by Th1 cells. IL-10 profoundly inhibited a broad spectrum of activated macrophage/monocyte functions, including monokine synthesis, NO production, and expression of class II MHC and costimulatory molecules such as IL-12 and CD80/CD86 [6]. These functions constitute important components of the anti-inflammatory and immunosuppressive effects of IL-10. Because of these immunosuppressive abilities, IL-10 has been considered to be an effective agent for treating Th1-mediated autoimmune diseases. In fact, numerous studies have described the correlations between autoimmune diabetes attenuation and increased IL-10 production, suggesting that IL-10 has a protective effect. In nonobese diabetic (NOD) mice, IL-10 disappeared from both islets and pancreatic exocrine tissue at the time when diabetes was first detected [7]. In high risk T1D first degree relatives, IL-10 production in vitro was decreased, supporting the hypothesis that IL-10 is involved in the pathogenesis of T1D [8]. Depending on the time and mode of administration (early vs. late, systemic vs. local), treatment with the IL-10 can inhibit the development of T1D in NOD as well as prevent the recurrence of disease. For example, early systemic treatment with exogenous murine IL-10 (mIL-10) by various means (e.g., rIL-10 protein, immunoglobulin fusion protein, or injected expression plasmid administration) can inhibit T1D [9–11]. Treatment with IL-10 also reduced the severity of insulitis and prevented cellular infiltration of islet cells. Due to a relatively short half-life, treatment with a recombinant IL-10 protein requires repeated or continuous administrations. Viral vectors are attractive gene transfer vehicles because they often mediate highly efficient gene transfer and stably express genes. The most commonly used viral vectors are those derived from human adenoviruses. Adenovirus vectors have been shown to transduce pancreatic islet cells efficiently with greater than 70% of human pancreatic endocrine cells and rodent islets being transduced ex vivo [12, 13]. The capacity of this virus to infect non-dividing cells allows insertion of cDNA into pancreatic islets [13].

Since side effects of systemic gene transfer strategies must be considered, the genetic modification of cells in vitro is an interesting option. There are few data regarding the direct effects of IL-10 on islet β cells function. To address these questions, we used adenoviral vectors carrying the enhanced green fluorescent protein (EGFP) reporter genes and transduced to insulin-secreting pancreatic islet β cells—RINm5F cells directly. To evaluate the efficacy of adenovirus vector-mediated mIL-10 gene transfer in vitro and to determine if mIL-10 could protect RINm5F cells from pro-inflamatory cytokines-IL-1β mediated inhibitory effects and apoptosis.

Materials and methods

Cell and cell culture

Human embryonic kidney cells (HEK 293, kindly provided by Prof. Bing Luo, Medical college of Qingdao University, China) were cultured in Dulbecco’s modified Eagle medium (Sigma–Aldrich, St. Louis, MO, USA). The insulin-producing rat β-cell line RINm5F (obtained from the Center of Experiment Animal of Sun Yat-sen University, China) was cultured in Rosewell Park Memorial Institute (RPMI) 1640 medium (Sigma-Aldrich). Both media were supplemented with 10% fetal bovine serum (FBS) and antibiotics.

Construction of recombinant adenoviral vector expressing mIL-10

Recombinant adenovirus containing the mIL-10 gene was prepared by use of the AdEasy System (also gifted by Prof. Bing Luo). Briefly, the sense primer (5′-GGCAGATCTATGCTTGGCTCAGCACTG-3′) and the antisense primer (5′-GCGATATCCCTGCAGTCCAGTAGACG-3′) were used for cloning the mIL-10 gene directly from RNA of mouse splenic cells by using RT-PCR. The underline represented shearing site of BglII (AGATCT) and EcoRV (ATATC). Both mIL-10 and the shuttle vector pAdTrack-CMV were digested by BglII and EcoRV. Subsequently, two DNA fragments were religated using T4 DNA ligase. The new plasmid, pAdTrack-CMV-mIL-10, was identified by gene sequencing and then cut by PmeI and the fragment of interest cloned by homologous recombination in the competent BJ5183 bacteria with pAdEasy-1. The pAdEasy-mIL-10 recombinant was identified by restriction digestion with enzymes BglII/EcoRV and PacI. The linear DNA fragment after digestion of pAdEasy-mIL-10 with PacI was isolated and transfected into HEK293 cells using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Transfected cells were monitored for EGFP expression and collected 7–10 days after transfection. The cells were lysed by four times of freezing and thawing (alternating between −80 and 37°C). Viral particles were purified by cesium chloride density gradient centrifugation. Recombinant adenovirus expressing EGFP (Ad-EGFP) was constructed similarly and used as a control virus in all experiments. All the viral titers were 5.5 × 1010 pfu/ml. Viral suspensions in 3% sucrose were stored at −80°C until thawed for use.

