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Endocrinology Vol. 141, No. 6 2003-2010
Copyright © 2000 by The Endocrine Society

ARTICLES

Cytokines Induce Both Necrosis and Apoptosis via a Common Bcl-2-Inhibitable Pathway in Rat Insulin-Producing Cells1

Johan Saldeen

Department of Medical Cell Biology, Uppsala University, S-751 23 Uppsala, Sweden

Address all correspondence and requests for reprints to: Dr. J. Saldeen, Department of Medical Cell Biology, Biomedicum, Uppsala University, P.O. Box 571, S-751 23 Uppsala, Sweden. E-mail: johan.saldeen{at}medcellbiol.uu.se


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The presence of activated macrophages within pancreatic islets in insulin-dependent diabetes mellitus suggests an involvement of ß-cell death by necrosis. The aim of this study was to investigate the frequencies and mechanisms of cytokine-induced ß-cell apoptosis and necrosis and the possible protection mediated by the antiapoptotic gene bcl-2. A combination of interleukin-1ß, interferon-{gamma}, and tumor necrosis factor-{alpha} increased both necrosis (17% of cells) and apoptosis (5% of cells) in isolated whole rat islets, as determined by vital staining and fluorescence microscopy. Hyperexpression of Bcl-2, achieved by stable transfection using a multicopy viral vector containing a bcl-2 complementary DNA in rat insulin-producing RINm5F cells, counteracted both apoptosis and necrosis. Cytokine-induced cleavage of the caspase-3 substrate poly(ADP-ribose) polymerase (which, in other cell types, may occur downstream or independently of a Bcl-2-preventable mitochondrial permeability transition) was observed in control- but neither in bcl-2-transfected cells nor in the presence of the iNOS inhibitor NG-methyl-L-arginine. Tumor necrosis factor-{alpha} alone did not clearly induce cell death or poly(ADP-ribose) polymerase-cleavage. These findings suggest that cytokines induce both necrosis and apoptosis in insulin-producing cells via a common Bcl-2-preventable nitric oxide-dependent pathway, which may involve mitochondrial permeability transition. The necrosis:apoptosis ratio might be increased by a relative lack of caspase activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN-DEPENDENT diabetes mellitus (IDDM) is regarded as a chronic autoimmune disease resulting from progressive destruction of the insulin-producing ß-cells in the pancreas (1). Studies in animal models of IDDM have shown that macrophages are the first cells to infiltrate the islets of Langerhans, followed by lymphocytes. An increased expression of cytokines in the insulitis has been reported, and different combinations of cytokines have been shown to decrease the function and viability of ß-cells in vitro, partially through the production of nitric oxide and/or reactive oxygen species by the ß-cells themselves or by cells in their vicinity, such as macrophages and endothelial cells (2). It has been hypothesized that initial damage to ß-cells could result in the release of intracellular components, leading to inflammation and activation of a ß-cell directed immune response, followed by destruction of all insulin-producing cells (2).

It is generally believed that programmed cell death has evolved in multicellular organisms as a means to dispose of unwanted or damaged cells by a controlled manner (reviewed in Refs. 3, 4). The implementation of the apoptotic program, which is dependent on the activation of caspases, results in the dismantling of the cell and recognition and ingestion by phagocytocing cells without eliciting an inflammatory response (3, 4). By contrast, cell death by necrosis is known to result in activation of macrophages and to provoke inflammation and immune responses (3, 4). It has recently become clear that many forms of both apoptosis and necrosis share a common effector phase of mitochondrial permeability transition (MPT) (reviewed in Ref. 5). MPT may result from the opening of a multiprotein complex pore located at the contact site between the inner and outer mitochondrial membrane, which leads to disruption of the mitochondrial membrane potential ({triangleup}{Psi}m), uncoupling of the respiratory chain with cessation of ATP production, hyperproduction of reactive oxygen species (ROS), depletion of reduced glutathione, and release of Ca2+, some of which may provoke necrosis (5). However, MPT is also associated with the release of apoptosis inducing factor and cytochrome c from mitochondria, which may result in activation of caspases involved in the degradation phase of apoptosis, such as caspase-3, which cleaves poly(ADP-ribose) polymerase (PARP) and other substrates (reviewed in Refs. 4, 6). The principal role of the antiapoptotic protein Bcl-2 in preventing cell death was recently shown to be the inhibition of the MPT-pore opening (5, 7, 8). Moreover, receptors such as the tumor necrosis factor (TNF)R1 and CD95/Fas, which share a death signaling pathway involving FADD/MORT1 (reviewed in Ref. 9), may activate the initiation phase of apoptosis and are both known to induce MPT (5, 10, 11, 12). However, in some (but not all) cell types, signaling via FADD/MORT1, in addition to MPT, rapidly activates the degradation phase of apoptosis with cleavage of PARP through a pathway that seems independent of MPT (11). The latter pathway may depend on a direct activation of caspase-8 (FLICE) upon recruitment of FADD/MORT1, by so-called induced proximity, followed by activation of downstream caspases independently of MPT (5, 11).

