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Endocrinology Vol. 143, No. 12 4809-4819
Copyright © 2002 by The Endocrine Society

ARTICLE

1,25-Dihydroxyvitamin D3 Protects RINm5F and Human Islet Cells against Cytokine-Induced Apoptosis: Implication of the Antiapoptotic Protein A20

Rita Riachy, Brigitte Vandewalle, Julie Kerr Conte, Ericka Moerman, Paola Sacchetti, Bruno Lukowiak, Valery Gmyr, Thomas Bouckenooghe, Mathilde Dubois and François Pattou

Cellular Therapy of Diabetes, Institut National de la Santé et de la Recherche Médicale, Equipe de Recherche et d’Innovation Méthodologique 0106 (R.R., B.V., J.K.C., B.L., V.G., T.B., M.D., F.P.), and Institut National de la Santé et de la Recherche Médicale Unité 459 (E.M., P.S.), Faculté de Médecine, 59045 Lille, France

Address all correspondence and requests for reprints to: B. Vandewalle, Thérapie Cellulaire du Diabète, Institut National de la Santé et de la Recherche Médicale, Equipe de Recherche et d’Innovation Méthodologique 0106, Faculté de Médecine, Place de Verdun, 59045 Lille, France. E-mail: bvandewalle{at}univ-lille2.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Transplantation of islets of Langerhans is a potential cure for type 1 diabetes, but its success is hampered by destruction of the islets. The data presented herein suggest that the active metabolite of vitamin D3 [1,25-(OH)2D3] may promote islet cell survival by modulating the effects of inflammatory cytokines, which contribute to ß-cell demise. We investigated some of the mechanisms triggering the apoptotic machinery in rat insulinoma RINm5F cells and human islets treated with IL-1ß plus interferon-{gamma} plus TNF{alpha} and assessed the effects of 1,25-(OH)2D3 in these processes. Mitochondrial transmembrane permeability and apoptotic features, determined by percentage of sub-G1 cells, quantitation of DNA strand breaks, and Hoechst staining, were significantly increased by cytokines and reverted toward control values by 1,25-(OH)2D3 cotreatment. The cytoprotection of cells correlated with the abrogation of cytokine-induced nitric oxide production. The activation of nuclear factor-{kappa}B plays a key role in the different pathways implicated in nitric oxide generation. We demonstrated for the first time, in both RINm5F cells and human islets, that 1,25-(OH)2D3 was able to induce and maintain high levels of A20, an antiapoptotic protein known to block nuclear factor-{kappa}B activation. Our study showed a clear efficiency of 1,25-(OH)2D3 on the apoptotic machinery triggered by cytokines in ß-cells and suggests that 1,25-(OH)2D3 could help overcome a major obstacle encountered in the cellular therapy of diabetes, such as nonfunction in the immediate posttransplantation period.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TRANSPLANTATION OF PANCREATIC islets of Langerhans is a promising therapy for type 1 diabetes, potentially restoring physiologic insulin secretion. In experimental models of islet transplantation, a loss of grafted ß-cell mass has been observed and attributed, at least in part, to ß-cell apoptosis (1). Indeed, apoptotic activity within islet cells has been shown to increase in the immediate period post ovulation (2, 3). In addition, the inflammatory response activates cell death within the islet and adversely influences engraftment. Impaired function and destruction of ß-cells result from direct contact with islet-infiltrating macrophages and T lymphocytes and/or exposure to inflammatory products of the islet-infiltrating cells such as free radicals and cytokines.

Inflammatory cytokines IL-1ß, interferon (IFN)-{gamma}, and TNF{alpha} are cytotoxic to ß-cells in vitro. They have been shown to induce apoptosis in purified primary human (4), rat (5), and mouse (6) ß-cells, possibly by stimulating the synthesis of inducible nitric oxide synthase (7) and nitric oxide (NO) (8). NO is highly efficient in inducing mitochondrial permeability transition, thereby causing the liberation of apoptogenic factors from mitochondria, which can induce DNA fragmentation in pancreatic ß-cells (9).

The induction of inducible nitric oxide synthase in ß-cells is predominantly regulated by the nuclear factor-{kappa}B (NF{kappa}B) (10). NF{kappa}B is normally inactive and sequestered in the cytoplasm as a heterodimer comprising two polypeptides of 50 and 65 kDa noncovalently complexed with a cytoplasmic inhibitory protein, inhibitor of {kappa}B (I{kappa}B) (11). In response to a variety of stimuli such as cytokines, I{kappa}B proteins are rapidly phosphorylated by the I{kappa}B kinase (IKK). This results in the liberation of NF{kappa}B from I{kappa}B and subsequent translocation of NF{kappa}B to the nucleus in which it regulates gene transcription. The antiapoptotic A20 gene blocks the activation of NF{kappa}B upstream from the kinase cascade leading to I{kappa}B degradation (12). Consequently, A20 is a candidate gene potentially able to down-regulate the synthesis of cytotoxic mediators.

