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¹ Hannover Medical School, Institute of Clinical Biochemistry, 30625 Hannover, Germany² Department of Cell Biology and Genetics, University of Maringá, 87020-900 Maringá, Brazil

(Correspondence should be addressed to E Gurgul-Convey; Email: Gurgul-Convey.Ewa{at}mh_hannover.de)

(Present address of K L A Souza is the Department of Physiology and Biophysics, University of Campinas, Brazil)

^* (K L A Souza and E Gurgul-Convey equally contributed to this work)

	Abstract

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Pro-inflammatory cytokines cause β-cell dysfunction and death. The aim of this study was to investigate the interactions between different pro- and anti-inflammatory cytokines and their effects on apoptotic β-cell death pathways. Insulin-producing RINm5F cells were exposed to different combinations of cytokines. Gene expression analyses of manganese superoxide dismutase (MnSOD) and inducible nitric oxide synthase (iNOS) were performed by real-time RT-PCR. Cell viability was measured by the MTT assay, NFB activation using a SEAP reporter gene assay, protein expression by western blotting and caspase-3 activity using the DEVD cleavage method. IL-1β, tumour necrosis factor (TNF) and a combination of all three pro-inflammatory cytokines increased while IFN alone did not affect NFB activity and iNOS gene and protein expression. Interestingly, the anti-inflammatory cytokines IL-4, IL-13 and IL-10 decreased IL-1β-stimulated NFB activation and iNOS expression. IL-1β, TNF and the pro-inflammatory cytokine combination also increased MnSOD gene and protein expression. But IL-4, IL-13 and IL-10 did not affect MnSOD expression and did not modulate IL-1β-stimulated MnSOD expression. Caspase-3 activity was increased by IL-1β and the pro-inflammatory cytokine combination, and to a lesser extent by TNF. In contrast, IFN had no effect on caspase-3 activity. IL-4, IL-13 and IL-10 decreased caspase-3 activity and increased viability of insulin-producing cells treated with pro-inflammatory cytokines. The anti-inflammatory cytokines counteracted the cytotoxic effects of pro-inflammatory cytokines in insulin-producing cells. This was achieved through the reduction of nitrosative stress. Thus, a balance between the anti-inflammatory and the pro-inflammatory cytokines is of crucial importance for the prevention of pancreatic β-cell destruction.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The pro-inflammatory cytokines, such as IL-1β, tumour necrosis factor (TNF) and IFN, are putative mediators of the progressive loss of pancreatic β-cells in type 1 diabetes mellitus. These cytokines are released by macrophages and T cells in infiltrated islets of Langerhans and cause impaired function and ultimately cell death by apoptosis or necrosis (Eizirik & Mandrup-Poulsen 2001). On the other hand, the release of anti-inflammatory cytokines, in particular IL-4, IL-13 and IL-10, is related to the protection of pancreatic β-cells (Zaccone et al. 1999, Kaminski et al. 2007) and the prevention of destructive insulitis (Rabinovitch & Suarez-Pinzon 2003). Cytokines acting alone or in combination induce various transcription factors and signal transduction pathways within β-cells. One of the most important signalling events is the activation of the transcription factor, nuclear factor kappa B (NFB; Eizirik et al. 1996b), which plays the role of a master switch in β-cells, activating transcription of a number of genes involved in cytokine-mediated toxicity. Of great importance for cytokine toxicity in β-cells are in particular the generation of nitric oxide (NO) via induction of the inducible nitric oxide synthase (iNOS) and production of reactive oxygen species. Cytokine-induced nitrosative and oxidative stresses trigger eventually β-cell death (Eizirik et al. 1996a,b, Eizirik & Mandrup-Poulsen 2001, Bast et al. 2002). β-Cells produce a large amount of NO when exposed to IL-1β or a combination of pro-inflammatory cytokines (Cetkovic-Cvrlje & Eizirik 1994, Eizirik & Leijerstam 1994, Eizirik et al. 1996b, Flodstrom et al. 1996, Darville & Eizirik 1998). However, it is not clear whether NO is crucial for apoptosis in β-cells, since conflicting results have been published (Wright et al. 1999, Saldeen 2000, Zumsteg et al. 2000, Kuhn et al. 2003). The pro-inflammatory cytokines induce also the expression of manganese superoxide dismutase (MnSOD) in β-cells, the enzyme that resides in the mitochondria and generates there toxic hydrogen peroxide, which cannot be removed in face of lack of appropriate detoxifying enzymes such as catalase and glutathione peroxidase (Gpx) in insulin-producing cells (Lenzen et al. 1996, Tiedge et al. 1997). The activation of MnSOD seems to play an important role in cytokine-mediated oxidative stress in β-cells (Lortz et al. 2005).

