0021-972X/00/$03.00/0 The Journal of Clinical Endocrinology & Metabolism Copyright © 2000 by The Endocrine Society
Vol. 85, No. 2 Printed in U.S.A.
Interferon-␥ Induces Interleukin-1 Converting Enzyme Expression in Pancreatic Islets by an Interferon Regulatory Factor-1-Dependent Mechanism* ALLAN E. KARLSEN, DEJAN PAVLOVIC, KARIN NIELSEN, JAN JENSEN, HENRIK U. ANDERSEN, FLEMMING POCIOT, THOMAS MANDRUP-POULSEN, ´ CIO L. EIZIRIK, AND JØRN NERUP DE Steno Diabetes Center and Hagedorn Research Institute (A.E.K., K.N., J.J., H.U.A., F.P., T.M.-P., J.N.), 2820 Gentofte, Denmark; and Diabetes Research Center, Vrije Universiteit Brussel (D.P., D.L.E.), B-1090 Brussels, Belgium kines but not to IFN␥ or TNF␣ ⫹ IFN␥. Cytokine-induced NO-independent ICE transcription was confirmed using iNOS inhibitors. Exposure of rat and mouse islets, or rat insulinoma cells, for 24 h to IFN␥ alone or in combination with the two other cytokines also resulted in a highly significant ICE mRNA expression. ICE transcription was not inducible in islets from IFN regulatory factor-1 knock-out mice, suggesting a key-role of this transcription-factor in cytokine-mediated ICE expression in pancreatic islets. In conclusion, cytokines and IFN␥ in particular increase ICE mRNA expression in pancreatic islet cells and -cell lines, independently of NO synthesis, suggesting that ICE up-regulation may be involved in cytokine-induced NO-independent apoptosis of human islets. (J Clin Endocrinol Metab 85: 830 – 836, 2000)
ABSTRACT Whereas nitric oxide (NO) production is associated with the toxic effect of cytokines on rodent pancreatic -cells, cytokine-induced apoptosis in human islets may occur independently of NO. The cysteine protease interleukin (IL)-1 converting enzyme (ICE) is a key proapoptotic caspase. Our aim was therefore to analyze the effect of cytokines on ICE expression in human, rat, and mouse islets and rat insulinoma cells. ICE messenger RNA (mRNA) expression was highly up-regulated after 6-, 24-, and 72-h exposure of human islets to interferon (IFN)␥, tumor necrosis factor (TNF)␣ ⫹ IFN␥ or IL-1 ⫹ TNF␣ ⫹ IFN␥, paralleled by increased iNOS (the inducible form of NO synthase) expression and NO production after exposure to the combined cyto-
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NSULIN-DEPENDENT diabetes mellitus (IDDM) is associated with infiltration of the islets of Langerhans with autoreactive lymphocytes and specific destruction of the insulin-producing -cells. Cytokines, in particular interleukin-1 (IL-1), and up-regulation of the inducible form of nitric oxide synthase (iNOS) and consequent nitric oxide (NO) production have been associated with -cell destruction (reviewed in Ref. 1). Although NO may play an important role in cytokine-mediated destruction of rat -cells and rodent -cell lines, its role in cytokine-mediated destruction of human islet cells is less clear. Whereas both rat islets and rodent -cell lines are readily induced to produce NO by IL-1 alone, and toxicity may be observed as early as after 24 h, human islets seem more resistant to IL-1  alone and require exposure to combinations of cytokines to produce NO (1). Prolonged exposure (6 –9 days) of human islet cells to a combination of IL-1, tumor necrosis factor (TNF)␣, and interferon (IFN)␥ induces apoptosis in an NO-independent way (2).