ß-cell death by apoptosis has been demonstrated to occur in response to various treatments, including cytokines, cytokine-induced production of nitric oxide (13, 14, 15) or Fas-FasL interactions (16). Furthermore, apoptotic ß-cell death induced by cytokines has been shown to be prevented by overexpression of Bcl-2 (17, 18, 19). Some of the methods employed to demonstrate apoptosis, however, do not exclude a concomitant increase in necrotic cell death. Indeed, the early presence of activated macrophages within the islets of Langerhans during the initiation of IDDM may suggest an involvement of ß-cell necrosis. The aim of the present study was to investigate the frequencies and mechanisms of apoptosis and necrosis in insulin-producing cells in response to cytokines and the possible protection mediated by hyperexpression of Bcl-2. For this purpose, the rat insulin-producing ß-cell line RINm5F was transfected to stably express high levels of Bcl-2. Control-transfected RIN cells and RIN cells hyperexpressing Bcl-2, as well as isolated whole rat islets, were exposed to cytokines, after which the frequencies of apoptosis and necrosis were determined using fluorescence microscopy after staining with bisbenzimide and propidium iodide. To address the issue of whether cytokines or cytokine-induced nitric oxide induce caspase activation dependently or independently of the Bcl-2-preventable pathway, their effect upon cleavage of PARP in non-, control-, or bcl-2-transfected RIN cells, in the presence or absence of the iNOS inhibitor NG-methyl-L-arginine, was determined.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
The chemicals were obtained from the following sources: RPMI 1640, FBS, L-glutamine, HBSS, trypsin-EDTA, G418 (geneticin), NG-methyl-L-arginine, bisbenzimide (Hoechst 33342), and propidium iodide from Sigma (St. Louis, MO); Lipofectamine from Life Technologies, Inc. (Gaithersburg, MD); proteinase K from Roche Molecular Biochemicals (Mannheim, Germany); recombinant mouse TNF-{alpha} (biological activity 10 U/ng) from R&D Systems (Abingdon, UK); recombinant mouse interferon (IFN)-{gamma} (biological activity 10 U/ng) from AMS Biotechnology (Täby, Sweden). Human recombinant interleukin (IL)-1ß was kindly provided by Dr. K. Bendtzen (Laboratory of Medical Immunology, Rigs-hospitalet, Copenhagen, Denmark). The cytokine was produced by Immunex Corp. (Seattle, WA) and had a biological activity of 50 U/ng, as compared with an interim international standard rIL-1ß preparation (NIBSC, London, UK). All other chemicals of analytical grade were obtained from E. Merck & Co., Inc.(Darmstadt, Germany).

Rat pancreatic islet and RINm5F cell culture
Rat islets were isolated from male 3-month-old Sprague Dawley rats (local Uppsala colony), as previously described (20). Islets were cultured in groups of 150 islets per 50-mm well in RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, benzylpenicillin (100 U/ml) and streptomycin (0.1 mg/ml) at 37 C in humidified air with 5% CO2. RINm5F cells were trypsinized every 3–5 days and subcultured (1 x 105 cells per 10-mm well or 5 x 105 cells per 50-mm well) under the conditions described above.

Generation of RINm5F cell clones hyperexpressing Bcl-2
A vector harboring the mouse bcl-2 complementary DNA (cDNA) was kindly provided by Dr. E. Podack (Miami, FL) (21). To generate pancreatic ß cells hyperexpressing recombinant protein, the bovine papilloma virus (BPV)-derived BMG neo multicopy vector (22), which replicates episomaly, was used for transfection of RINm5F cells. In this vector, the mouse bcl-2 cDNA is inserted downstream of a metallothionein promoter. RINm5F cells, at a density of 3 x 105 cells per 50-mm well, were transfected with this or an empty BPV-derived neo-containing vector by liposome-mediated gene transfer using Lipofectamine as described (23). At 48 h after the start of transfection, cells were cultured in the presence of 150 µg/ml G418. Stable clones were isolated and characterized by Western blot analysis for their expression of Bcl-2 protein.

Bisbenzimide/propidium iodide staining and fluorescence microscopy
After exposure to cytokines, RINm5F cells (free-floating cells pooled with cells detached by mild trypsinization) or rat islets were incubated in PBS containing 20 µg/ml bisbenzimide and 10 µg/ml propidium iodide for 10 min at 37 C, as described (24, 25). Islets or cells were then washed with PBS, examined with fluorescence microscopy using a UV-2A filter with excitation at 330–380 nm, and photographed. The percentage of apoptotic and necrotic cells was determined by differential counting of 200–300 (RIN) or 1000–1500 (islet) cells in each experimental group. Blinded differential counting was performed by an independent examiner.

Western blot analysis
RINm5F cells were harvested and washed in cold PBS and sonicated in 100 µl Tris-EDTA. An aliquot was taken for total protein determination according to Bradford (26). Proteins were precipitated by the addition of 3 vol of cold acetone and pelleted by centrifugation at 12,000 x g for 10 min. Pellets were solubilized in SDS-ß-mercaptoethanol sample buffer by boiling for 4 min. Equal amounts of protein (~20 µg) were run on 7.5–12% SDS-polyacrylamide gels and electrically transferred to nitrocellulose filters, which were then incubated with rabbit anti Bcl-2 antibody (Santa Cruz Biothechnology, Inc., Santa Cruz, CA) or rabbit antimouse PARP antibody (a kind gift from Dr. Koichiro Yoshihara, Kashihara Nara, Japan), both diluted 1:1000 in PBS + 5% fat free milk-powder. Horseradish peroxidase-linked goat antirabbit Ig was used as a second layer. Immunodetection was performed as described for the Amersham International ECL immunoblotting detection system (Amersham Pharmacia Biotech, Buckinghamshire, UK).