Transduction of RINm5F cells with Ad-mIL-10

To assess the optimal multiplicity of infection (MOI) for maximal transgene expression, exponentially growing RINm5F cells were transduced with Ad-mIL-10 at various MOIs for 1 h with frequent gentle shaking and then incubated with complete medium for the experiment. Two days after culturing, fluorescence microscopy was used to observe the expression of GFP in infected cells. Cells in the control group were mock infected. Mock-infected cells underwent a similar procedure, but were not exposed to the virus during the incubation period and were not transduced with any vector. All the functional assays described below were performed in triplicate on at least three different occasions unless otherwise indicated.

mIL-10 sequencing assay of transductant

Exactly 72 h after infection, total RNA was extracted separately from RINm5F cells. Expression of IL-10 mRNA was detected by RT-PCR. The PCR conditions were: (1) 94°C for 5 min; (2) 35 cycles of 30 s at 94°C, 1 min at 55°C, and 1 min at 72°C; and (3) 72°C for 10 min. Sequence of DNA was assayed by Sangon Company (Shanghai, China).

Western blotting analysis

RINm5F cells treated with Ad-mIL-10, Ad-EGFP or mock infected for 48 h were harvested for western blotting analysis. Total cell lysates were resolved by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked by incubation for 2 h at room temperature with 3% nonfat dry milk in phosphate-buffered saline (PBS). The membrane was incubated with primary antibody anti-IL-10 (1:2000) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), for 2 h in blocking solution at room temperature. All of the membranes were then washed and incubated for 1 h at room temperature, and incubated overnight with alkaline phosphatase conjugated secondary antibody (1:10,000, Sigma-Aldrich).

Enzyme-linked immunosorbent assay (ELISA) for mIL-10 protein expression

RINm5F cells were seeded at 1 × 105 in a 24-well plate and cultured in growth medium. Cells were transduced with 100 MOI of Ad-mIL-10 or Ad-EGFP and incubated for 48 h. IL-10 in the cell culture supernatants were measured by ELISA kit (R&D Systems, Abigdon, U.K.).

MTT assay for cell viability

Cells were treated with Ad-mIL-10 or with Ad-EGFP then were continued to culture for 24, 48, 72, and 96 h. At the end of the treatment, cells were incubated with MTT (Sigma-Aldrich). After 4 h, the supernatant was removed and replaced with 100 μl dimethyl sulfoxide (DMSO). After 20 min, cells were centrifuged at 4°C at 14,000 rpm. The absorbance was measured at 490 nm using a microplate reader.

Evaluation of β cell function and nitrite concentration induced by IL-1β

To assess the effects of IL-1β on β-cell function, we used glucose-stimulated insulin secretion as a functional assay. RINm5F cells were pretreated with Ad-mIL-10, Ad-EGFP or mock infected for 24 h, and then IL-1β (10 ng/ml) were added for 12 h. The IL-1β-containing medium was removed and the cells were washed twice with Kregs–Ringer bicarbonate buffer (KRB) supplemented with 10 mmol/l HEPES (KRBH) and 2 mg/ml BSA. Incubation was carried out at 37°C in KRBH buffer for 30 min followed by an additional incubation for 30 min in the presence of 1.67 or 16.7 mmol/l glucose. Insulin concentrations were measured by ELISA Kit (R&D Systems).

NO production was measured as nitrite accumulation in conditioned media determined by the Griess reaction. To measure nitrite, 100 μl aliquots were removed from conditioned medium and incubated with an equal volume of Griess reagent at room temperature for 10 min. The absorbance at 540 nm was determined using microplate reader. NO was determined by using sodium nitrite as a standard.