Several death signals, triggered by different exogenous stimuli, may result in apoptosis. A major pathway in the apoptotic cascade is controlled by the cysteine proteases of the IL-1-converting enzyme (ICE)-like family, classified as caspases (reviewed in Ref. 3). One of the major proapoptotic caspases is the interleukin-converting enzyme (ICE, or caspase 1). In T lymphocytes, mitogen induced ICE expression, and resulting apoptosis has been demonstrated to be dependent on the transcription factor IFN regulatory factor-1 (IRF-1) (4). In serum-depleted vascular smooth muscle cells, a similar IRF-1-dependent ICE up-regulation and resulting apoptosis have been reported (5). Against this background, we presently aimed to clarify whether proinflammatory cytokines induce ICE expression in human and rodent islets, and in insulinoma cell lines, and whether such expression is dependent on cytokine-induced iNOS and IRF-1 activation.
Received August 26, 1998. Revision received July 13, 1999. Accepted November 2, 1999. Address correspondence and requests for reprints to: Dr. Allan E. Karlsen, Steno Diabetes Center, Niels Steensensvej 2, 2820 Gentofte, Denmark. E-mail:
[email protected]. * This work was supported by the Juvenile Diabetes Foundation International (1–1998-4), the Danish Diabetes Association, The Danish Research Council, Novo Nordisk A/S, and the Belgian Fonds voor Wetenschappelijk Onderzoek (3.0057.94), Flemish Scientific Research Foundation (FWO, G.0216.99), and by a Shared Cost Action in Medical and Health Research of the European Community (BMH4-CT98 –3448).
Cytokines used for the human islet experiments were recombinant human (rh) IL-1 (105 U/g), rh IFN␥ (4.7 ⫻ 104 U/g) from Genzyme Corporation (Cambridge, MA), and recombinant murine (rm) TNF␣ (1.5 ⫻ 105 U/g) from Innogenetics (Gent, Belgium). The cytokines used for the mouse islets were rm IFN␥ (104 U/g, Holland Biotechnology, Leiden, The Netherlands), rh IL-1 (3.8 ⫻ 104 U/g, a kind gift of Dr. C. W. Reynolds from the National Cancer Institute, Bethesda, MD), and rm TNF␣ (2.2 ⫻ 105 U/g, Innogenetics). The cytokines used for the rat islets, rat insulinoma (RIN), and MSL cell experiments were rh IL-1 (4 ⫻ 105 U/g, Novo Nordisk Ltd., Bagsværd, Denmark), rh TNF␣ (1.43 ⫻ 105 U/g), and rm IFN␥ (1.14 ⫻ 104 U/g) (Genzyme). NG-monometh-
Materials and Methods Materials
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IFN-␥ INDUCES ICE EXPRESSION IN ISLETS yl-l-arginine (NMMA) was purchased from Alexis Corporation (San Diego, CA).
Islet isolation, cell culture, and cytokine treatment Islets from 11 human donors (mean age ⫾ sd, 41 ⫾ 4 yr; range, 25– 60 yr) were isolated and cultured at the Central Unit of the -Cell Transplant Program (Vrije Universiteit Brussel), as previously described (6). Light microscopic examination of the immunocytochemically stained islets indicated the prevalence of insulin- and glucagon-positive cells to be 50 ⫾ 4% and 12 ⫾ 2% (mean ⫾ sd), respectively. The remaining cells are mostly ductal cells and other endocrine cells, as previously described (7). The human islets were cultured in the presence or absence of the cytokines IL-1 (50 U/mL), TNF␣ (1000 U/mL), and IFN␥ (1000 U/mL) alone or in combination for 6, 24, or 72 h, concentrations derived from our previous experiments (2, 8); 105 ⫺ 2 ⫻ 105 cells per analysis from 5 different donors were divided among the different experimental groups in the first series of experiments. In a second set of experiments, human islet preparations were exposed for 72 h to cytokines (n ⫽ 4); and the culture medium was collected for determination of nitrite, as a measure of NO production, by the Griess reaction (9). In a third set of human islet analyses, islet preparations were exposed to IL-1 or IFN␥ alone or in combination, for 6 or 24 