Gel electrophoresis of low-molecular-weight DNA
RIN cells, growing at a density of 1.5 x 106 cells per 50-mm well, were exposed to cytokines for 24 h, after which both free-floating and attached cells were harvested and washed in cold PBS, pelleted, and resuspended in 400 µl of lysis buffer (100 mM Tris-HCl (pH 8.5), 200 mM NaCl, 5 mM EDTA, 100 µg/ml proteinase K, 0.2% SDS) (27). The samples were agitated at 37 C overnight, followed by addition of 320 µl of isopropanol. High molecular weight DNA was immediately removed upon precipitation, followed by addition of 40 µl of 3 M NaAc (pH 5.2) and 680 µl ethanol to precipitate the low-molecular-weight DNA. Samples were kept at -20 C for 1 h and then centrifuged for 10 min at 12,000 x g. In some cases, a small amount of high molecular weight DNA remained in the sample. This should not, however, influence the amount of fragmented DNA. Pellets were dissolved in water and treated with ribonuclease for 15 min at 37 C. Samples were run on 1.5% agarose gels and visualized by ethidium bromide staining.

Nitrite determination
Duplicate samples (2 x 80 µl) of media from RINm5F cells, cultured (at 50–80% confluency) for 6 h in the presence or absence of cytokines and 5 mM NG-methyl-L-arginine, were taken for nitrite determination, as previously described (28).

Statistical analysis
Statistical analysis was performed using StatView Student v1.0 software (Abacus Concepts, Inc., Berkeley, CA). Data were expressed as means ± SEM and compared using Student’s t test or ANOVA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of cytokines on necrosis and apoptosis in rat islets
Dissociation of islet cells may induce artifacts on cell function and viability. Moreover, ß-cell purification implies that nonendocrine cells, which could contribute to cytokine- induced ß-cell destruction (2), may be lost. Therefore, whole rat islets were exposed to cytokines for 24 h and then stained with bisbenzimide and propidium iodide and examined with fluorescence microscopy. Using this technique, apoptotic cells are identified by their highly condensed or fragmented nuclei, which are only bisbenzimide positive (resulting in blue fluorescence) representing early apoptosis, or both bisbenzimide and propidium iodide positive (resulting in pink fluorescence) representing late apoptosis (24, 25). Intact, round nuclei are regarded as viable if only bisbenzimide positive or necrotic if both bisbenzimide and propidium iodide positive (24, 25). A magnification of 400x was routinely used and allowed for the characterization of visualized cell nuclei as intact, fragmented, or highly condensed (with approximate diameter <= 2/3 of an average diameter of intact control cell nuclei) staining positively for propidium iodide and/or bisbenzimide. These cell nuclei were differentially counted to gain an estimate of the number of apoptotic, necrotic, and viable cells, respectively. In cultured rat islets, highly condensed nuclei were sometimes observed very centrally, especially within large islets, possibly representing what is commonly referred to as central necrosis. The number of these cells did not seem to increase in response to cytokines and were therefore not counted. In rat islets exposed for 24 h to the combination of IL-1ß (25 U/ml), IFN-{gamma} (1000 U/ml), and TNF-{alpha} (1000 U/ml), there was a significant increase in predominantly necrosis (17 ± 2.2% of cells), whereas apoptosis was increased to a lesser extent (5.4 ± 0.8% of cells) (Figs. 1AGo and 2Go). When cytokine-exposed pancreatic islets were viewed at lower magnification (100x), the propidium iodide positive cells seemed to be evenly distributed within the islets, indicating a predominance of ß-cells among the dead cells (Fig. 1BGo). The region for counting was chosen if it was found to be representative of the islet as a whole. However, that the propidium positive cells in general appeared evenly distributed suggested that it was of little importance which region was chosen for counting. It cannot be excluded that some nuclei regarded as necrotic were slightly condensed and that apoptosis in such cells may have been initiated but apparently not completed before plasma membrane integrity was lost. In control islet cells, only a few apoptotic or necrotic cells were observed (Fig. 2Go). Exposure to the combination of IL-1ß and IFN-{gamma} resulted in a similar or slightly smaller increase of necrosis (9.0 ± 2.0% of cells) and apoptosis (4.3 ± 1.6% of cells) (Fig. 2Go). Exposure of rat islets to the combination IL-1ß, IFN-{gamma}, and TNF-{alpha} in the presence of the iNOS inhibitor methyl-L-arginine (5 mM) did not seem to increase either type of cell death after 24 h; neither did exposure to TNF-{alpha} (1000 U/ml) alone (Fig. 2Go).



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  		Figure 1. Micrographs showing the effect of cytokines on apoptosis and necrosis in pancreatic islets. After exposure to the combination of IL-1ß (25 U/ml), IFN-{gamma} (1000 U/ml), and TNF-{alpha} (1000 U/ml), whole isolated islets were stained with bisbenzimide and propidium iodide and examined with fluorescence microscopy. Highly condensed or fragmented nuclei represent early (bisbenzimide positive, appearing blue) or late (propidium iodide positive, appearing pink) apoptosis. Intact nuclei represent viable (bisbenzimide positive) and necrotic (propidium iodide positive) cells, respectively. A, Rat islet exposed to cytokines for 24 h; Arrows, examples of apoptotic (a), necrotic (n), or viable (v) cells. B, Cytokine-exposed islet viewed at lower magnification. The magnification is 400x (A) or 100x (B).