Morphological changes of apoptotic cells by Hoechst 33258

Cells were fixed in 3.7% formaldehyde for 5 min at room temperature (RT) and the membranes were permeabilized with methanol for an additional 5 min at RT. Cells were then rinsed with PBS and incubated with Hoechst 33258 (Sigma-Aldrich) for 15 min at RT. Images were collected on fluorescence microscopy.

Fas expression detected by flow cytometric analysis

For determination of Fas-expressing cells, the cells were harvested with trypsin, washed twice with PBS, and were stained with a polyclonal rabbit anti-Fas antibody (Stress Gene, Victoria, BC, Canada) in 75 μl cold FACS buffer (1× PBS, 1% FBS, 0.1% sodium azide). After washing with PBS, the cells were incubated for 30 min on ice with 10 μg/ml FITC-conjugated anti-Fas mAb (BD Pharmingen, San Diego, CA, USA), or a FITC-conjugated isotype-matched control Ab in 100 μl of FACS buffer. The supernatant was discarded. Cells were resuspended with 500 μl of 2% FBS/PBS and analyzed by FACS.

Measurement of caspase-3 activity

Caspase-3 activity was measured in lysates of RINm5F cells using the caspase-3 colorimetric assay protease kit (Keygen Biotech. Co., LTD, Nanjing, China) following the instructions of manufacturer. In brief, the cells (5 × 106) were lysed with 50 μl of chilled cell lysis buffer on ice for 20 min. After centrifugation for 5 min at 10,000×g, supernatant was transferred to a fresh tube. Protein levels in the lysates were measured by the Bradford assay and equalized accordingly to obtain 200 μg of cytosolic extract per sample. Samples were incubated with 5 μl caspase-3 substrate at 37°C for 4 h. Samples were analyzed at 405 nm in a microtiter plate reader. The results are presented as % of untreated cells.

Statistical analysis

Statistical analysis was performed with SPSS 11.5 software. Data were expressed as mean ± standard deviation (SD). Statistically significant differences were determined by Student’s t test and one-way analysis of variance (ANOVA) as appropriate. A value of P < 0.05 was considered statistically significant.

Results

Generating recombinant adenovirus with mIL-10

We have generated an adenovirus vector, Ad-mIL-10, which carries the coding sequence of the mIL-10 gene. The recombinant adenoviral vector was validated by restriction endonuclease analysis (Fig. 1a). Under fluorescence microscopy, green fluorescence indicating expression of EGFP was strong in HEK 293 cells (Fig. 1b). We concluded that the recombinant adenoviral vector Ad-mIL-10 was constructed successfully.

Fig. 1
figure 1

Construction of recombinant adenoviral vector Ad-mIL-10 and determination of GFP expression under fluorescence microscopy. a Verification of adenoviral plasmid vector pAd-mIL-10 by means of the restriction endonucleases PacI. DNA marker DL15000 (M), pAd-mIL-10 digested by Pacl (lane 1). b Transfection of recombinant adenovirus in HEK293 cells. Uninfected control group (panel ①), infected with Ad-EGFP group (panel ②), infected with Ad-mIL-10 group (panel ③)

Full size image

Ad-mIL-10 gene transfer to RIN cells results in high IL-10 expression

At an MOI of 100 and higher, more than 70% of the RINm5F cells transfected with Ad-mIL-10 were GFP positive (Fig. 2a). We also found that MOI between 50 and 300 did not affect the viability of RINm5F, which remained in excess of 95% (as determined by trypan blue exclusion staining, data not shown). Therefore, an MOI of 100 was selected as the optimal dose for transfection of RINm5F cells.