h, in the presence or absence of 1.0 mmol/L of the iNOS inhibitor L-NMMA. We have previously observed that this concentration of NMMA prevents cytokine-induced nitrite production by human pancreatic islets (10). Islets from 3- to 6-day-old Wistar rats (Møllegård, Lille Skensved, Denmark) were isolated by hand-picking after collagenase digestion of the pancreata. After isolation, the islets were kept in preculture for 3–7 days at 37 C in atmospheric humidified air in RPMI 1640 ⫹ 10% FCS (Life Technologies, Inc., Rockville, MD) as previously detailed (11, 12). After preculture, a total of 150 islets per condition were set up in 300 L RPMI 1640 ⫹ 0.5% normal human serum, as previously described detailed (12). Rat insulinoma (RIN-5AH-T2B) and MSL cells, another well-described beta-cell line (13), were cultured in RPMI 1640 ⫹ 10% FCS, as previously described (14, 15). The islets and cells were cultured in the presence or absence of the cytokines IL-1 (150 U/mL), TNF␣ (200 U/mL), and IFN␥ (200 U/mL) alone or in combination for 24 or 72 h, with or without NMMA. At the end of the experiment, culture media were collected for nitrite determination (as a measure on NO production), and the pelleted islets/cells were snap-frozen and kept at ⫺80 C until RNA isolation. The cytokine concentrations used for these experiments are based on our previous experiments and, by titration, to obtain significant levels of cytotoxicity in the cell-lines after 24 –72 h of culture. For NO and messenger RNA (mRNA) analyses, the RIN and MSL cells were set up in 6-well tissue-culture plates (Costar, Cambridge, MA) at 1.5 ⫻ 106 cells/ well and allowed to settle for 24 hr before cytokine exposure for an additional 24 h. The IRF-1⫺/⫺ mice (16) were a generous gift from Dr. Tak Mak of the Ontario Cancer Institute (Ontario, Canada). The IRF-1⫺/⫺ mice were backcrossed into a C57BL/6 background, and wild-type (wt) C57BL/6 were used as controls for the IRF-1⫺/⫺ mice. The animals were bred and maintained under filter hoods at the experimental animal facility of the Catholic University of Leuven. Wt C57BL/6 mice were purchased from Harland Nederland, Horst, The Netherlands, and maintained under similar conditions as the IRF-1⫺/⫺ mice. The mouse islets were also isolated by collagenase digestion of the pancreas, followed by filtration over 500 mol/L pore mesh nylon screen and hand-picking. Cell culture was performed in Ham’s F-10 medium supplemented with 10 mmol/L glucose, 50 mol/L 3-isobutyl-1-methylxanthin and 1% BSA, as previously described (17). After a 24-h preculture, culture was continued for an additional 24 h in the presence or absence of IFN␥ (1000 U/mL), IL-1 (50 U/mL), and/or TNF␣ (1000 U/mL) for 24 h, with 150 islets per experimental setup. For the IRF-1⫺/⫺ islets, four independent experiments were performed; and for the wt, one or two were performed. The homozygosity of the IRF-1⫺/⫺ mice was confirmed in islet and spleen tissue by the absence of IRF-1 mRNA expression after IFN␥ exposure. Islets isolated from wt mice presented a high IRF-1 expression (data not shown).
Proliferation assay The Cell Titer 96 nonradioactive cell proliferation assay (Promega Corp., Madison, WI), also known as the MTT [3-(4,5-dimethylthiazol-
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2-yl)-2,5-diphenyltetrazolium bromide] assay, was used as an assay of viability based on mitochondria activity (18 –20) in control vs. cytokineexposed RIN cells; 104 RIN cells were set up in 96-well tissue-culture plates (Costar), allowed to settle for 24 h, before cytokine exposure for additional 3 days. The assay is based on cellular conversion of a tetrazolium salt (MTT) to a blue formazan product by the mitochondrial enzyme succinate dehydrogenase, the resulting color-reaction read at A570 nm (18, 21).