 


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  		Figure 2. Effect of cytokines on the relative number of apoptotic or necrotic cells in rat islets. Islets exposed for 24 h to the indicated combinations of IL-1ß, IFN-{gamma}, or TNF-{alpha}, in the absence or presence of 5 mM methyl-L-asinine (MA), were vital stained and examined by fluorescence microscopy, as described in Fig. 1Go; and the number of cell nuclei exhibiting viable, apoptotic, or necrotic features were counted. Data are means ± SEM of two to nine observations. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. corresponding control using Student’s paired t test.

 
Hyperexpression of Bcl-2 in RINm5F cells
To investigate the possibility that hyperexpression of Bcl-2 might increase resistance to both apoptosis and necrosis in ß-cells and to reveal the level of induction of caspase activation by cytokines, insulin-producing rat pancreatic cell line RINm5F was stably transfected with a vector containing the mouse bcl-2 cDNA using liposomal gene delivery. The vector used is a BPV-derived vector, which replicates episomaly, often reaching numbers of 20–100 copies per cell, thus resulting in high levels of recombinant protein (22). In this vector, the bcl-2 cDNA (21) is inserted downstream of a metallothionein promoter. Selection with G418 yielded cell clones hyperexpressing Bcl-2 protein, as assessed by Western blot analysis (Fig. 3Go). Cells transfected with an empty BPV-derived neo-containing vector expressed low levels of Bcl-2 (Fig. 3Go). The clone expressing the highest level of Bcl-2 (4), a mixture of clones expressing intermediate levels of the protein (2456) and a control-(neo) transfected clone, were used in subsequent experiments (Fig. 3Go).



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  		Figure 3. Expression of Bcl-2 in control- (neo) or bcl-2-transfected RINm5F cell clones. RINm5F cells were transfected with BPV- derived vectors containing or not containing the mouse bcl-2 cDNA. Stable clones were selected using G418, and the expression of Bcl-2 protein was determined by Western blot analysis. The level of Bcl-2 expression in control (neo)-transfected cells, bcl-2-transfected cell clones 2–6, and a mixture of cell clones 2–6 (2456) is shown. The figure is representative of two separate experiments.

 
Effect of Bcl-2 on cytokine-induced necrosis and apoptosis in RINm5F cells
To determine whether Bcl-2 might increase the resistance to both apoptosis and necrosis in ß-cells, Bcl-2-hyperexpressing, and control-transfected RINm5F cells were exposed to different combinations of cytokines and then gently trypsinized, stained with bisbenzimide and propidium iodide, and examined with fluorescence microscopy. As for whole rat islets, the number of apoptotic cells was low and comparable (1.2% of cells) in neo- and bcl-2-transfected cells not exposed to cytokines, whereas the number of necrotic cells seemed higher (5.2% of cells) (see Fig. 5Go). A 24- to 26-h exposure to the combination of IL-1ß (25 U/ml), IFN-{gamma} (1000 U/ml), and TNF-{alpha} (1000 U/ml) in control (neo)-transfected cells resulted in a significant increase in predominantly necrosis (33 ± 8.1% of cells), whereas the number of apoptotic cells was smaller (12 ± 2.8% of cells) (Figs. 4AGo and 5Go). Hyperexpression of Bcl-2 significantly protected against both necrosis and apoptosis induced by this combination of cytokines (the corresponding values were 12.1 ± 1.6% and 3.1 ± 0.6%, respectively) (Figs. 4BGo and 5Go). To exclude the possibility of clonal variation as an explanation for the protection against cytokine-induced cell death, a mixture of four different RIN-bcl-2 cell clones, designated 2456 (Fig. 3Go), was exposed to the same combination of cytokines and was found to exhibit a similar degree of protection against cytokine-induced cell death, with 14.3 ± 2.3% necrotic (P < 0.05 vs. corresponding neo using ANOVA) and 3.8 ± 1.9% apoptotic cells (n = 4–11). In control (neo)-transfected cells, a 24-h exposure to the combination of only IL-1ß and IFN-{gamma} resulted in a similar increase in necrosis (29 ± 8.4% of cells) and apoptosis (12 ± 2.8% of cells) as when TNF-{alpha} was present in the cytokine combination (Fig. 5Go). In bcl-2-transfected cells, however, cell death remained low and Bcl-2 significantly protected against necrosis induced by this combination of cytokines (Fig. 5Go). As assessed by nitrite determination, exposure to cytokines for 6 h (a time period during which no obvious sign of cell death could be observed) resulted in production of similar amounts of nitric oxide by both control- and bcl-2-transfected RINm5F cells (Table 1Go).



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  		Figure 5. Effect of Bcl-2 on the relative number of apoptotic or necrotic RIN cells. Control- or bcl-2-transfected RINm5 cell clones were exposed for 24–26 h to the indicated combinations of IL-1ß, IFN-{gamma}, and TNF-{alpha}, followed by vital staining and fluorescence microscopy, as given in Fig. 1Go. Differential counting of apoptotic, necrotic cells and viable cells was performed. Data are means ± SEM of 9–11 separate experiments. *, P < 0.05 vs. corresponding control (neo); {ddagger}, P < 0.05 vs. corresponding neo using two-way ANOVA with the Bonferroni test.

 


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  		Figure 4. Micrographs showing the effect of Bcl-2 on cytokine-induced apoptosis and necrosis in RINm5F cells. Control-transfected (A) or bcl-2-transfected (B) RINm5 cell clones were exposed for 24–26 h to the combination of IL-1ß, IFN-{gamma}, and TNF-{alpha}, followed by vital staining and fluorescence microscopy, as given in Fig. 1Go. Cells staining positively for bisbenzimide appear dark, whereas cells staining positively for both bisbenzimide and propidium appear bright in the figure.