Frequent suboptimal primary islet function on transplantation has drawn attention to the factors promoting islet engraftment and minimizing graft cell injury. The active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], is a secosteroid hormone that not only regulates bone and calcium/phosphate metabolism but also exerts a number of other biological activities including immunomodulation (13) and insulinotropic effect in pancreatic ß-cells (14) and vitamin D-deficient rats (15). 1,25-(OH)2D3 and its analogs also have been shown to reduce the incidence of diabetes (16) and prolong graft survival in spontaneously diabetic nonobese diabetic mice (17). We have previously shown that 1,25-(OH)2D3 might reduce the vulnerability of the human pancreatic islets cells to in vitro cytotoxic cytokine challenge by decreasing major histocompatibility complex class I induction, IL-6 production, and nitrite release, a reflection of NO production (18).

In the present study, we investigated the early mechanisms implied in ß-cell death by first focusing on the rat insulin-producing RINm5F ß-cell line, a model currently used for the study of pancreatic cell death (10, 19, 20, 21, 22, 23, 24). We particularly studied the disruption of the mitochondrial transmembrane potential ({Delta}{psi}m) and apoptotic features to ascertain the counteracting action of 1,25-(OH)2D3 on the mechanistic targets implied in the death processes such as NF{kappa}B activation and NO production. We then focused on the effect of 1,25-(OH)2D3 on the expression of the antiapoptotic A20 gene and its induction by cytokines in RINm5F cells, and we subsequently extended these experiments to human pancreatic islet cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
The rat insulin-producing ß-cells RINm5F obtained from ATCC (Manassas, VA) were trypsinized every 5 d and subcultured (1 x 105cells/cm2) at 37 C in 5% CO2 and maintained for 24 h in RPMI 1640 medium supplemented with 10% fetal calf serum and antibiotics. Cell processing consisted of treatments for 48 h in different media including cytokine-treated medium: basal medium plus IL-1ß (50 IU/ml), IFN-{gamma} (1000 IU/ml), and TNF{alpha} (1000 IU/ml; cytokines from Valbiotech, AbCys, Paris, France) and cytokine-treated medium containing 1,25-(OH)2D3 dissolved in ethanol and used at physiological (10-8 M) and pharmacological (10-6 M) concentrations. Alcohol concentration did not exceed 0.01% in the culture media and was without measurable effect on cell cultures. 1,25-(OH)2D3 was a generous gift from Hoffmann la Roche AG (Basel, Switzerland).

In some experiments, cells were incubated with the NO synthase inhibitor NG-methyl-L-arginine (NOi, 5 mm; Sigma-Aldrich Chemicals, Saint Quentin Fallavier, France), added for 1 h before stimulation with cytokines. After incubation, analyses were performed on free-floating cells pooled with the cells detached by mild trypsinization.

Human islet processing
Human pancreases (mean age 38 ± 6 yr, n = 5) were harvested from adult brain-dead donors in accord with French regulations and the local Institutional Ethical Committee. Pancreatic islets were isolated after ductal distension of the pancreas and digestion of the tissue with liberase (Roche Molecular Biochemicals, Mannheim, Germany). Semipurification was achieved with Euro-Ficoll discontinuous density gradient. Islet number was determined on samples of each preparation after dithizone staining and expressed as equivalent number of islets. Preparations used in this study exhibited a 75 ± 3% purity and an average yield of 4091 ± 1127 equivalent number of islets per gram pancreas. Semipurified islets were cultured for 24 h in CMRL-1066 medium with 2% Ultroser G (Life Technologies, Inc., Cergy Pontoise, France). Human ß-cell processing consisted of a 48-h treatment at 37 C with cytokines added or not with 1,25-(OH)2D3 used in the same concentrations as for RINm5F cells.

Estimation of metabolic activity of cells
RINm5F cells were treated for 48 h with cytokines with or without 1,25-(OH)2D3. Insulin released in the culture media by cells was stored at -80 C before we performed the insulin assay using the RIA kit CT from CIS-Bio International (Gif-sur-Yvette, France).

As described previously (25), we used the fluorescent and nontoxic REDOX indicator Alamar blue (Biosource Technologies, Inc., Clinisciences Montrouge, France) for evaluation of cell viability. It was directly added (10% vol/vol) to cell culture medium for 2 h at 37 C in an atmosphere of air: CO2 (95:5). The fluorescence was read at 544 nm (excitation wavelength) and 590 nm (emission wavelength).

After exposure of cells for 48 h to the different experimental conditions, duplicate samples (2 x 120 µl) of media were taken for nitrite determination. Nitrite assay is based on the reaction of nitrite with 2,3-diaminonaphtalene (Sigma-Aldrich Chemicals) to form the fluorescent product 1-(H)-naphtotriazole, as previously described (25).