Although the action of anti-inflammatory cytokines has been studied in different cell types during recent years (Perretti et al. 1995, Berkman et al. 1996, Laffranchi & Spinas 1996, Ledeboer et al. 2000, Lubberts et al. 2000, Nishisaka et al. 2001, Plunkett et al. 2001, Lieb et al. 2003, Serafin et al. 2004, Raz et al. 2005, Pennock & Grencis 2006, Verri et al. 2006, Walsh 2006), little is known about the effects of these cytokines in pancreatic β-cells. It is known from the studies in different cell models that IL-4 is able to counteract many of the IL-1β effects, and reduced NO production has been considered an important element for this beneficial effect (Perretti et al. 1995, Ledeboer et al. 2000, Lubberts et al. 2000, Nishisaka et al. 2001). The biological effects of IL-13 may be achieved through binding to the IL-4 receptor- (Callard et al. 1996, Kotowicz et al. 1996, Kraich et al. 2006), and therefore it is generally assumed that these two cytokines overlap in the biological effects. The effects of IL-10 on the nitric oxide pathway are unclear and the opposite findings have been reported (Berkman et al. 1996, Laffranchi & Spinas 1996, Plunkett et al. 2001, Lieb et al. 2003, Serafin et al. 2004). Some studies on anti-inflammatory cytokine action in insulin-producing cells have been published recently (Kaminski et al. 2007), but opposite effects with respect to β-cell survival have been reported and the underlying molecular mechanisms of the action of anti-inflammatory cytokines still remain unknown.

The pro-inflammatory cytokines may directly activate caspases, the main effectors of programmed cell death. Nuclear chromatin condensation and DNA fragmentation (classical characteristics of programmed cell death), sequential activation of caspases and segmentation in apoptotic bodies have already been described for insulin-producing cells exposed to cytokines (Delaney et al. 1997, Kurrer et al. 1997). Cell death by apoptosis is ultimately accomplished by effector caspases, i.e. caspases 3, 6 and 7 (Joshi & Sahni 2003, Joshi et al. 2003). Previous studies have shown that TNF activates caspases 3, 8 and 9 in different cell types (Alikhani et al. 2004) and that exposure of insulin-producing cells to IFN causes activation of caspase-1 (Karlsen et al. 2000). IL-1β is able to activate caspase-3 in β-cells and this effect is possibly linked to NO production (Chen et al. 2003, Veluthakal et al. 2004). However, also in this case, the differential effects of cytokines are unknown, and no data regarding the combined action of pro- and anti-inflammatory cytokines on caspase-3 activation in insulin-producing cells are available.

Since cytokines may vary in their mechanisms of action, we decided to perform a detailed analysis of the effects of single pro- and anti-inflammatory cytokines as well as of different combinations, in order to analyse potential crosstalk. We studied the different pathways and mechanisms of pro-inflammatory cytokine-mediated β-cell death as well as their interactions with anti-inflammatory cytokines with particular emphasis upon the early changes in the death signalling pathways.

	Materials and Methods

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Chemicals

Cytokines and the dNTP mixture were obtained from PromoCell (Heidelberg, Germany). The jetPEI transfection reagent was purchased from Qbiogene (Heidelberg, Germany) and Biotherm Taq polymerase from GeneCraft (Münster, Germany). The SuperScript II RT reverse transcriptase and all tissue culture equipment were purchased from Invitrogen. All primers were from MWG-Biotech (Ebersberg, Germany). The Ac-DEVD-AMC caspase-3 substrate and Ac-DEVD-CHO₂ caspase-3 inhibitor were from Biosource International (Carnarillo, CA, USA). SNAP (S-nitroso-n-acetylpenicillamine) was from Alexis Biochemicals (San Diego, CA, USA). Hybond N nylon membranes, ECL detection system and autoradiography films were from Amersham Biosciences and Immobilon-P polyvinylidene difluoride (PVDF) membranes from Millipore (Bedford, MA, USA). All other reagents were from Sigma Chemicals (München, Germany).