 

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  		Table 1. Effects of cytokines, in the absence or presence of monomethyl-L-arginine, on nitrite production in RIN cells not transfected or transfected (with neo or bcl-2)

 
Effect of Bcl-2 or methyl-L-arginine on cytokine-induced cleavage of PARP in RINm5F cells
To investigate whether cytokines result in caspase activation and whether this occurs downstream of a Bcl-2 preventable pathway, control-transfected and bcl-2-transfected cells were exposed for 24 h to cytokines, after which the cleavage, in intact cells, of PARP, a well-known substrate of caspase-3 and -7, was determined by Western blot analysis using a PARP antibody. The combination of IL-1ß plus IFN-{gamma} or IL-1ß plus IFN-{gamma} plus TNF-{alpha} both induced the appearance of the 85-kDa cleavage fragment in control-transfected cells but not in bcl-2-transfected cells, indicating that cytokine-induced caspase activation occurs downstream of the Bcl-2 preventable pathway (Fig. 6Go). To study how rapidly cytokines induce cleavage of PARP and the impact of nitric oxide in this event, RINm5F cells were exposed to TNF-{alpha} (1000 U/ml), IFN-{gamma} (1000 U/ml) or IL-1ß (25 U/ml). None of these cytokines, when present alone for 24 h, induced any consistent cleavage of PARP (Fig. 7Go). In combination, these cytokines did not induce any consistent effect on PARP cleavage when present for 6 h (results not shown). When assessed at 24 h, however, there was appearance of the 85-kDa cleavage fragment band in RINm5F cells exposed to the combination of cytokines (Fig. 7Go). PARP cleavage, in response to a 24-h exposure to cytokines, was counteracted in the presence of 5 mM methyl-L-arginine (Fig. 7Go).



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  		Figure 6. Effect of Bcl-2 on cytokine-induced cleavage of PARP. Control- or bcl-2-transfected RINm5 cell clones were exposed for 24 h to the indicated combinations of IL-1ß, IFN-{gamma}, and TNF-{alpha}. Western blot analysis was performed using a PARP-specific antibody. The intact 116-kDa band, as well as the apoptotic 85-kDa cleavage fragment of PARP, are indicated. The figure is representative of two separate experiments. ctrl, Control.

 


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  		Figure 7. Effect of methyl-L-arginine on cytokine-induced cleavage of PARP. RINm5 cells were exposed for 24 h to IL-1ß (25 U/ml), IFN-{gamma} (1000 U/ml), and TNF-{alpha} (1000 U/ml), alone or a combination thereof (cyt), in the absence or presence of 5 mM MA, as given in the figure. Western blot analysis was performed using a PARP-specific antibody. The figure is representative of three separate experiments.

 
Role of nitric oxide in cytokine-induced DNA ladder formation in RINm5F cells
To further determine the effect of the combination of cytokines and the contribution of nitric oxide to apoptosis, RINm5F cells were exposed to the combination of IL-1ß, IFN-{gamma}, and TNF-{alpha} for 24 h, in the presence or absence of methyl-L-arginine, and the effect upon apoptotic DNA fragmentation was assessed. Cytokines induced the formation of a DNA ladder, which was counteracted by the simultaneous addition of 5 mM methyl-L-arginine to the culture medium (Fig. 8Go). As determined by nitrite measurement, the same combination of cytokines induced an increase in nitric oxide production, which was significantly inhibited by 5 mM NG-methyl-L-arginine (Table 1Go).



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  		Figure 8. Effect of methyl-L-arginine on cytokine-induced DNA ladder formation in RINm5F. RINm5 cells were exposed for 24 h to the combination of IL-1ß (25 U/ml), IFN-{gamma} (1000 U/ml), and TNF-{alpha} (1000 U/ml), in the presence or absence of 5 mM MA. Low-molecular-weight DNA was extracted and run together with molecular weight standard (St) on 1.5% agarose gels. The figure is representative of two separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Nonendocrine cells may contribute to cytokine-induced ß-cell destruction (2), and such cells may not be present in purified ß-cell preparations. Using fluorescence microscopy on vital stained whole rat islets of Langerhans, this study demonstrates that cytokines induce an increase in both necrotic and apoptotic cell death in isolated pancreatic islets. This method was recently validated in single ß-cells using electron microscopy (29). It is possible that some nuclei regarded as necrotic were slightly condensed. Thus, in some of these cells, the apoptotic process may have been initiated, but plasma membrane integrity was apparently lost before apoptosis was completed. However, the notion of apoptosis as a fast process, once activated, does not favor the possibility that islet cells, presently regarded as necrotic, given a longer time of incubation, would have proceeded to an apoptotic morphology.

The finding that the same treatment can provoke either necrosis or apoptosis may imply a common pathway for these types of cell death, induced by cytokines in ß-cells. A possible explanation was provided by the recent finding that many forms of both apoptosis and necrosis share an upstream effector phase of MPT (5, 7). The principal action of Bcl-2, as well as some related proteins, was recently shown to be the inhibition of the opening of the multiprotein complex pore leading to MPT, thereby preventing also necrosis induced by various treatments in different cell types (5, 24). Based on these notions, the finding that Bcl-2 can prevent both necrosis and apoptosis in RIN cells may indicate that cytokine-induced ß-cell death involves MPT as a common effector mechanism. Several molecules have been implicated in the induction of MPT in other cell systems, including ceramide (10), ROS (5, 30), and peroxynitrite (30, 31). Nitric oxide may also increase the sensitivity to (32) or induce MPT (33). In the present study, cell death induced by a 24-h exposure to cytokines seemed to be counteracted by inhibition of iNOS. This indicates an essential role of nitric oxide in induction of cell death by this combination of cytokines in rat pancreatic ß-cells. However, it does by no means exclude an important contribution of ROS or other molecules, which may be induced by cytokines in ß-cells or by cells in their vicinity (2, 34).