Detection of {Delta}{psi}m
The {Delta}{psi}m was measured by incubating RINm5F cells (5 x 105/ml) during 1 h at 37 C with 0.1 µM lipophilic cationic fluorochrome tetramethyl rhodamine methyl ester (TMRM; Molecular Probes, Inc. Europe, Leiden, The Netherlands), which accumulates in the mitochondrial matrix. Cells were analyzed by flow cytometry (Epics XL-MCL Coulter flow cytometer, Beckman, Margency, France) at 575 nm. A reduction in fluorescence intensity as measured by cytofluorometry is interpreted as an increase of mitochondrial permeability transition and a dissipation of {Delta}{psi}m.

Cell cycle analysis
RINm5F cells were fixed with 70% cold methanol and were treated with RNase (10 µg/ml, 30 min, 4 C; Sigma-Aldrich Chemicals). DNA content was measured by staining cells with the intercalating DNA dye propidium iodide (50 µg/ml, 15 min at room temperature; Sigma-Aldrich Chemicals). Apoptotic cells show a low DNA stainability resulting in a distinct, quantifiable region below the Go/G1 peak and analyzed by flow cytometry at 620 nm.

Determination of DNA fragmentation
RINm5F cells (1.0 x 106) were trypsinized and collected by centrifugation at 200 g for 5 min. After cell lysis, the lysates were centrifuged at 20,000 x g for 10 min. The cytoplasmic fractions were prediluted 1:10 with incubation buffer and tested for nucleosomes by Cell Death Detection ELISA Kit from Roche Molecular Biochemicals. The kit is based on a quantitative sandwich enzyme-immunoassay using mouse monoclonal antibodies directed against DNA and histones. This allows the specific determination of mono- and oligonucleosomes in the cytoplasmic fraction of cell lysates.

Nuclear chromatin staining
Bisbenzimide (Hoechst 33342, 10 µg/ml; France Biochem, Meudon, France) , which enters cells with intact or damaged membranes and stains DNA, was used to detect differences between normal and apoptotic nuclei in both RINm5F and human islet cells. After stimulation with cytokines ± 1,25-(OH)2D3, RINm5F cells grown on glass coverslips were directly stained for 10 min at 37 C, and human islets were dissociated with trypsin-EDTA, as described (26), and cytocentrifuged at 700 rpm before being stained. Cells were examined by fluorescence microscopy (excitation 340–380 nm). Apoptotic cells, identified by the presence of condensed or fragmented nuclei, were estimated by differential counting of 300–400 cells in each experimental condition.

Immunohistochemical characterization of IKK{alpha}/ß
RINm5F and human islet cells were incubated for 48 h with 1,25-(OH)2D3 and then treated with cytokines for 5 min. After stimulation, immunohistolabeling was performed on RINm5F cell cytospins fixed in acetone for 5 min or on fixed, paraffin-embedded human islets. After washings in PBS, fixed cells or deparaffinized sections were permeabilized for 10 min with 0.1% saponin, and nonspecific sites were blocked with 10% normal goat serum in PBS. Incubation for 90 min at room temperature with the primary antibody (rabbit polyclonal antibody to IKK{alpha}/ß, 1:50 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was followed by a second incubation with EnVision system, using goat antirabbit immunoglobulin conjugated to alkaline phosphatase labeled-dextran polymer (DAKO Corp., Trappes, France) revealed with PhtaloRed from Kirkegaard & Perry (Gaithersburg, MD). Controls included replacing the primary antibody with PBS containing 1% BSA. Nuclei were counterstained with Carazzi’s hematoxylin.

Nuclear extract preparation and EMSA
RINm5F cells (7.106/condition) were incubated for 48 h with 1,25-(OH)2D3 and then treated with cytokines for 1 h. After stimulation, cells were washed with PBS and nuclear protein fraction extracted, as described (27). Protein concentration was determined using the Bradford reagent. For the EMSA, a double-stranded oligonucleotide containing the {kappa}B-binding site: 5'-CTTCAGAGGGGACTTTCCGAGA, was end labeled with [{gamma}32P]dATP (3000 Ci-mmol) and T4 polynucleotide kinase and used as a probe. A 50-fold molar excess of nonlabeled oligonucleotide was used as a negative control. The nuclear fractions (10 µg) were allowed to react with the probe for 30 min on ice in a binding buffer containing 10 mM Tris (pH 8.0), 50 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol, 1 µg polydeoxyinosinic acid, and 0.1 ng DNA (15,000 cpm). The samples were then separated at 4 C on 5% nondenaturing polyacrylamide gels in 0.25x Tris-borate EDTA. After electrophoresis, gels were dried for autoradiography or PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA) quantification.

Cell lysis and Western blotting
After being stimulated, human islet cells were lysed for 30 min on ice in 50 µl lysis buffer (20 mM Tris acetate, pH 7.0; 0.27 M sucrose; 1 mM EDTA; 1 mM EGTA; 1 mM sodium vanadate; 50 mM sodium fluoride; 1% Triton X-100; 5 mM sodium pyrophosphate; 10 mM ß-glycerophosphate; 1 mM dithiothreitol; 1 mM benzamidine; and 4 µg/ml leupeptin; all reagents were from Fluka, Sigma). The detergent-insoluble material was pelleted by centrifugation at 15,000 rpm for 5 min at 4 C. The supernatants containing whole-cell lysate were either immediately used for Western blotting or stored at -80 C.