Cell culture and cytokine incubation

Insulin-producing RINm5F cells were cultured as described (Lortz et al. 2000) in RPMI1640 medium, supplemented with 10 mM glucose, 10% (v/v) fetal calf serum (FCS), penicillin and streptomycin in a humidified atmosphere at 37 °C and 5% CO₂. For RNA extraction and protein analysis, cells were platted at a density of 2x10⁶ per 90 mm plastic dish and grown to confluence within 2 days. Thereafter, cells were exposed to the desired concentration of pro- and/or anti-inflammatory cytokines, the nitric oxide-donor SNAP, the iNOS blocker N-nitro-L-arginine (L-NOARG) and camptothecin. The concentrations of cytokines were 60 U/ml IL-1β (4.4 ng/ml); 600 U/ml IL-1β (44 ng/ml); 185 U/ml TNF (8.7 ng/ml); 1850 U/ml TNF (87 ng/ml); 14 U/ml IFN (10.3 ng/ml); 140 U/ml IFN (103 ng/ml); 500 U/ml IL-4 (50 ng/ml); 500 U/ml IL-13 (50 ng/ml) and 500 U/ml IL-10 (50 ng/ml). Control RINm5F cells were grown in the absence of test compounds. The lower concentrations of pro-inflammatory cytokines are commonly used in β-cell research (Mandrup-Poulsen et al. 1987, 1990, Nerup et al. 1994, Lortz et al. 2000). The anti-inflammatory cytokine concentrations were chosen on the basis of preliminary concentration dependencies as the best suited for counteracting the effects of pro-inflammatory cytokines (data not shown).

MTT cell viability assay

In all sets of experiments, the viability of the cells was determined after a 24 h or 48 h incubation period using a microplate-based MTT assay (Mosmann 1983). The viability was expressed as % of the MTT absorbance at 562/650 nm in the absence of test compounds.

iNOS and MnSOD western blot analyses

RINm5F cells were incubated for 24 h with cytokines, washed with ice-cold PBS and homogenised in ice-cold buffer (50 mM Tris–HCl, 1% Triton T100, 5% glycerol, pH 7.5) using short bursts (Braun-Sonic 125 Homogenisator, Quigley-Rochester, Inc., Rochester, NY, USA) and then heated up to 94 °C for 3 min. Cell homogenates were centrifuged at 9000 g and 4 °C for 10 min and the supernatants were used for western blot analyses. Protein content was determined by the BCA assay (Pierce, Rockford, IL, USA). For the assays, 20 µg (for MnSOD) or 40 µg (for iNOS) total protein were resolved in SDS polyacrylamide gel electrophoresis and then electroblotted onto membranes. The Ponceau staining was used as a loading control. Immunodetection was performed by specific primary antibodies against MnSOD (rabbit polyclonal, a kind gift from Dr. K Dobashi, Japan) or iNOS (NOS2 rabbit polyclonal IgG, Santa Cruz Biotechnology, Heidelberg, Germany) followed by exposition to secondary peroxidase-conjugated AffiniPure donkey anti-rabbit IgG (H+L) (Dianova, Hamburg, Germany). The hybrids were visualised through chemiluminescence using the ECL detection system after short exposure (2–3 min) to autoradiography films. The intensity of the bands was quantified through densitometry with the Gel-Pro Analyzer 4.0 program (Media Cybernetics, Silver Spring, MD, USA).

Reporter gene assay

For the NFB enhancer element activity studies, 2x10⁴ cells/well were seeded in 96-well plates 24 h before transient transfection was performed (jetPEI transfection reagent) and 48 h before addition of tested compounds for 6 h. The pSEAP-NF-B construct was used as described in detail earlier (Azevedo-Martins et al. 2003). Secreted alkaline phosphatase expression was measured using the Phospha-Light System kit (Applera, Darmstadt, Germany).

Caspase-3 activity assay

The fluorimetric method of cleavage of the DEVD was employed. Caspase-3 cleaves the DEVD (a tetrapeptide composed of Asp-Glu-Val-Asp) in the C side of the aspartate residuum. In case of the cytokine incubations, control and treated incubated RINm5F cells were washed with ice-cold PBS and harvested using a cell scraper and incubated in hypotonic lysis buffer containing Chaps and TritonX for 10 min in microfuge tubes on ice. In contrast to pro-inflammatory cytokines, the chemical compounds used in this study caused significant cell death even after short-time exposures. Therefore, in order to measure caspase-3 activity in all cells, also the dying cells, after indicated time period the cells were directly harvested using a cell scraper, centrifuged at 800 g at 4 °C for 5 min and then the cell pellet was washed with ice-cold PBS and lysed as written for cytokines. Thereafter, the tubes were centrifuged at 12 500 g at 4 °C for 10 min and the supernatants were stored at –80 °C for measurement. For the assay, 20 µl lysate, 10 µl substrate and 70 µl assay buffer were placed in 96-well plates in duplicate, and the fluorescence at 360/460 nm excitation/emission was measured every 5 min for 2 h at 37 °C. Total protein content was determined by the Bradford assay. Calculation of the specific activity of caspase-3 was done adjusting the delta fluorescence value at 60 min– 0 min (FU) to the protein content and dividing the obtained value by the slope of the standard curve. All caspase-3 activity curves were linear in the time window chosen. The amount of fluorescence produced was calculated against a standard curve of AMC over the time. The threshold for the enzyme activity was set at 1000 FU/h. The results are presented as % of untreated cells.