It is well known that many stimuli, at low concentrations, can cause apoptosis but, at higher concentrations, may lead to necrosis. In line with this, it has recently been demonstrated that massive MPT can result in cell death with loss of plasma membrane integrity (i.e. necrosis), as a result of the ensuing bioenergetic catastrophe, before apoptogenic proteins have been able to process their substrates (8). Furthermore, it has been demonstrated that efficient apoptotic processing relies on availability of ATP, because the degradation phase of apoptosis is an energy-requiring process. Hence, low energy levels of ATP will promote necrosis (35). It is noteworthy that cytokine-induced production of nitric oxide leads to inhibition of mitochondrial enzyme aconitase (28) and fall in ATP-production (36), which could contribute to the promotion of necrotic cell death. Indeed, RINm5F cell necrosis, in response to high levels of prooxidants, was recently found to be preceded by depletion of glutathione, ATP, and NAD+ (37). Inhibition of caspases responsible for the apoptotic degradation phase will also favor necrosis (38). Interestingly, nitric oxide may also inhibit apoptotic caspases (39), which might further promote necrotic cell death. In view of these notions, it may also be speculated that whether ß-cells death occurs by apoptosis or necrosis could depend on the intercellular differences in sensitivity to cytokines or their intracellular mediators and the ability to successfully implement the degradation phase of apoptosis.

The cytokines IL-1ß, IFN-{gamma}, and TNF-{alpha}, when present alone for 24 h, did not induce PARP cleavage, a sensitive measure of caspase activation that has been shown to be involved in ß-cell apoptosis (25). It cannot be excluded that extending the incubation period would have resulted in a small increase in apoptosis, as has been shown in other studies (40, 41). In recently described so-called type I cells, recruitment of FADD/MORT1 with activation of caspase-8 (as may occur in response to TNF-{alpha} or Fas/CD95 receptor stimulation) results in activation of caspase-3 with cleavage of PARP within 30 min, whereas the same events in type II cells are slower and dependent upon MPT (11, 12). Apoptosis induced by receptor cross-linking is accordingly prevented by Bcl-2 in the latter, but not the former, cell type (11, 12). Regardless of the nature and relative contribution of the factors responsible for cytokine-induced ß-cell death, the results of the present study indicate that none of the presently used cytokines, at least not in the RINm5F cell line, efficiently induce any obvious sign of apoptosis independent of the Bcl-2-preventable pathway. Indeed, Bcl-2 inhibited cytokine-induced cleavage of PARP, a well recognized substrate of caspase-3 and -7, which, depending on cell type, may be processed downstream of either MPT or TNFR1- or Fas/CD95-activated caspases in the MPT-independent pathway (5, 11). Whether this finding applies also to native rat islets and in vivo remains to be determined. Interestingly, cytokines have also been shown to up-regulate Fas/CD95 in primary ß-cells (16), and nitric oxide has been demonstrated to induce ß-cell death via Fas-FasL interactions (42). It is noteworthy, however, that exposure of ß-cells to cytokines at the same time potently induces activation of NF-{kappa}B (43), which has, in other cell types, been associated with increased resistance to apoptosis (9, 44, 45).

It is widely believed that programmed cell death and apoptosis have evolved as a means to rapidly and efficiently eliminate unwanted cells, without eliciting inflammation and immune responses (3, 4). By contrast, a cell dying by necrosis, having lost its plasma membrane integrity, will leak intracellular contents to the exterior, thereby provoking inflammation and activation of macrophages (3, 4), a scenario consistent with the insulitis observed early in IDDM. The present study, in conclusion, demonstrates that death by necrosis may be increased in parallel to apoptosis in rat insulin-producing cells, in response to cytokines. An increased necrosis:apoptosis ratio might result from a relative lack of apoptotic caspase activity and could have implications for the pathogenesis of IDDM. Cytokine-induced ß-cell death, either by necrosis or apoptosis, seems dependent on a common Bcl-2-preventable pathway, which may involve MPT. These findings indicate that controlling the Bcl-2-preventable pathway of cell death, by the use of small molecules or by genetic engineering, if applied for the prevention or treatment of IDDM, might enhance ß-cell survival and possibly act to diminish inflammation.


   Acknowledgments
 
The skillful technical assistance by I.-B. Hallgren is gratefully acknowledged. Valuable suggestions and critical reviewing of the manuscript by Dr. Nils Welsh are gratefully appreciated.