For Western blotting, 20 µl (50 µg) protein were added to 20 µl SDS sample buffer (125 mM Tris-HCl, pH 6.8; 25% glycerol; 5% ß-mercaptoethanol; and 0.02% bromophenol blue) and boiled for 5 min. SDS-PAGE (10%) was performed and Western blotting carried out according to standard protocols (28, 29). Goat polyclonal antibody to A20, 1:200 dilution, (Santa Cruz Biotechnology, Inc.) was used. Western blotting detection was achieved using the enhanced chemoluminescence plus reagent (ECLplus, Amersham Pharmacia Biotech, Orsay, France).

Analysis of A20 mRNA expression
After being treated with 1,25-(OH)2D3 alone, cytokines alone or a combination of both for different time periods, RINm5F cells, and islet cells were lysed in a 1% ß-mercaptoethanol containing buffer obtained from an RNA extraction kit (Macherey Nagel, Hoerdt, France). RNA was extracted as described by the manufacturer and cDNA synthesized using random hexamers (Superscript preamplification system for first-strand cDNA synthesis, Life Technologies, Inc.). Semiquantitative, noncompetitive RT-PCR was performed with AmpliTaqGold (Perkin-Elmer Applied Biosystems, Courtaboeuf, France), which allowed simultaneous coamplification of the housekeeping gene (ß-actin) and the target gene.

Primer set for A20 was sense: 5'-TTTGAGCAATATGCGGAAAGC-3', and antisense: 5'-AGTTGTCCCATTCGTCATTCC-3' (Perkin Elmer-Applied Biosystems) resulting in a 479-bp product. PCR was performed with 1 µl cDNA from each sample in the presence of 200 µM deoxynucleotide triphosphate, 1.5 mM MgCl2, 5 U AmpliTaq DNA polymerase, and 25 pM of each primer. PCR was carried out in a thermal cycler (2400 Perkin Elmer-Applied Biosystems) with each cycle consisting of denaturation at 94 C for 60 sec, annealing at 54 C for 60 sec, and polymerization at 72 C for 30 sec. The last PCR step was a final extension at 75 C for 7 min. The PCR products were electrophoresed in 2% agarose gel and bands were scanned with an integration camera CDD (COHU 4912, Clara Vision, Orsay, France) and analyzed with GelAnalysts 3.01 FR software (GreyStone-Iconix, MEB Electronique, Paris, France). Band intensity was expressed in arbitrary units and A20 expression indexed to ß-actin mRNA expression.

Statistical analysis
Data were presented as means ± SEM. The statistical differences between the groups were determined by ANOVA followed by multiple t tests using the Fisher’s least significant difference test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of 1,25-(OH)2D3 on altered metabolic functions in cytokine-treated RINm5F cells
As expected, insulin secretion and cell viability were significantly decreased by cytokines in RINm5F cells. The addition of 1,25-(OH)2D3 resulted in significantly higher values demonstrating a return toward control values (Fig. 1Go).



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  		Figure 1. Functional activity of RINm5F cells determined after 48 h of treatment with cytokines (IL-1ß, 50 IU/ml; TNF{alpha}, 1000 IU/ml; IFN{gamma}, 1000 IU/ml) ± 1,25-(OH)2D3. Values represent percentages of control cells (means ± SEM). A, Insulin secretion (n = 8), control value: 870 ± 217 ng/106 cells. B, Metabolic activity (n = 7) assessed using the REDOX indicator Alamar blue as described in Materials and Methods. *, P < 0.05; **, P < 0.001; ***, P < 0.0001.

 
Conversely, nitrite released in the culture media was markedly increased by cytokine treatment, and significant counteracting effects of 1,25-(OH)2D3 were noted. The addition of NOi during 1 h before stimulation with cytokines resulted in a near complete abolition of nitrite overproduction (Table 1Go).


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  		Table 1. Effect of 1,25-(OH)2D3 on nitrite production in cytokine-treated RINm5F cells preincubated or not with NO synthase inhibitor NOi (5 mM)

 
Effect of 1,25-(OH)2D3 on cytokine-induced loss of mitochondrial transmembrane permeability in RINm5F cells
TMRM is a cationic green fluorochrome that, as a consequence of the transmembrane potential of the mitochondria (active proton pump), is taken up by these organelles. Live nonapoptotic cells with an intact plasma membrane and active (charged) mitochondria concentrate TMRM and exhibit strong fluorescence. As cells undergo apoptosis, their plasma membrane integrity is preserved for some period of time. However, they lose their {Delta}{psi}m and hence the ability to stain with TMRM.