RNA isolation and semiquantitative RT-PCR analysis Total RNA from the islets was extracted by a modification of the 8 mol/L guanidine method (22), and complementary DNA (cDNA) (Invitrogen Corp. cDNA cycle Kit, Carlsbad, CA) was prepared using oligo(dT) as primer (23). For the RIN and MSL cells, total RNA was extracted after culture in 6-well plates (Costar) by the RNAzol method (RNAzol, Campro Scientific, Veenendaal, Netherlands); and oligo(dT)primed cDNA synthesis was performed on 1 g total RNA (Invitrogen cDNA cycle Kit). Semiquantitative RT-PCR was done for 25–29 cycles, as previously described (24), using deoxycycidine triphosphate as the 33 P-labeled nucleotide. After separation by 6% PAGE, the transcription products were scanned and quantified on a PhosphorImager using the ImageQuant version 3.3 software (Molecular Dynamics, Inc., Sunnyvale, CA). To compensate for variations in cDNA concentration and PCR efficiency between tubes, internal standards were included in each amplification for normalization. No contamination with genomic DNA was observed. As internal standards, TATA-binding protein (TBP), cyclophilin, and -glucoronidase were used based on their linear amplification in the same range as for the mRNAs of interest (ICE and iNOS) and their unresponsiveness to the cytokine treatment. The primers used (amplicon-size in base pairs and GENEbank accession number included) were: 1) RT-PCR primers used with human islets: ICE (5⬘aaatctcactgcttcggacat, 5⬘gggcagttcttggtattcaac; 201 bp; no. M87507); iNOS (5⬘tctgctggcttcctgctttc, 5⬘actgggtcttggggcttca; 197 bp; no. D26525); Cyclophilin (5⬘caagatcgaggtggagaagc, 5⬘gtccgctccaccagatgccag; 147 bp; no. M60857); and TBP (5⬘gccagcttcggagagttctg, 5⬘tgaaaatcagtgccgtggtt; 185 bp; no. M55654); and 2) RT-PCR primers used with RIN/MSL cells and rat and mouse islets: ICE (5⬘aagttgctgctggaggatct, 5⬘gtcccacatattccctcctg; 170 bp; no. L28095); iNOS (5⬘cagcaatgggcagactct, 5⬘cacaggctgcccccggaaggtttg; 247 bp; no. U26686); TBP (5⬘acccttcaccaatgactcctatg, 5⬘atgatgactgcagcaaatcgc; 190bp; no. D01034); and -glucoronidase (5⬘gtgatgtggtctgtggccaa, 5⬘tctgctccatactcgctctg; 301 bp; no. M13962).
Statistical analysis Results are presented as means ⫾ sd. When multiple comparisons were performed, the data were compared by one-way ANOVA. Twotailed Student’s paired t tests were used for statistical analysis of difference between groups. The level of significance was chosen at P ⬍ 0.05.
Results
ICE transcription was barely detectable in human islets in the absence of cytokines or after exposure to IL-1 alone (Fig. 1). Using cyclophilin for normalization, ANOVA analysis clearly demonstrated that cytokines indeed influenced ICE mRNA expression after both 24-h (F3 ⫽ 25.9, P ⬍ 0.0001, n ⫽ 5) and 72-h (F3 ⫽ 15.4, P ⬍ 0.0001, n ⫽ 5) exposure. Using t tests for comparison between the groups, a highly significant up-regulation of ICE mRNA expression was observed after 24-h or 72-h exposure of human islets to TNF␣ ⫹ IFN␥ (P ⬍ 0.01 vs. controls) or to IL-1 ⫹ TNF␣ ⫹ IFN␥ (Mix, P ⬍ 0.01 vs. control). ICE expression induced by 24 h exposure to TNF␣ ⫹ IFN␥ was significantly reduced when IL-1 was also present (Mix) (P ⬍ 0.004). Similar data for relative ICE expression was obtained using TBP as internal standard (data not shown). To evaluate whether cytokine-induced ICE mRNA expression in human islets was paralleled by increased iNOS
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FIG. 1. ICE and iNOS mRNA expression in human islets (relative to cyclophilin mRNA). A shows ICE mRNA expression, and B shows iNOS mRNA expression, after 24 h (black bars) or 72 h (white bars) of culture in the absence or presence of cytokines. Data are means ⫾ SD, n ⫽ 5. Mix indicates IL-1 (50U/mL) ⫹ TNF␣ (1000 U/mL) ⫹ IFN␥ (1000 U/ml). The level of significance is shown relative to the values obtained by TNF␣ ⫹ IFN␥. **, P ⬍ 0.01; ***, P ⬍ 0.005.