   Footnotes
 
1 This work was supported, in part, by Swedish Medical Research Council Grants (72X-11564-05 C, 72P-12995–02B), the Swedish Diabetes Association, the Nordic Insulin Fund, the Juvenile Diabetes Foundation International, the Wallenberg Fund, the Swedish Society of Medicine, and the Family Ernfors Fund. Back

Received August 5, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Bach J-F 1994 Insulin-dependent diabetes mellitus as an autoimmune disease. Endocr Rev 15:516–542[Abstract]
  2. Mandrup-Poulsen T 1996 The role of interleukin-1 in the pathogenesis of IDDM. Diabetologia 39:1005–1029[Medline]
  3. Raff M 1992 Social controls on cell survival and cell death. Nature 356:397–400[CrossRef][Medline]
  4. Salvesen G, Dixit MV 1997 Caspases: intracellular signaling by proteolysis. Cell 91:443–446[CrossRef][Medline]
  5. Green D, Kroemer G 1998 The central executioners of apoptosis: caspases or mitochondria? Trends Cell Biol 8:267–271[CrossRef][Medline]
  6. Reed JC 1997 Cytochrome C: can’t live with it—can’t live without it. Cell 91:559–562[CrossRef][Medline]
  7. Marzo I, Brenner C, Zamzami N, Susin SA, Beutner G, Brdiczka D, Remy R, Xie ZH, Reed JC, Kroemer G 1998 The permeability transition pore complex: a target for apoptosis regulation by caspases and bcl-2-related proteins. J Exp Med 187:1261–1271[Abstract/Free Full Text]
  8. Hirsch T, Marchetti P, Susin SA, Dallaporta B, Zamzami N, Marzo I, Geuskens M, Kroemer G 1997 The apoptosis-necrosis paradox. Apoptogenic proteases activated after mitochondrial permeability transition determine the mode of cell death. Oncogene 13:1573–1581
  9. Yuan J 1997 Transducing signals of life and death. Curr Opin Cell Biol 9:247–251[CrossRef][Medline]
  10. Pastorino JG, Simbula G, Yamamoto K, Glascott Jr PA, Rothman RJ, Farber JL 1996 The cytotoxicity of tumor necrosis factor depends on induction of the mitochondrial permeability transition. J Biol Chem 271:29792–29798[Abstract/Free Full Text]
  11. Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ, Debatin KM, Krammer PH, Peter ME 1998 Two CD95 (APO-1/Fas) signaling pathways. EMBO J 17:1675–1687[CrossRef][Medline]
  12. Li H, Zhu H, Xu C-J Yuan J 1998 Cleavage of BID by caspase 8 mediates mitochondrial damage in the Fas pathway of apoptosis. Cell 94:491–501[CrossRef][Medline]
  13. Ankarcrona M, Dypbukt JM, Brüne B, Nicotera P 1994 Interleukin-1-ß- induced nitric oxide production activates apoptosis in pancreatic RINm5F cells. Exp Cell Res 213:172–177[CrossRef][Medline]
  14. Kaneto H, Fujii J, Seo HG, Suzuki K, Matsuoka T-A, Nakamura M, Tatsumi H, Yamasaki Y, Kamada T, Taniguchi N 1995 Apoptotic cell death triggered by nitric oxide in pancreatic ß cells. Diabetes 44:733–738[Abstract]
  15. Delaney C, Pavlovic D, Hoorens A, Pipeleers D, Eizirik DL 1997 Cytokines induce deoxyribonucleic acid strand breaks and apoptosis in human pancreatic islet cells. Endocrinology 138:2610–2614[Abstract/Free Full Text]
  16. Yamada K, Takane-gyotoku N, Yuan X, Ichikawa F, Inada C, Nonaka K 1996 Mouse islet cell lysis mediated by interleukin-1-induced Fas. Diabetologia 39:1306–1312[CrossRef][Medline]
  17. Iwahashi H, Hanafusa T, Eguchi Y, Nakajima H, Miyagawa J, Itoh N, Tomita K, Namba M, Kuwajima M, Noguchi T, Tsujimoto Y, Matsuzawa Y 1996 Cytokine-induced apoptotic cell death in a mouse pancreatic ß-cell line: inhibition by Bcl-2. Diabetologia 39:530–536[Medline]
  18. Liu Y, Rabinovitch A, Suarez-Pinzon W, Muhkerjee B, Brownlee M, Edelstein D, Federoff HJ 1996 Expression of the bcl-2 gene from a defective HSV-1 amplicon vector protects pancreatic ß-cells from apoptosis. Hum Gene Ther 7:1719–1726[Medline]
  19. Rabinovitch A, Suarez-Pinzon W, Strynadka K, Ju Q, Edelstein D, Brownlee M, Korbutt GS, Rajotte RV 1999 Transfection of human pancreatic islets with an anti-apoptotic gene (bcl-2) protects beta-cells from cytokine-induced destruction. Diabetes 48:1223–1229[Abstract]
  20. Andersson A 1978 Isolated mouse pancreatic islets in culture: effects of serum and different culture media on the insulin production of the islets. Diabetologia. 14:397–404
  21. Deng G, Podack ER 1993 Suppression of apoptosis in a cytotoxic T-cell line by interleukin 2-mediated gene transcription and deregulated expression of the protooncogene bcl-2. Proc Natl Acad Sci USA 90:2189–2193[Abstract/Free Full Text]
  22. Karasuyama H, Melchers F 1988 Establishment of mouse cell lines which constitutively secrete large quantities of interleukin 2, 3, 4 or 5, using modified cDNA expression vectors. Eur J Immunol 18:97–104[Medline]
  23. Saldeen J, Curiel DT, Eizirik DL, Andersson A, Strandell E, Buschard K, Welsh N 1996 Efficient gene transfer to dispersed human pancreatic islet cells in vitro using adenovirus-polylysine/DNA complexes or polycationic liposomes. Diabetes 45:1197–1203[Abstract]