RNA isolation and cDNA preparation

Total RNA was obtained using the guanidine thiocyanate method (Chomczynski & Sacchi 1987). The quality of the total RNA was verified in agarose gel electrophoresis. RNA was quantified spectrophotometrically at 260/280 nm. Thereafter, 2 µg RNA was reverse transcribed into complementary DNA using an oligo-dT18TNV primer and a reverse transcriptase.

Real-time RT-PCR

The QuantiTect SYBR Green technology (QIAGEN), which uses a fluorescent dye that binds only double-stranded DNA, was employed. The reactions were performed by the DNA Engine Opticon Sequence Detection System (Biozym Diagnostik, Hess. Oldendorf, Germany). A total volume of 25 µl was used for the PCRs. Samples were first denatured at 94 °C for 2 min followed by up to 30 PCR cycles. Each PCR cycle comprised melting at 94 °C for 30 s, annealing at 61–63 °C for 30 s and extension at 72 °C for 30 s. Each PCR amplification was performed in triplicate. The optimal parameters for the PCRs were empirically defined. The purity of the amplified PCR products was verified by melting curves. The standard curves for each gene were generated using the specific amplified fragment cloned into a plasmid vector (pCR II-TOPO, Invitrogen), and the amounts determined using a fluorimetric assay (PicoGreen DNA Quantitation Kit, Invitrogen). The primers used in this study are shown in Table 1.

Analyses of the real-time RT-PCR data and of the standard curve for the genes were performed by the Opticon Monitor v. 1.07 (MJ Research Inc., Waltham, MA, USA). Densitometry for the immunoblottings was done using specific software (Gel-Pro Analyzer v. 4.0, Media Cybernetics, Silver Spring, MD, USA). All data are expressed as means±S.E.M. Statistical analyses for each group were performed by the Prism analysis program (Graphpad, San Diego, CA, USA).

	Results

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Effects of cytokines on NFB enhancer element activation

To study the effects of cytokines on early events in the cytokine-induced cell death signalling cascade, the activation of the transcription factor NFB was analyzed. Incubation of insulin-producing RINm5F cells for 6 h with IL-1β resulted in a significant, concentration-dependent activation of the NFB enhancer element (Table 2). TNF and to a lesser extent IFN enhanced IL-1β-stimulated NFB induction. A combination of all three pro-inflammatory cytokines (60 U/ml IL-1β, 185 U/ml TNF, 14 U/ml IFN) strongly stimulated NFB activation. TNF (185 U/ml) alone increased NFB activity slightly, while IFN (140 U/ml) alone did not significantly activate NFB (Table 2). The anti-inflammatory cytokines IL-4, IL-13 and IL-10 did not induce NFB activation, but rather decreased IL-1β-stimulated NFB activation (Table 2). The pro-apoptotic caspase-3 activator camptothecin failed to activate the NFB enhancer element (Table 2).

View this table: [in this window] [in a new window]   Table 2 Effects of pro- or anti-inflammatory cytokines in insulin-producing RINm5F cells on NFB activation and the inducible nitric oxide synthase (iNOS) and manganese superoxide dismutase (MnSOD) gene expression. Insulin-producing RINm5F cells were exposed for 6 h to the indicated compounds. Results are presented as fold increase comparing against control values obtained from cells incubated without the compounds. Normalisation of the real-time PCR curves was done against GAPDH. Data are mean values for the indicated number (n) of independent experiments, in each measured in at least three repetitions

The induction of the iNOS gene in insulin-producing RINm5F cells was basically an IL-1β-mediated event (Table 2). IL-1β-stimulated iNOS gene expression achieved a maximum 5 h after addition of the cytokine (Fig. 2). The effect of IL-1β on iNOS gene expression was concentration dependent (Table 2). The pro-inflammatory cytokines TNF and IFN neither alone nor in combination significantly induced iNOS gene expression (Table 2). However, a combination of all three pro-inflammatory cytokines induced a more potent stimulatory effect on iNOS gene expression than 60 U/ml IL-1β alone (Table 2). Interestingly, TNF in combination with IL-1β also showed an additive effect (Table 2).

View larger version (13K): [in this window] [in a new window]   Figure 2 Time-dependent effect of IL-1β on the iNOS gene expression in insulin-producing cells. RINm5F cells were seeded 24 h before the incubation with 600 U/ml IL-1β. The mRNA was obtained using the guanidine–thiocyanate method. After reverse transcription, gene expression was measured by quantitative real-time PCR at 0 (no cytokines added, control), 1, 2, 3, 4, 5, 6, 8 and 24 h after incubation. The means of absolute values from four to eight independent experiments are shown. *P<0.05, ANOVA followed by Bonferroni.