mRNA expression, this transcript was also quantified (Fig. 1B). Whereas iNOS transcription was barely detectable in control islets or islets exposed to IL-1 alone or to TNF␣ ⫹ IFN␥, the combination of the three cytokines induced a highly significant iNOS expression after 24 and 72 h, when compared with the other three culture conditions (P ⬍ 0.008). Analysis of the resulting NO production, determined as nitrite production after 72-h cytokine exposure, revealed that only the mixture of the three cytokines induced a significantly increased NO production (pmol/g DNA ⫻ h; mean ⫾ sd, n ⫽ 4): control, 5.9 ⫾ 6.3; IL-1, 6.1 ⫾ 6.4; TNF␣ ⫹ IFN␥, 3.4 ⫾ 3.3; IL-1 ⫹ TNF␣ ⫹ IFN␥, 46.3 ⫾ 16.6 (P ⬍ 0.02 vs. controls). To evaluate the influence of IFN␥ alone on ICE mRNA expression in human islets and the role for NO in this phenomenon, islets were exposed for 6 or 24 h to IFN␥ or IL-1 alone or in combination, in the presence or absence of NMMA. We have previously observed that a combination of IL-1 ⫹ IFN-␥ induces a nitrite production similar to that induced by IL-1 ⫹ IFN-␥ ⫹ TNF-␣ (10). Using cyclophilin for normalization, ANOVA analyses demonstrated that both 6-h (F4 ⫽ 9.1, P ⬍ 0.003, n ⫽ 3) and 24-h (F4 ⫽ 16.2, P ⬍ 0.002, n ⫽ 3) cytokine exposure influenced ICE expression, and (in agreement with the data in Fig. 1) t test analysis compared with the expression level in control islets revealed no significant change in ICE expression in IL-1-exposed islets (control vs. IL-1; 6 h: 2.9 ⫾ 1,1 vs. 1.6 ⫾ 0.9; 24 h: 1.2 ⫾ 0.6
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vs. 1.3 ⫾ 0.9) or in islets exposed to NMMA alone (data not shown). In contrast, exposure to IFN␥ alone resulted in a clear increase in ICE mRNA expression already after 6 h of exposure (10.9 ⫾ 3.2, P ⬍ 0.03 vs. control), which was maintained for 24 h (13.8 ⫾ 2.7, P ⬍ 0.02 vs. control). Whereas ICE mRNA expression after exposure to the combination of IL-1 and IFN␥ was also increased (6 h: 5.9 ⫾ 3.0 and 24 h: 7.3 ⫾ 2.0), it was not statistically different from the level in control islets; however, coincubation with the iNOS inhibitor NMMA resulted in a borderline-significant up-regulation of ICE expression after both 6 and 24 h (P ⫽ 0.05 vs. control). Although not statistically significant, IL-1 decreased IFN␥induced ICE expression; but when these cells were exposed to IL-1 ⫹ IFN-␥ in the presence of NMMA, ICE expression was similar to that observed with IFN␥ alone (Fig. 2). Taken together, these data suggest that NO is not required for cytokine-induced ICE expression. On the contrary, this radical apparently has an inhibitory effect on ICE expression, which may explain, at least in part, the inhibitory effect of IL-1 on ICE expression induced by the combination of TNF␣ and IFN␥, seen in Fig. 1, but the small number of experiments performed precluded a clear conclusion. In a second series of experiments, we evaluated ICE expression in the clonal rat insulin-producing RIN cells. Similar to human islets, the RIN cells are sensitive to cytokines (25, 26). First, in our analyses using an MTT assay as an indirect measure of cell growth and viability (18, 20), we demonstrate that both NO-dependent (involving IL-1) and NO-independent (involving IFN␥) cell-death may be induced by cytokines (Fig. 3). Thus, the combination of IL-1, TNF␣, and IFN␥ (resulting in 7– 8 nmol/L nitrite accumulation per well, as a measure of NO) had a profound effect on viability (Fig. 3). In contrast, exposure to IFN␥ or TNF␣ alone or in combination did not induce any NO production above the detection limit of our assay (1 nmol/L). Despite this, also here a markedly decreased cell viability was observed after exposure to any of the tested IFN␥ concentrations, whereas TNF␣ alone did not reduce the cell survival at any of the concentrations tested, and barely potentiated the effect of IFN␥. These data suggest that IFN␥ is the main inducer of NO-independent RIN cell death, which correlates well with
FIG. 2. ICE mRNA expression in three separate human islet isolations (relative to cyclophilin mRNA) after 6- or 24-h exposure to IFN␥ alone or in combination with IL-1, in the presence or absence of 1 mM NMMA.