As shown in Fig. 2Go, a clear increase in weakly fluorescent cells vs. control cells was noted after cytokine treatment. Significant restoration of fluorescence intensity was achieved by 1,25-(OH)2D3 treatment. In the presence of NOi, the effect of cytokine treatment was less marked and no longer reached statistical significance (Table 2Go).



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  		Figure 2. Effect of 1,25-(OH)2D3 on cytokine-induced loss of mitochondrial transmembrane permeability. Typical flow cytometry histograms showing, in RINm5F cells stained with TMRM, the difference in fluorescence between control cells and cells treated with cytokines (IL-1ß, 50 IU/ml; TNF{alpha}, 1000 IU/ml; IFN{gamma}, 1000 IU/ml) ± 1,25-(OH)2D3 for 48 h, as described in Materials and Methods. As an additional control, cells were also exposed to 1,25-(OH)2D3 alone. By setting markers on these histograms, an increase in the percentage of low fluorescent cells induced by cytokines was noted in region B. These data with corresponding replicates were used to estimate the percentage of cells with depolarized mitochondria (Table 2Go).

 

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  		Table 2. Effect of 1,25-(OH)2D3 on mitochondrial transmembrane potential ({Delta}{Psi}m) in cytokine-treated RINm5F cells preincubated or not with NO synthase inhibitor NOi (5 mM)

 
Effect of 1,25-(OH)2D3 on DNA fragmentation and chromatin condensation in cytokine-treated RINm5F and human islet cells
Single-parameter flow cytometric measurement of apoptosis was determined on DNA frequency histograms of RINm5F cells, in which apoptotic cells, containing less DNA, stain less intensely with the DNA fluorochrome. Apoptotic cells are therefore located to the left of the G1 peak and constitute the sub-G1 cells. Figure 3Go shows that a significant increase of sub-G1 cells was induced by cytokines but significantly reduced by 1,25-(OH)2D3 cotreatment (Table 3Go). The addition of NOi almost completely abolished the efficiency of cytokines to induce apoptosis, whereas the addition of 1,25-(OH)2D3 seemed to enhance the process. Interestingly, when we compared the cells stimulated in these various conditions, a clear correlation (r = 0.85, P = 0.0001) was found between the percentages of sub-G1 cells and nitrite release in the culture media (Fig. 4Go).



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  		Figure 3. Effect of 1,25-(OH)2D3, on DNA content in cytokine-treated RINm5F cells. Typical flow cytometry histograms showing, in cells stained with propidium iodide, the difference in fluorescence between control cells and cells treated with cytokines (IL-1ß, 50 IU/ml; TNF{alpha}, 1000 IU/ml; IFN{gamma}, 1000 IU/ml) ± 1,25-(OH)2D3. As an additional control, cells were also exposed to 1,25-(OH)2D3 alone. Apoptotic cells show a low DNA stainability, resulting in a distinct, quantifiable region below the Go/G1 (sub-G1 cells). These data, with corresponding replicates, were used to estimate the percentage of apoptotic cells (Table 3Go).

 

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  		Table 3. Effect of 1,25-(OH)2D3 on DNA content below the G0/G1 peak in cytokine-treated RINm5F cells preincubated or not with NO synthase inhibitor NOi (5 mM)

 


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  		Figure 4. Analysis of regression between percentage of RINm5F apoptotic cells and nitrite released in culture media. The regression equation was: y = 0.237x + 5.04, r = 0.85, P = 0.0001. Dotted line, 95% confidence interval.

 
Cytoplasmic oligonucleosome enrichment, as determined by ELISA, was also found to be significantly higher in cytokine-treated RINm5F cells than in control cells. Results obtained with additional 1,25-(OH)2D3 confirmed the return toward control values as previously measured by flow cytometric DNA analyses (Fig. 5Go).



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  		Figure 5. Effect of 1,25-(OH)2D3 on DNA fragmentation in cytokine-treated RINm5F cells. The enrichment of nucleosomes in the cytoplasm of RINm5F cells treated for 48 h with cytokines (IL-1ß, 50 IU/ml; TNF{alpha}, 1000 IU/ml; IFN{gamma}, 1000 IU/ml) ± 1,25-(OH)2D3 was determined by ELISA as described in Materials and Methods. The absorbance measurements of the samples were used to calculate the enrichment factor (absorbance of the treated sample/absorbance of the corresponding control). Values are expressed as percentages of control cells, they are means ± SEM of five independent experiments. *, P < 0.005.

 
Cells stained with Hoechst dye were analyzed in both RINm5F and human islet cells. Apoptotic features such as chromatin condensation, which often aggregates peripherally under the nuclear membrane, and extensive surface blebbing followed by formation of characteristic apoptotic bodies were particularly obvious (2- to 3-fold increase) after 48 h of cytokine treatment for RINm5F and 144 h for human islet cells. In both cases, cotreatment with 1,25-(OH)2D3 resulted in a return toward control levels (Fig. 6Go).