  24. Shimizu S, Eguchi Y, Kamiike W, Waguri S, Uchiyama Y, Matsuda H, Tsujimoto Y 1996 Retardation of chemical hypoxia-induced necrotic cell death by Bcl-2 and ICE inhibitors: possible involvement of common mediators in apoptotic and necrotic signal transductions. Oncogene 12:2045–2050[Medline]
  25. Saldeen J, Welsh N 1998 Nicotinamide-induced apoptosis in insulin-producing cells is associated with cleavage of poly(ADP-ribose) polymerase. Mol Cell Endocrinol 139:99–107[CrossRef][Medline]
  26. Bradford MM 1976 A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254[CrossRef][Medline]
  27. Laird PW, Zijderveld A, Linders K, Rudnicki MA, Jaenisch R, Berns A 1991 Simplified mammalian DNA isolation procedure. Nucleic Acids Res 19:4293[Free Full Text]
  28. Welsh N, Eizirik DL, Bendtzen K, Sandler S 1991 Interleukin-1 ß-induced nitric oxide production in isolated rat pancreatic islets requires gene transcription and may lead to inhibition of the Krebs cycle enzyme aconitase. Endocrinology 129:3167–3173[Abstract]
  29. Hoorens A, Van de Casteele M, Klöppel G, Pipeleers D 1996 Glucose promotes survival of rat pancreatic ß cells by activating synthesis of proteins which suppress a constitutive apoptotic program. J Clin Invest 98:1568–1574[Medline]
  30. Bernardi P 1996 The permeability transition pore. Control points of a cyclosporin A-sensitive mitochondrial channel involved in cell death. Biochim Biophys Acta 1275:5–9[Medline]
  31. Packer MA, Murphy MP 1994 Peroxynitrite causes calcium efflux from mitochondria which is prevented by cyclosporin A. FEBS Lett 345:237–240[CrossRef][Medline]
  32. Balakirev MY, Khramtsov VV, Zimmer G 1997 Modulation of the mitochondrial permeability transition by nitric oxide. Eur J Biochem 246:710–718[Medline]
  33. Hortelano S, Dallaporta B, Zamzami N, Hirsch T, Susin SA, Marzo I, Bosca L, Kroemer G 1997 Nitric oxide induces apoptosis via triggering mitochondrial permeability transition. FEBS Lett 410:373–377[CrossRef][Medline]
  34. Rabinovitch A, Suarez WL, Thomas PD, Strynadka K, Simpson I 1992 Cytotoxic effects of cytokines on rat islets: evidence for involvement of free radicals and lipid peroxidation. Diabetologia. 35:409–413
  35. Leist M, Single B, Castoldi AF, Kühnle S, Nicotera P 1997 Intracellular adenosine triphosphate (ATP) concentration: a switch in the decision between apoptosis and necrosis. J Exp Med 185:1481–1486[Abstract/Free Full Text]
  36. Sandler S, Bendtzen K, Eizirik DL, Strandell E, Welsh M, Welsh N 1990 Metabolism and ß-cell function of rat pancreatic islets exposed to human interleukin-1 ß in the presence of a high glucose concentration. Immunol Lett 26:245–251[CrossRef][Medline]
  37. Dypbukt JM, Ankarcrona M, Burkitt M, Sjöholm A, Ström K, Orrenius S, Nicotera P 1994 Different prooxidant levels stimulate growth, trigger apoptosis, or produce necrosis of insulin-secreting RINm5F cells. The role of intracellular polyamines. J Biol Chem 269:30553–30560[Abstract/Free Full Text]
  38. Xiang J, Chao DT, Korsmeyer SJ 1996 Bax-induced cell death may not require interleukin 1 ß-converting enzyme-like proteases. Proc Natl Acad Sci USA 93:14559–14563[Abstract/Free Full Text]
  39. Dimmeler S, Haendele J, Nehls M, Zeiher AM 1997 Suppression of apoptosis by nitric oxide via inhibition of interleukin-1 ß-converting enzyme (ICE)-like and cysteine protease protein (CPP)-32-like proteases. J Exp Med 185:601–607[Abstract/Free Full Text]
  40. Soldevila G, Buscema M, Doshi M, James RF, Bottazzo GF, Pujol-Borell R 1991 Cytotoxic effect of IFN-{gamma} plus TNF-{alpha} on human islet cells. J Autoimmun 4:291–306[CrossRef][Medline]
  41. Laffranchi R, Spinas G 1997 Interferon-{gamma} inhibits insulin release and induces cell death in the pancreatic ß-cell line INS-1 independently of nitric oxide production. Exp Cell Res 237:217–222[CrossRef][Medline]
  42. Stassi G, De Maria R, Trucco G, Rudert W, Testi R, Galluzo A, Giordano C, Trucco M 1997 Nitric oxide primes pancreatic ß cells for Fas-mediated destruction in insulin-dependent diabetes mellitus. J Exp Med 186:1193–1200[Abstract/Free Full Text]
  43. Saldeen J, Welsh N 1994 Interleukin-1ß induced activation of NF-{kappa}B in insulin producing RINm5F cells is prevented by the protease inhibitor N{alpha}-p-tosyl-L-lysine chloromethylketone. Biochem Biophys Res Commun 203:149–155[CrossRef][Medline]
  44. Boldin MP, Goncharov TM, Goltsev YV, Wallach D 1996 Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death. Cell 85:803–815[CrossRef][Medline]
  45. Lipton S 1997 Janus faces of NF-{kappa}B: neurodestruction versus neuroprotection. Nat Med 3:20–21[CrossRef][Medline]



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