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FIG. 3. MTT activity (as a measure of viability) was determined in RIN cells after 3 days culture in the absence or presence of different concentrations of TNF␣ (at the x-axis) and IFN␥ (different histogrampatterns) found not to induce measurable NO production. As a positive control, the well-described, cytotoxic NO-inducing mixture of IL-1, TNF␣, and IFN␥ (last column) was included. Mean data from one typical experiment, performed in triplicate, are shown.
the need for IFN␥ in NO-independent apoptosis of human islets (2). In accordance with the data from the human islets, ICE transcription in the RIN cells was barely detectable in the absence of cytokines, or in response to IL-1 or TNF␣ alone or in combination, whereas exposure to IFN␥ alone or in combination with the other cytokines induced a marked ICE expression (Fig. 4A). In addition, the RIN mRNA data confirmed the dissociation between ICE and iNOS expression observed in the human islets (Fig. 4, A and B). In line with the data on human islets, IFN␥ induced ICE expression in the absence of an NO production (Fig. 4C), and inhibition of NO production by addition of NMMA potentiated the IL-1 ⫹ TNF␣ ⫹ IFN␥-induced ICE expression (P ⬍ 0.001, Fig. 4A). We also analyzed mRNA samples from another rat -cell line, the MSL-G2 cells (27), as well as isolated rat islets. Exposure of these cells for 24 h to IL-1 (150 U/mL), TNF␣ (185 U/mL), and IFN␥ (14 U/mL) showed an up-regulation of ICE mRNA expression (ICE expressed as per cent of TBP, mean ⫾ sd), MSL cells: control: 2.7 ⫾ 3.7 and 3 cytokines: 26.4 ⫾ 12.7, n ⫽ 4; and rat islets: control:1.1 ⫾ 1.0 and 3 cytokines: 70.7 ⫾ 33.0, n ⫽ 3. From the data described above, it seems clear that IFN␥ is the main cytokine responsible for cytokine-induced ICE expression. To test whether the transcription factor IRF-1 mediates this effect of IFN␥, we exposed islets isolated from IRF-1⫺/⫺ or wt mice to different combinations of cytokines (Fig. 5). In agreement with the human islet and RIN cell data, IFN␥ alone and in combination with IL-1 and/or TNF␣ induced ICE expression in wt mouse islets (expression relative to TBP in the wt-control islets: 8.5% vs. 103–349% in the cytokine-exposed islets). In contrast, neither IFN␥ alone nor a combination of IFN␥ with the other cytokines was able to induce a significant ICE up-regulation in islets from IRF1⫺/⫺ mice (expression in control islets: 4.1% vs. 4.2–5.8% in the cytokine exposed islets, see also Fig. 5) This indicates that IRF-1 is critically involved in cytokine-induced, ICE expression in islet cells.
FIG. 4. Cytokine-induced ICE and iNOS mRNA expression (relative to the internal standard TBP), as well as accumulated NO production in RIN cells after 24 h of culture in the absence or presence of cytokines and/or NMMA. Mean ⫾ SD values from four to six experiments are shown. Similar data were obtained using -glucoronidase as the internal standard. The level of significance is shown relative to the values obtained by IFN␥. *, P ⬍ 0.05; ***, P ⬍ 0.005.