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  		Figure 6. Micrograhs showing the effect of cytokines on apoptosis in RINm5F and human islet cells. Hoechst-stained RINm5F and human islet cells were exposed to cytokines (IL-1ß, 50 IU/ml; TNF{alpha}, 1000 IU/ml; IFN{gamma}, 1000 IU/ml) for 48 and 144 h, respectively, and examined with fluorescence microscopy as described in Materials and Methods. Highly condensed or fragmented nuclei represent cytokine-treated cells. A cotreatment with 1,25-(OH)2D3 diminished the levels of positively stained cells. Percentages of apoptotic cells reached 37.6% in cytokine-treated RINm5F cells and 15.8% and 14% in cells cotreated with 10-8 M and 10-6 M 1,25-(OH)2D3, respectively (control RINm5F cells: 8.7%). In human islet cells, percentages of apoptotic cells reached 10.5% in control cells, 22.9% in cytokine-treated cells, and 13.1% and 13.8% in cells treated with cytokines plus 10-8 M or 10-6 M 1,25-(OH)2D3, respectively. Countings are the means of two independent experiments assessed on 300–400 cells per condition. Scale bar, 10 µm.

 
Inhibition of cytokine-induced increase of I{kappa}B kinase activity and NF{kappa}B translocation by 1,25-(OH)2D3
As shown in Fig. 7Go, cytokine treatment increased intensity and number of IKK{alpha}/ß-stained cells in both RINm5F and human islets. A pretreatment of cells with 1,25-(OH)2D3 reduced the stimulating effect of cytokines. To further demonstrate the action of 1,25-(OH)2D3 on the cytokine-stimulated NF{kappa}B pathway, we examined the translocation of NF{kappa}B from the cytosol to the nucleus by EMSA. As shown in Fig. 8Go, nuclear extracts from cytokine-treated RINm5F cells contained higher NF{kappa}B-binding activity than extracts from cells that had been treated for 48 h by 1,25-(OH)2D3 before cytokine stimulation.



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  		Figure 7. Immunohistochemical localization of IKK{alpha}/ß (red staining) in cytoplasm of RINm5F and human islet cells treated for 5 min with cytokines. RINm5F cytospins and paraffin-embedded human islet sections were immunolabeled as described in Materials and Methods. A pretreatment with 1,25-(OH)2D3 diminished the levels of positive staining in cytokine-stimulated cells. A counting on about 500 cells per culture condition led to the following results: RINm5F cells, positive staining in 66% of control cells, 85% of cytokine-treated cells, and in 64% and 62% of cells pretreated with 10-8 M and 10-6 M 1,25-(OH)2D3, respectively, before cytokine stimulation; human islet cells, positive staining in 23% of control cells, 69% of cytokine-treated cells, and 46% and 34% of cells pretreated with 10-8 M and 10-6 M 1,25-(OH)2D3, respectively, before cytokine stimulation. Countings are the means of two independent experiments. Scale bar, 50 µm.

 


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  		Figure 8. Effect of 1,25-(OH)2D3 on cytokine-induced NF{kappa}B activation in RINm5F cells. Cells were incubated for 48 h with 1,25-(OH)2D3 and then stimulated for 1 h with cytokines (IL-1ß, 50 IU/ml; TNF{alpha}, 1000 IU/ml; IFN{gamma}, 1000 IU/ml). At the end of incubation, nuclear proteins were extracted, and NF{kappa}B activation was determined by gel shift analysis as described in Materials and Methods. Lane 1, Free probe; 2, control; 3, cytokines; 4, cytokines plus 1,25-(OH)2D3, 10-8 M; 5, cytokines plus 1,25-(OH)2D3, 10-6 M; 6, cytokines in the presence of 50x excess of unlabeled probe. The figure is representative of two independent experiments.

 
Modulation of A20 mRNA and protein expression by 1,25-(OH)2D3 in RINm5F and human islet cells
In RINm5F cells, we found, and our results corroborate those reported by Grey et al. (12), that cytokines induced a rapid (15–60 min) expression of the antiapoptotic A20 gene. This expression was, however, transient and gradually declined. 1,25-(OH)2D3 alone induced a more belated expression of A20 because it was obvious after 24 h, when cytokine-stimulated A20 expression had vanished and was even weaker than in controls. The addition of 1,25-(OH)2D3 to cytokines maintained high levels of A20 (Fig. 9Go).



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  		Figure 9. Analysis of A20 expression in RINm5F cells. A20 mRNA was determined by RT-PCR in RINm5F cells treated for different periods of time with 1,25-(OH)2D3 or cytokines (IL-1ß, 50 IU/ml; TNF{alpha}, 1000 IU/ml; IFN{gamma}, 1000 IU/ml) or the combination of both, as described in Materials and Methods. The figure is representative of three different experiments.