Fig. 2
figure 2

Expression of mIL-10 in RIN cells following infection by Ad-mIL-10. a GFP reporter expression in adenoviral transduced cells under phase contrast microscopy (panel ①), under fluorescence microscopy (panel ②). At 48 h after Ad-mIL-10(MOI = 100) transduction, fluorescence microscopy was used to observe the expression of GFP in infected cells. b RT-PCR analysis of mIL-10 gene expression in RIN cells. PCR Marker DL2000 (M), negative control (lane 1), PCR amplification product (lanes 2 and 3). Total mRNA samples were isolated from RIN cells 72 h after the transduction of Ad-mIL-10 or Ad-EGFP. After the reverse transcriptase reaction, mIL-10 mRNA levels were measured by semiquantitative RT–PCR assay. c The detection of IL-10 protein expression of RINm-5F cells by western blotting. α-Tublin was used as an internal control. d mIL-10 expression measured by ELISA. IL-10 levels were increased significantly accompanying with increased MOI and peak expression was seen on 100 MOI, however, mIL-10 expression decreased when increased the viral dose. e MTT assay was used to measure the effects of Ad-mIL-10 on the growth of RIN cells in vitro. Growth of Ad-mIL-10 infected cells were no difference compared with Ad-EGFP infected cells and mock-infected RIN cells

Full size image

IL-10 expression in RIN cells

RT-PCR was used to detect the mIL-10 expression at the transcriptional level. The results of the RT-PCR analysis demonstrated that IL-10 mRNA was stably expressed in RINm5F cells infected with Ad-mIL-10 (Fig. 2b) and cDNA sequence was consistent with the GenBank sequence (NM010548). We next examined the expression of mIL-10 protein via western blotting analysis. Expression of mIL-10 was observed only in the Ad-mIL-10-transfected RINm5F cells (Fig. 2c), demonstrating the Ad-mIL-10-mediated effective infection and specific expression of mIL-10 gene in RINm5F cells. IL-10 protein was also detected in the culture supernatant using a commercially available ELISA kit. IL-10 in the media of uninfected cells and Ad-EGFP infected cell were undetectable, however, in Ad-mIL-10-infected cells, a significantly higher level of IL-10 was detected (Fig. 2d). ELISA analysis revealed that islet cell could effectively express and secrete IL-10 after being transduced with Ad-mIL-10.

The viability of cells in vitro was measured by MTT assay. Relative cell number was evaluated by comparing the absorbance in each cell at 24, 48, 72, and 96 h. As shown in Fig. 2e, there were no statistically significant difference between the growth of adenovirus infected cells and mock-infected RINm5F cells, suggesting that adenovirus infection did not influence their viability.

Prevention of IL-1β-induced suppression of glucose-stimulated insulin release as well as nitric oxide production with adenoviral gene transfer of IL-10 to RIN cells in vitro

To determine if IL-10 is able to block the effects of IL-1β on islet β cell function, RIN cells were treated with recombinant IL-1β. The effects of Ad-mIL-10 on IL-1β-induced inhibition of insulin secretion are shown in Fig. 3a. After 24 h incubation, IL-1β completely inhibits glucose-stimulated insulin secretion, and this effect is attenuated by Ad-mIL-10.

Fig. 3
figure 3

Effect of Ad-mIL-10 transduction on glucose-stimulated insulin release (a) and NO production (b) incubation with IL-1β. RIN cells were pretreated with Ad-EGFP, Ad-mIL-10, or mock infected for 24 h and then exposed to 10 ng/ml of IL-1β for 12 h. a Glucose-stimulated insulin release assay and insulin was measured in the supernatant by ELISA. RIN cells were incubated for 60 min at 1.67 mmol/l glucose followed by a second 60 min incubation at 16.7 mmol/l glucose. After the incubations, the insulin content was measured. Data are means ± SD from three independent experiments. * P < 0.01 vs. control; ** P < 0.05 vs. IL-1β-treated group. b NO released in the medium was quantitated using Griess reagent. Data are means ± SD from three independent experiments. * P < 0.01 vs. control; ** P < 0.05 vs. IL-1β-treated group

Full size image

It has been reported that IL-1β-mediated destruction of β cells is caused by an increase of NO. Incubation of RIN cells with IL-1β for 12 h resulted in significant production of nitrite (a stable oxidized product of NO) by these cells. In the presence of IL-1β, however, Ad-mIL-10-infected cells displayed a significant decrease in NO production compared with Ad-EGFP-infected and mock-infected cells (P < 0.05). These results demonstrate that IL-10 is able to block the production of NO in response to IL-1β (Fig. 3b).