Discussion
Combinations of two to three cytokines (TNF␣ ⫹ IFN␥, IL-1 ⫹ IFN␥, or IL-1 ⫹ TNF␣ ⫹ IFN␥), but not IL-1 alone, induce apoptosis in human -cells after 6 –9 days of exposure (Ref. 2; Hoorens et al., manuscript in preparation). These effects are not prevented by iNOS blockers (2), suggesting that cytokine-induced apoptosis in human islets may depend
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FIG. 5. ICE mRNA expression in wt and IRF-1⫺/⫺ mouse islets after 24-h culture in the absence or presence of cytokines. Data are representative for 1– 4 independent experiments. To illustrate the difference in ICE expression between the wt and IRF-1⫺/⫺ mouse islets, data from a 30-cycle PCR reaction is shown. Thus, the lower bands seen in some of the lanes represent renaturation of the PCR products in the gel, attributable to the large amount of amplicon. Semiquantitative calculation of expression level (see text) was done after 28 PCR cycles.
on activation of alternative, non-NO-dependent pathways. In the present study, we substantiate the hypothesis that both NO-dependent and NO-independent death-pathways may be induced in beta-cells/islets by different combinations of cytokines. Furthermore, we demonstrate that combinations of cytokines that are shown to induce human -cell apoptosis also induce up-regulation of ICE mRNA expression. It is noteworthy that IL-1 alone, at a concentration reported not to lead to human -cell apoptosis (Ref. 2; Hoorens et al., manuscript in preparation), also fails to increase ICE expression. Although we cannot exclude that ICE transcription may be induced by cytokines in the non--cells present in the human islets preparations (6, 7), the present finding that similar cytokine treatment induced ICE expression in the insulin-producing clonal RIN and MSL cells suggests the -cell association of this transcription. Furthermore, analyses of the RIN cells revealed that exposure to IFN␥ alone significantly reduced the viability and increased ICE mRNA expression in the absence of induced NO production, whereas TNF␣ alone did not influence either viability or ICE expression. While the present experiments were being performed [part of the present data has been previously published in abstract form (28, 29)], up-regulated ICE protein expression was demonstrated in IL-1-exposed mouse islets (30). However, this is, to our knowledge, the first demonstration of cytokine up-regulated ICE expression in human and rat pancreatic islets. In the human islets, the combination of cytokines (TNF␣ ⫹ IFN␥) which induced the strongest increase in ICE expression did not induce NO production, whereas the combination of IL-1 ⫹ TNF␣ ⫹ IFN␥ induced both iNOS and NO production but less ICE up-regulation. In the RIN cells, IFN␥ alone, or in combination with TNF␣ and/or IL-1, also induced ICE expression. It is noteworthy that, in the human islets, addition of IL-1 to TNF␣ ⫹ IFN␥ seems to have an NO-dependent inhibitory effect on ICE mRNA expression, as
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suggested by the observation that the iNOS inhibitor NMMA further potentiates induction of ICE by the three cytokines. IL-1 may have a potentiating effect on ICE mRNA expression in RIN cells, which is, nevertheless, inhibited by NO. Species differences in promoter regions, signal transduction, and the use of transformed cell-line vs. primary islet cells may explain these differences. In support of NO as an inhibitor of ICE, this radical has been shown to inhibit both ICE activity, and thus IL-1 release in macrophages (31), and ICE-mediated apoptosis in different cell-lines, by S-nitrosylation at the active cysteine-site of ICE (32, 33). Taken together, our data suggest that, in human islets and RIN cells, IL-1 is necessary for iNOS and NO expression, and NO seems to have a inhibitory effect on cytokine-induced ICE mRNA expression. ICE expression is involved in apoptosis resulting from different pathways, such as granzyme B-induced apoptosis (34), DNA damage and IRF-1-mediated apoptosis (4), and degradation of extracellular matrix-induced apoptosis in epithelial cells (35). ICE expression is also involved in Fasmediated apoptosis in several cell systems (3, 36), including pancreatic -cells (37, 38). Constitutive expression of the caspases ICE, Cpp32, and Ich is required