MnSOD gene expression was also affected by cytokine treatment (Table 2). After exposure to IL-1β (60 U/ml) insulin-producing RINm5F cells exhibited a significant increase in MnSOD gene expression when compared with control cells (Table 2). Higher concentrations of IL-1β (600 U/ml) further increased this effect. While a low concentration of TNF (185 U/ml) did not significantly affect MnSOD gene expression, a higher concentration of this cytokine (1850 U/ml) caused a significant increase of MnSOD gene expression. IFN (14 and 140 U/ml) did not affect the expression of MnSOD and did not induce any synergistic effect in combination with IL-1β or TNF. The combination of three pro-inflammatory cytokines increased MnSOD expression to a level similar to that found after exposure to IL-1β plus TNF. IL-4 (500 U/ml), IL-13 (500 U/ml) and IL-10 (500 U/ml) did not affect MnSOD gene expression. Moreover, combinations of IL-4, IL-13 or IL-10 plus IL-1β did not show a significant additive effect. Again, camptothecin did not modulate MnSOD expression.

The caspase-3 gene expression levels were not affected by any of the different cytokine treatments after a 6 h incubation time (data not shown). A time-dependent effect to the exposure to a high concentration of IL-1β (600 U/ml) revealed a small significant increase in caspase-3 gene expression only after a 4 h incubation time (Fig. 1).

View larger version (12K): [in this window] [in a new window]   Figure 1 Time-dependent effect of IL-1β on the caspase-3 gene expression in insulin-producing cells. RINm5F cells were incubated with 600 U/ml IL-1β. The mRNA was obtained using the guanidine–thiocyanate method. After reverse transcription, gene expression was measured by quantitative real-time PCR at 0 (no cytokines added, control), 1, 2, 3, 4, 5, 6, 8 and 24 h after incubation. n=4. *P<0.05, ANOVA followed by Bonferroni.

There was no iNOS protein detectable in untreated insulin-producing RINm5F cells (Table 3 and Fig. 3). Induction of iNOS protein was basically an IL-1β-mediated effect, as shown in Table 3 and Fig. 3. A time course analysis of iNOS protein expression showed a delayed increase of protein expression in comparison with gene expression. The expression of iNOS protein became evident after a 3- to 4 h exposure and achieved a maximal expression level after an 8 h exposure to IL-1β (600 U/ml), which remained stable up to 24 h (Fig. 3). Thus, maximal iNOS protein expression was delayed by around 3 h when compared with maximal iNOS gene expression (Figs 2 and 3). iNOS protein was not detected after 24 h incubation of the cells with TNF at both low and high concentrations (185 and 1850 U/ml) and also after exposure of the cells to both low and high concentrations of IFN (14 and 140 U/ml; Table 3). TNF showed a synergistic effect with IL-1β (Table 3). The combination of IL-1β with IFN did not have an additive effect when compared with IL-1β alone (Table 3). The combination of TNF and IFN did not induce iNOS protein expression (Table 3). A combination of all three pro-inflammatory cytokines (60 U/ml IL-1β, 185 U/ml TNF, 14 U/ml IFN) strongly induced iNOS protein expression. IL-4 (500 U/ml), IL-13 (500 U/ml) and IL-10 (500 U/ml) did not affect iNOS protein expression. However, all three anti-inflammatory cytokines significantly decreased IL-1β-induced iNOS protein expression, an effect that was particularly evident with IL-4 (Table 3).

View larger version (29K): [in this window] [in a new window]   Figure 3 Time-dependent effect of IL-1β on the iNOS protein expression in insulin-producing cells. RINm5F cells were seeded 24 h before the incubation with 600 U/ml IL-1β. The cells were lysed and used for western blot analyses at 0 (no cytokines added, lane C, control), 1, 2, 3, 4, 5, 6, 8 and 24 h after incubation. #not detectable. A representative blot of four independent experiments is shown.

Effect of cytokines on cell viability

Incubation of control RINm5F cells with the pro-inflammatory cytokines IL-1β, TNF and IFN for 24 and 48 h caused a loss of cell viability in the MTT assay, which was more pronounced in the case of the 48 h incubation (Table 4).