Effect of IL-10 overexpression on IL-1β-induced apoptosis

To test whether Ad-mIL-10 can protect pancreatic β-cells from IL-1β-induced apoptosis, the effects of Ad-mIL-10 were investigated in the presence of IL-1β. Micrographs showing the effect of cytokines on apoptosis in RINm5F cells were checked by Hochest 33258 stain. Highly condensed or fragmented nuclei represent apoptosis (Fig. 4a). Culturing cells with IL-1β in the presence of Ad-mIL-10 suppressed IL-1β-induced apoptosis.

Fig. 4
figure 4

Adenovirus-mediated IL-10 production inhibits IL-1β-induced apoptosis, Fas expression, and caspase-3 activity in insulin-producing RIN cells. RIN cells were infected with Ad-EGFP, Ad-mIL-10 or mock infected for 24 h and then exposed to 10 ng/ml of IL-1β for 12 h. a Apoptosis in RIN cells by Hoescht 33258 staining. Representative images of RIN cells in different groups: Control group (panel ①), Ad-EGFP group (panel ②), Ad-mIL-10 group (panel ③), IL-1β group (panel ④), Ad-EGFP + IL-1β group (panel ⑤), Ad-mIL-10 + IL-1β group (panel ⑥). Nuclear fragmentation due to IL-1β is shown by arrows. A total of six different fields per dish were counted to quantify the number of apoptotic cells. Study was done in duplicate for each condition tested. Data are mean ± SD from three independent preparations. In the histograms, * P < 0.01 vs. control; # P < 0.01 vs. when compared with the IL-1β-treated group. b Flow cytometric analysis of surface Fas expression in RIN cells. Results shown are means ± SD of three experiments. * P < 0.01 vs. controls, # P < 0.01 vs. IL-1β-treated group. c IL-10 prevents the increase in caspase-3 activity evoked by IL-1β. Caspase-3 activity was measured by caspase-3 colorimetric assay protease kit. Data are the mean ± SD of three independent preparations (n = 8 each group). * P < 0.01 vs. control; # P < 0.01 vs. IL-1β-treated group

Full size image

As the Fas/FasL-pathway of apoptosis is thought to be involved in autoimmune destruction of β-cells, we determined the expression of Fas (CD95) in control and IL-1β-treated cells by flow cytometry. Flow cytometry analysis of Fas expression showed that untreated RIN cells expressed low levels of Fas. However, IL-1β alone strongly induced expression of Fas on RINm5F cells. Following 24 h exposure of non- or Ad-EGFP transduced RIN cells to IL-1β, an increase in the number of Fas-positive cells was observed. Compared with this, the induction of apoptosis was reduced when the cells were transduced with the Ad-mIL-10 (Fig. 4b).

Caspase-3 is known to play an important role in the signaling and execution of apoptosis. To test the effects of Ad-mIL-10 on the viability of IL-1β-treated RIN cells, we investigated the activity of caspase-3. Incubation of control RINm5F cells with the pro-inflammatory cytokines IL-1β for 12 h caused increase of caspase-3 activity. Ad-mIL-10 or Ad-EGFP treatment alone did not show any effect on caspase-3 activation. However, the cells pretreated with Ad-mIL-10 significantly decreased IL-1β induced caspase-3 activity (Fig. 4c).

Discussion

Ex vivo gene transfer to pancreatic endocrine cells is an effective approach to gene therapy for T1D. The use of local gene expression is advantageous over systemic virus vector administration given that it circumvents the off-target side effects that are associated with systemic gene therapy and systemic expression of the therapeutic protein [14]. The genetic modification of islets is an interesting option. Therefore, we used the gene expression approach in vitro to transfer mIL-10 gene to insulin-producing cell to investigate weather it could reserve the insulin release and protect pancreatic β cells from destruction directly.

In the present study, we have demonstrated that adenoviral gene transfer of the mIL-10 gene to the rat pancreatic β cell can express both mRNA and protein in vitro effectively. It is important to note that adenoviral infection of RINm5F cells in culture does not change their viability or functional characteristics at multiplicities of infection we used. Then we demonstrated that Ad-mIL-10 infection was able to block the inhibitory effects of IL-1β on glucose-stimulated insulin secretion, NO production, Fas-dependent apoptosis activation, and caspase-3 activity.