for TNF␣-induced apoptosis in human fibroblast cell lines (39). In several cell systems, elevated cellular ICE expression, as induced either by ICE gene transfection (40) or by cytokines (41), leads to cell death by apoptosis. The presence of IFN␥ is required for IL-1- or TNF␣induced apoptosis in human islets (2), and IFN␥ is the most important cytokine for the induction of ICE expression in these cells (present data). IFN␥ induces ICE expression in different cell types via activation of the transcription factors STAT-1 and IRF-1 (41, 42). We have previously shown that IFN␥ and (to a minor extent) IL-1 induce IRF-1 expression in human islets and RIN cells (43), and existing evidence supports that these cytokines also induce STAT-1 activation and binding to the nucleus of rodent -cells (44). Thus, it is conceivable that the cytokines used in the present experiments increase ICE expression via activation of STAT-1 and IRF-1. To test this hypothesis, we used islets from wt and IRF-1⫺/⫺ mice, and we observed that none of the cytokines tested induced ICE expression in the IRF-1⫺/⫺ mice islets. On the other hand, IFN␥ alone, or in combination with the other cytokines, induced a severalfold increase in ICE expression in the islets isolated from wt mice. This confirms an essential role for IRF-1 in this process. In this context, it is of interest to note that IRF-1 has been suggested to play a key role in promoting inflammation and autoimmunity in type II collagen-induced arthritis and experimental allergic encephalomyelitis (45) and recently in a mouse model of the neurodegenerative disorder Huntington’s disease (46). ICE interacts with a network of several other pro- and antiapoptotic proteins, including the apoptosis-inhibiting protein bcl-2, the combined action of which determines whether the cell will eventually undergo apoptosis (reviewed in Refs. 38 and 47). Overexpression of bcl-2 in mouse pancreatic betacell lines and primary islets protected them against cytokinemediated apoptosis (48 –50). Based on the present and previous data, the apoptosis observed in rat islets and RIN cells, after short-term incubation with IL-1 alone or in combination with TNF␣ and
IFN-␥ INDUCES ICE EXPRESSION IN ISLETS
IFN␥, seems to be related to NO production (reviewed in Ref. 1). However, here we show that an NO-independent beta-cell destruction may also occur in RIN cells after IFN␥ exposure, a process associated with up-regulated ICE mRNA expression. As a whole, our data suggest that induced ICE expression may be an important effector mechanism involved in the NO-independent apoptosis reported in human islets after 6 days of cytokine exposure (2). Furthermore, our data support the view that cytokines may induce apoptosis or necrosis by several different interacting pathways, dependent on cytokine profile, concentration, exposure-time, islet-species, metabolic state, and degree of transformation. This view is supported by the recent demonstration that cytokine exposure may result in recovery, apoptosis, or necrosis in Jurkat cells, regulated by NO at two ATP-dependent steps (51). The degree of activation and level of interaction between these different pathways may be responsible for the reported differences in cytokine response among human, rat, and mouse islets and different beta-cell lines. In conclusion, it is conceivable that ICE expression is critically involved in cytokine-induced NO-independent human islet cell apoptosis. ICE expression is induced in an IRF-1dependent fashion, and it is, at least in part, inhibited by NO. Acknowledgments The expert technical skills of Susanne Munch, Rikke Bonne, Erna Engholm Petersen, Ruth Leemans, Eveline Verheugen, and the technical staff providing rat and human islets is greatly appreciated. We are grateful to Dr. Chantal Mathieu, Laboratory for Experimental Medicine and Endocrinology, Katholieke Universiteit Leuven, for the maintenance and breeding of the IRF-1⫺/⫺ mice in her Animal Care Facility. We are grateful to Prof. D. G. Pipeleers, Coordinator of the -Cell Transplant Program, for providing access to the human islet preparations and information on the cell composition, financially supported by a Shared Cost Action in Medical and Health Research of the European Community (BMH CT 95–1561).
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