After 48 h, IL-1β (600 U/ml) reduced the cell viability by around 30% (Table 4). IL-1β, TNF and IFN added in low concentrations (60, 185 and 14 U/ml respectively) to the incubation medium each decreased the viability by around 5–15% (Table 4). TNF and IFN each slightly potentiated IL-1β-induced β-cell loss leading to a loss of 20–25% (Table 4). Treatment of insulin-producing cells with a combination of TNF and IFN reduced the cell viability by around 35% (Table 4). As expected the most toxic combination of cytokines was the mixture of IL-1β, TNF and IFN (60, 185 and 14 U/ml, respectively) as evident from a roughly 50% reduction of the cell viability when compared with untreated cells (Table 4).

The anti-inflammatory cytokines IL-4, IL-13 and IL-10 did not affect β-cell viability either after 48 h (Table 4). A combination of anti-inflammatory cytokines with IL-1β (60 U/ml) again, like in case of a 24 h incubation, did not significantly affect cell viability when compared with IL-1β alone (Table 4). Each of the anti-inflammatory cytokines alone was able to partially prevent the loss of cell viability in insulin-producing cells caused by a cytokine combination consisting of IL-1β, TNF and IFN (60, 185 and 14 U/ml respectively; Table 4). The mixture of all three anti-inflammatory cytokines did not affect cell viability after 48 h incubation (Table 4). ANTI-mix significantly protected against IL-1β-induced β-cell loss, failed, however, to counteract the toxicity of the pro-inflammatory cytokine mixture (Table 4).

Exposure of insulin-producing RINm5F cells to cytotoxic chemical compounds resulted in impaired cell viability as shown in Table 5. This was especially evident after treatment with a high concentration of the nitric oxide-donor SNAP (1 mM) and with the pro-apoptotic caspase-3 activator camptothecin (0.5 µM). Incubation of insulin-producing cells with the iNOS blocker L-NOARG (5 mmol/l) significantly decreased toxicity induced by the combination of all three pro-inflammatory cytokines (Table 4).

The time dependent effect on caspase-3 enzyme activity in response to pro-inflammatory cytokines and camptothecin is shown in Fig. 4. High concentrations of IL-1β strongly increased caspase-3 activity starting at 8 h incubation (Fig. 4).

View larger version (20K): [in this window] [in a new window]   Figure 4 Time-response curve for caspase-3 activity after exposure of insulin-producing cells to cytokines. Insulin-producing RINm5F cells were exposed to cytokines in the indicated concentration for different periods of incubation. After lysis of the cells and extraction of the total intracellular protein, the activity of caspase-3 was measured using the DEVD cleavage method. n=4–8. *P<0.05, ANOVA followed by Bonferroni. Mix, 60 U/ml IL-1β, 185 U/ml TNF, 14 U/ml IFN.

Additionally, a comprehensive panel of incubations for 8 h with different combinations of cytokines is shown in Table 4. Incubation of insulin-producing RINm5F cells with a low concentration of IL-1β (60 U/ml) also resulted in increased caspase-3 activity (Table 4). Co-incubation of cells with IL-1β (60 U/ml) and either IL-4 (500 U/ml), IL-13 (500 U/ml) or IL-10 (500 U/ml) decreased the activation of caspase-3 (Table 4). This antagonistic effect of the anti-inflammatory cytokines IL-4, IL-13 and IL-10 was more evident when the cells were co-incubated with the pro-inflammatory cytokine mixture (Table 4). While the treatment with the three pro-inflammatory cytokines resulted in a more than 400% increase in caspase-3 activity, co-incubation with IL-13 or IL-10 decreased the activity to half this level and co-incubation with IL-4 reduced the activity to levels near the control group (Table 4). Co-incubations of IL-1β alone or of pro-inflammatory cytokine mixture with the ANTI-mix completely prevented caspase-3 activation after 8 h (Table 4).

Use of the iNOS blocker L-NOARG (5 mmol/l) in the absence of pro-inflammatory cytokines did not affect caspase-3 activity. Co-incubation of the cells with IL-1β (60 U/ml or 600 U/ml) plus the iNOS blocker L-NOARG significantly decreased caspase-3 activity when compared with IL-1β alone. Co-incubation with iNOS blocker failed, however, to prevent caspase-3 activation caused by the mixture of the three pro-inflammatory cytokines (Table 5). Camptothecin and a NO-donor SNAP significantly and very pronouncedly induced caspase-3 activity in the present study (Table 5). It is important to mention that the high cell death rate caused by these very toxic chemical compounds even after 8 h incubation forced us to change the cell sample preparation procedure for caspase-3 in order to avoid washing away of dying and dead cells, which decrease the values for caspase-3 significantly.