Pro-inflammatory cytokines, especially IL-1β, play an important role in β-cell destruction. In rodent β cells and insulinoma cell line (RIN), IL-1β alone or in combination with IFNγ induces the expression of inducible nitric oxide synthase (iNOS) resulting in production of NO [15]. IL-1β acts on β-cells via IL-1 receptors. The signal transduction pathways involve activation of the transcription factor nuclear factor-κB (NF-κB) pathway, which is essential for regulation of multiple pro-apoptotic genes, including iNOS [16]. We observed significant decrease in NO production in Ad-mIL-10-infected cells exposed to IL-1β compared with uninfected cells. In contrast, Ad-EGFP infection followed by IL-1β treatment, resulted in a dramatic increase in NO production. Cultures of RIN cells showed a marked inhibition of glucose-induced insulin release after exposure to IL-1β. Exposed to IL-1β, insulin secretion of transduced cells in response to basal glucose concentration resulted in expected low values. In response to high glucose, Ad-mIL-10 transduced cells showed an increased insulin secretion comparable with that of uninfected cells and Ad-EGFP-transduced cells. IL-1β inhibits insulin secretion by inducing the expression of iNOS and the production of NO. A reduction of IL-1-induced NO production due to the action of IL-10 has been previously observed with rat islets and RIN cells [17, 18], even though the mechanisms remain unknown. This study indicated that Ad-mIL-10 can inhibit IL-1β induced producing NO and increase insulin secretion.

There is increasing evidence that apoptosis is the main mode of β-cell death leading to T1D. IL-1β induced NO production, which was responsible for mitochondrial damage, caspase activation, and apoptosis in rat insulinoma (RIN) cells [15]. Moreover, NO contributes to cytokine-induced apoptosis via potentiation of c-Jun N-terminal kinase (JNK) activity, a member of the mitogen-activated protein kinase (MAPK) family, and suppression of Akt [19]. IL-1β is able to activate caspase-3 in β-cells and this effect is possibly linked to NO production [20]. The pro-inflammatory cytokine-stimulated increase in caspase-3 activity was also substantially blocked by pretreated with Ad-mIL-10, which reduced iNOS expression. However, there are studies demonstrating that cytokine-induced β-cell death can be NO-independent [21]. Fas is a potential mechanism of pancreatic β cell death in T1D. Pro-inflammatory cytokines have a direct, NO- and Fas-independent effect on β-cell viability [21]. So, we test Fas expression on the surface of β-cells by flow cytometry analysis. Although normal pancreatic β-cells do not express Fas on their surface, exposure of these cells to IL-1 or IL-1 plus INF induces Fas expression [22]. Increased Fas expression was clearly observed on β cells during accelerated diabetes caused by transfer of diabetogenic splenocytes or in several TCR-transgenic models [23]. In this study, we showed that IL-1β induced an increase in Fas expression on RIN cell, most important, IL-10 treatment of these cells diminished IL-1β-induced apoptosis and decreased cytokine-induced Fas expression. IL-10 might act on either of the NO or Fas pathway. We demonstrated a potent effect of IL-10 in decreasing inflammatory cytokine-induced apoptosis of RIN cell. IL-10 is a potent inhibitor of the production of the proinflammatory cytokines associated with the development of Th1-type responses. It has been shown to modulate various inflammatory processes and regulates cell survival in various cell systems [24]. Regulation of apoptosis by IL-10 has been shown in promyeloid cell, chondrocyte, and intestinal epithelial cell systems [25–27]. IL-10 might decrease Fas-mediated apoptosis by decreasing caspase-3 activity through the upregulation of FLIP and downregulation of caspase-8 activity [27].

In conclusion, we have demonstrated the feasibility of using an adenoviral vector encoding IL-10 to infect insulin-producing cells as a means of preventing IL-1β-induced impairment of β cell function. Additionally, our results demonstrate that mIL-10 production from genetically modified rat β-cells can suppress NO production and Fas-dependent apoptosis activation induced by IL-1β.