 
In human islet cells, we tested the expression of A20 levels after treatment for 48 h with 1,25-(OH)2D3 alone, cytokines, or the combination of both. As observed in RINm5F cells, our data confirmed that 1,25-(OH)2D3 up-regulates A20 mRNA and maintains high levels of A20 in cotreated cells, compared with cytokine-treated cells. Identical up-regulations were also observed on the protein level by Western blot (Fig. 10Go).



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  		Figure 10. Analysis of A20 expression in human islet cells. A, Typical example of A20 mRNA expression. A20 mRNA was determined by RT-PCR in human islet cells treated for 48 h with 1,25-(OH)2D3 or cytokines (IL-1ß, 50 IU/ml; TNF{alpha}, 1000 IU/ml; IFN{gamma}, 1000 IU/ml) or the combination of both, as described in Materials and Methods. The figure is representative of five independent experiments. B, Typical example of Western blot analysis performed on islet cell lysates from cells treated for 48 h with 1,25-(OH)2D3 or cytokines (IL-1ß, 50 IU/ml; TNF{alpha}, 1000 IU/ml; IFN{gamma}, 1000 IU/ml) or the combination of both, as described in Materials and Methods. A20 appears as an 86-kDa protein band. The figure is representative of two independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Islet graft injury by cytokines released from inflammatory cells that infiltrate the transplant is an important mechanism of early islet transplant dysfunction. The emphasis on preservation of islet function has drawn attention to the factors promoting islet engraftment or minimizing graft cell injury. We have previously shown that 1,25-(OH)2D3 contributed to restore some of the metabolic functions altered by cytokines in human pancreatic islets in vitro (18). In this study, we analyzed the effect of 1,25-(OH)2D3 on cytokine-induced pancreatic ß-cell apoptosis. In an attempt to determine the finer mechanisms implied in the protective effect of 1,25-(OH)2D3, we started by focusing on insulin-producing cells to obviate the mechanisms possibly induced by other cells enclosed in the islets. Our data, obtained in the RINm5F cells, a ß-cell model currently used for the study of pancreatic cell death, reproduced the beneficial effects of 1,25-(OH)2D3 on cytokine-induced insulin decrease and NO overproduction, which we previously described in human islet cells (18), establishing RINm5F as a suitable model. In these latter cells, we tested, as we did in human ß-cells, two concentrations of 1,25-(OH)2D3, a physiological (10-8 M) and a pharmacological one (10-6 M). No significant additional effects were observed using the higher concentration, suggesting a conceivable action of 1,25-(OH)2D3 via its specific high-affinity receptors (30, 31, 32). First we studied the possible alterations of the mitochondrial transmembrane permeability in RINm5F cells. Our results showed that cytokines clearly increased the proportion of cells having lost their {Delta}{psi}m, whereas 1,25-(OH)2D3 exhibited an efficient counteracting effect at this level. Changes in the permeability of the mitochondrial membrane lets certain proteins escape such as cytochrome C, which activates caspase-3, or other proteins like apoptosis-inducing factor implicated in apoptosis (33, 34).

Apoptotic RINm5F and human islet cells were counted based on nuclear apoptotic features in Hoechst-stained cells. Apoptotic features induced by cytokines were obvious after 2 d of treatment in RINm5F cells and 6 d in human islet cells. In both cases, cotreatment with 1,25-(OH)2D3 reduced apoptosis.

These results were firmly confirmed in RINm5F cells either by quantitation of DNA strand breaks or measurement of sub-G1 cell population by flow cytometry. They revealed a 2- to-3-fold increase of apoptosis in cytokine-induced cells. Apoptosis was significantly obviated by the addition of 1,25-(OH)2D3. A clear implication of NO in these apoptotic processes was highlighted by the fact that coincubation of cells with an NO synthase inhibitor reduced the effect of cytokines on the induction of sub-G1 cells and a clear correlation between the percentages of sub-G1 cells and nitrite levels was noted.

NO is known to mediate physiological processes via a reaction with superoxide or by complexing with a metal at the active sites of enzymes, and particularly on the Krebs cycle, ultimately leading to a severe decrease in glucose metabolism and ATP production (35). Cytokine-induced NO formation may also cause ß-cell toxicity by provoking DNA strand breaks, thereby activating DNA repair mechanisms, which can further cause cell death through depletion of cellular nicotinamide adenine dinucleotide.