	Discussion

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The pro-inflammatory cytokines induce cell death through complex signalling pathways (Eizirik et al. 1996b, Mandrup-Poulsen 1996, 2003a,b, Eizirik & Mandrup-Poulsen 2001, Cnop et al. 2005). Specific cell death pathways are activated depending on the individual pro-inflammatory cytokine as well as the composition of the cytokine combinations to which the cells are exposed. It is generally accepted that pro-inflammatory cytokines are major effectors of programmed cell death during the onset of type 1 diabetes mellitus (Eizirik 1988, 1991, Eizirik et al. 1991, 1994, Sandler et al. 1992, Mandrup-Poulsen 1996, Eizirik & Mandrup-Poulsen 2001, Cnop et al. 2005, Ronn et al. 2007). The mechanisms of action are only partially understood, but it is assumed that increased nitrosative and oxidative stresses (Lortz et al. 2000, Suarez-Pinzon et al. 2001, Bast et al. 2002, Lortz & Tiedge 2003, Gurgul et al. 2004, Storling et al. 2005) play a crucial role. Therefore, induction of nitrosative pathways, such as generation of NO by iNOS (Eizirik et al. 1992, 1996b, Eizirik & Leijerstam 1994, Eizirik & Pavlovic 1997, Tiedge et al. 1999), and oxidative stress by upregulation of MnSOD (Azevedo-Martins et al. 2003, Qadri et al. 2004, Lortz et al. 2005) might play a key role in the mechanisms of cytokine-mediated β-cell death. Upregulation of expression of these two genes leads always to increased protein expression and enzyme activity in insulin-producing cells (Tiedge et al. 1997). On the other hand, the anti-inflammatory cytokines IL-4, IL-13 and IL-10 may be involved in the protection of β-cells (Rapoport et al. 1993, Pennline et al. 1994, Racke et al. 1994, Kaminski et al. 2007) and other cell types (Paintlia et al. 2006).

IL-1β was the most potent inducer of NFB and iNOS and MnSOD gene expression, effects that could be potentiated by other pro-inflammatory cytokines. Our results showed that TNF also activated these genes, although to a much lesser extent, and therefore a significant effect was visible on the protein level only for MnSOD but not for iNOS. IFN (14 U/ml) by itself did not affect IL-1β- or TNF-induced iNOS or MnSOD gene and protein expression. However, a potentiation of the effects of IL-1β was observed in the presence of TNF. A potentiation by IFN was observed only in the combination with IL-1β or TNF. An induction of gene and protein expression by these pro-inflammatory cytokines correlated always with a loss of cell viability and an activation of caspase-3 as a sign of apoptotic cell death.

All anti-inflammatory cytokines tested in the present study (IL-4, IL-13 and IL-10) antagonised the effects of pro-inflammatory cytokines on the activation of NFB and the gene and protein expression of iNOS but not of MnSOD. In the same way, the effects of IL-1β and in particular of the pro-inflammatory cytokine mixture on caspase-3 activity and cytotoxicity were reduced. This supports the contention that the anti-inflammatory cytokines act in a protective way through antagonising the activation of the NFB-mediated pathway of cell death, thereby suppressing nitrosative stress. Moreover, our results confirm earlier observations in different cell types which have shown that anti-inflammatory cytokines decrease the pro-inflammatory cytokine stimulated iNOS gene and protein expression and reduce NO release (Sandler & Sternesjo 1995, Berkman et al. 1996, Plunkett et al. 2001, Paintlia et al. 2006). In case of a shorter 24 h incubation, the combination of three anti-inflammatory cytokines were also able to reduce pro-inflammatory cytokine toxicity. However, after 48 h, this reduction of cytotoxic effects was seen only in the case of IL-1β but not in the case of the pro-inflammatory cytokine mixture. This can be due to a very intense attack of pro-inflammatory cytokines which is hard to counteract especially in the face of a very low level of anti-inflammatory cytokine receptor expression in insulin-producing RINm5F cells (E Gurgul-Convey, unpublished observation).

The mechanisms underlying the reduction of NFB activation by anti-inflammatory cytokines, which may account for the decreased iNOS expression, range from de novo synthesis of IB (Molina-Holgado et al. 2002), interactions between STAT6 and upstream factors in signalling cascades activated by pro-inflammatory cytokines (Nelson et al. 2003) and transcription blockade by STAT6-mediated inhibition of NFB binding to the promoter regions of the genes coding NFB subunits (Bennett et al. 1997, Abu-Amer 2001) or prevention of NFB translocation to the nuclear compartment (Ohmori & Hamilton 2000, Molina-Holgado et al. 2002, Al-Ashy et al. 2006).

Previous studies have shown that TNF activates caspases 3, 8 and 9 in different cell types (Alikhani et al. 2004) and that exposure of insulin-producing cells to IFN causes activation of caspase-1 (Karlsen et al. 2000). IL-1β is able to activate caspase-3 in β-cells and this effect is possibly linked to NO production (Chen et al. 2003, Veluthakal et al. 2004).

Studies with NO donors have previously shown that NO is toxic to insulin-producing cells (Tiedge et al. 1999, Storling et al. 2005), an observation that was corroborated through the present study. However, the relevance of NO in triggering cytokine-induced apoptotic cell death in insulin-producing cells is still not entirely clear. While some studies have shown an absolute importance of the NO pathway in cytokine-mediated β-cell apoptosis (Delaney et al. 1996, Eizirik & Darville 2001, Storling et al. 2005), other experiments provided opposite results (Liu et al. 2000, Todaro et al. 2003, Zaitseva et al. 2006). From an in vitro point of view, our results indicate that the incubation of insulin-producing cells with pro-inflammatory cytokines and the NO-donor SNAP increased caspase-3 activity while the iNOS blocker L-NOARG reduced pro-inflammatory cytokine-induced caspase-3 activity. The pro-inflammatory cytokine-stimulated increase in caspase-3 activity was also substantially blocked by co-incubation with IL-4, IL-13 or IL-10, which reduced iNOS expression. Early activation of apoptotic pathways by pro-inflammatory cytokines seems thus to be NO dependent. Our results also indicate a clear time-dependent induction of caspase-3 by IL-1β or the pro-inflammatory cytokine combination at a very early stage. Notably, de novo synthesis of caspase-3 does not seem to be essential or required for its activity. Interestingly, maximal IL-1β-induced iNOS protein expression is reached at the same time point (8 h) as maximal IL-1β-induced caspase-3 activity, which is an additional indication for a link between NO and apoptotic pathways.

Insulin-producing cells are not well equipped with hydrogen peroxide detoxifying enzymes, especially in the mitochondrial compartment (Gurgul et al. 2004), but express significant levels of the superoxide dismutating enzymes CuZnSOD and MnSOD (Lenzen et al. 1996, Tiedge et al. 1997). MnSOD resides in the mitochondrial compartment and is responsible for dismutation of superoxide radicals into hydrogen peroxide. As shown in earlier studies, pro-inflammatory cytokines significantly stimulate the expression of MnSOD and this event appears to play a crucial role in triggering cytokine toxicity in insulin-producing cells (Lortz et al. 2005). Nonetheless, it appears from our present studies that induction of MnSOD in the early phase of cytokine toxicity does not account for cytokine-mediated cell damage. Oxidative stress caused by induction of MnSOD requires a prolonged time to cause damage to cellular components and to cause cytotoxic effects mediated by pro-inflammatory cytokines in insulin-producing cells (Lortz et al. 2005).

Interestingly, co-incubations with anti-inflammatory cytokines, although beneficial for β-cell survival, did not prevent MnSOD induction stimulated by the pro-inflammatory cytokine IL-1β. This is an additional indication that oxidative stress does not play a major role in the early phase of cytokine-mediated toxicity. Of importance, the effects of the combined action of pro- and anti-inflammatory cytokines upon MnSOD expression could not be explained by NFB activation. Our studies indicate that NFB although important for induction of the MnSOD expression in insulin-producing cells is not the only relevant transcription factor involved in the transcription of this gene. It is possible therefore that other transcription factors, such as AP1 or Sp1, are involved in the modulation of MnSOD expression in insulin-producing cells, as has been shown in other cell types (Kaneko et al. 2004, Qadri et al. 2004).

In conclusion, our results indicate that in insulin-producing cells exposed to cytokines, nitrosative stress plays an important role in activating apoptotic pathways in the early phase of cytokine toxicity. This might be true especially when the pro-inflammatory cytokine concentration is high. The use of IL-4, IL-13 and IL-10 to block pro-inflammatory cytokine-induced activation of caspase-3 may have important consequences for pancreatic β-cell protection and survival, in particular when the pro-inflammatory cytokine concentration is low or when only IL-1β is present. Thus, protection might be especially effective, when low concentrations of pro-inflammatory cytokines are released in the vicinity of the β-cells such as in the early phase of type 1 diabetes development or reach the β-cells via the circulation from distant sites of inflammation.

	Acknowledgements

 
This work was supported by a grant from the Ministry of Science and Culture of Lower Saxony (K S) and by the European Union (STREP SAVEBETA in the Framework programme 6 of the European Community). The skilful technical assistance of B Lueken and S Lippold is gratefully acknowledged. The authors are grateful to Dr K Dobashi (Kitakyushu, Japan) for the antibodies against rat MnSOD. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

	References

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Received in final form 20 December 2007
Accepted 4 February 2008
Made available online as an Accepted Preprint 5 February 2008

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