The processes leading to the inhibiting effect of 1,25-(OH)2D3 on nitrite production are worth being investigated. Cytokine-induced NO production has been reported to be mediated by NF{kappa}B (36). Our study indicated an incidence of 1,25-(OH)2D3 on activation of this transcription factor by cytokines: When analyzing the NF{kappa}B-binding activity in cytokine-treated RINm5F cells, we noted, as expected, a clear increase in the specific band intensity in contrast to the attenuation noted when cells had been pretreated with 1,25-(OH)2D3 for 48 h. IKK{alpha}/ß, the kinases that phosphorylate I{kappa}B on the sites that trigger its degradation, appear to be critical for NF{kappa}B activation in response to proinflammatory cytokines. Treatment of cells with 1,25-(OH)2D3 before cytokine stimulation resulted in an attenuated levels of IKK{alpha}/ß, suggesting that 1,25-(OH)2D3 may act upstream from NF{kappa}B activation. Such an effect has been demonstrated for the A20 antiapoptotic protein, reported to exert a cytoprotective effect that was dependent on the abrogation of cytokine-induced NO production (12). The inhibitory effect of A20 on cytokine-stimulated NO production was moreover caused by transcriptional blockade of NO synthase by inhibition of the activation of NF{kappa}B (37, 38). We therefore investigated A20 induction and confirmed the rapid increase of its expression after stimulation of RINm5F cells with cytokines. Although this effect vanished with time, that of cotreatment with 1,25-(OH)2D3 maintained high levels of A20 expression in cytokine-treated cells. 1,25-(OH)2D3 alone was able to stimulate A20 expression, the effects being noticeable after 24 h. In human islet cells, the study of A20 expression after 48 h of stimulation with cytokines with and without 1,25-(OH)2D3 clearly demonstrated that 1,25-(OH)2D3 up-regulated both the expression of A20 gene and its protein.

Overexpression of the A20 antiapoptotic gene by means of adenovirus-mediated gene transfer has recently been shown to protect human and rat islets against cytokine-induced apoptosis (12). Likewise, inhibition of NF{kappa}B activity and NO production by a dominant negative inhibitor of NF{kappa}B (39) or overexpression of I{kappa}B, an inhibitor of NF{kappa}B activity (40) was demonstrated to be cytoprotective for cytokine-induced cell death in the MIN6 ß-cell line and human islets, respectively. These studies indicate that cytokine-induced cell death in ß-cells involves mechanisms that are largely NF{kappa}B and NO dependent. NO reduction achieved by 1,25-(OH)2D3 in our cytokine-treated cells may be, at least in part, attributable to the inhibition of NF{kappa}B via expression and/or stabilization of the antiapoptotic gene A20.

Globally our results clearly indicated a protective effect of 1,25-(OH)2D3 on the apoptotic machinery triggered by proinflammatory cytokines in ß-cells. These cytokines released by activated mononuclear cells, activate ß-cells to up-regulate NO production that in turn mediates toxic mechanisms responsible for ß-cell dysfunction and apoptosis. In addition to apoptosis mediated by these soluble mediators, NO is known to induce Fas expression on ß-cells, priming them to T-lymphocyte-mediated killing through direct interactions using Fas/Fas ligand systems (41, 42, 43), a process we did not deal with in this study. Induction of Fas gene expression has recently been shown to require NF{kappa}B in RINm5F cells (20). 1,25-(OH)2D3 might therefore exert an additional antiapoptotic action by inhibiting Fas via NF{kappa}B repression whether related to NO reduction or not (20, 44).

Altogether, treatment of grafts with 1,25-(OH)2D3, as well as eventually the recipients, provided hypercalcemia is avoided by regional delivery (45) or use of nonhypercalcemic derivatives of 1,25-(OH)2D3 (46), could be beneficial at several levels: first, because 1,25-(OH)2D3 has known effects on the immunoreactivity of the recipients by decreasing alloreactive T lymphocytes (CD8+) and down-regulating cytokine-secreting cells (macrophages and monocytes) (47, 48); second, because it reduces the vulnerability of graft cells by decreasing cytokine-induced major histocompatibility complex class I overexpression and IL-6 production, as demonstrated earlier in rat and human islets (18, 48); and third, as shown herein, because 1,25-(OH)2D3 may protect ß-cells from cytokine-induced death via its antiapoptotic effect mainly mediated by NO reduction and attributable, at least in part, to induction of the antiapoptotic gene A20.


   Acknowledgments
 
The authors are most grateful to Dr. T. Idziorek (Institut National de la Santé et de la Recherche Médicale Unité 459) for fruitful discussion and Dr. M. d’Herbometz for insulin assays. We also acknowledge S. Belaïch, N. Jouy, and V. Thery for technical assistance and L. Touzet for proofreading. Part of this work was possible thanks to equipment provided by the Institut Fédératif de Recherche (IFR 114, Institut National de la Santé et de la Recherche Médicale).


   Footnotes
 
This work was supported by grants from Institut National de la Santé et de la Recherche Médicale, Juvenile Diabetes Foundation, and Etablissement Français des Greffes.

Abbreviations: IFN, Interferon; I{kappa}B, inhibitor of {kappa}B; IKK, inhibitor of {kappa}B kinase; {Delta}{psi}m, mitochondrial transmembrane potential; NF{kappa}B, nuclear factor-{kappa}B; NO, nitric oxide; NOi, NO synthase inhibitor NG-methyl-L-arginine; 1,25-(OH)2D3, active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3; TMRM, tetramethyl rhodamine methyl ester.

Received April 25, 2002.

Accepted for publication August 9, 2002.


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 Introduction
 Materials and Methods
 Results
 Discussion
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