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Effect of Proinflammatory Cytokines on Gene Expression of the Diabetes-Associated Autoantigen IA-2 in INS-1 Cells

German Diabetes Research Institute at the University of Duesseldorf, Duesseldorf D-40225, Germany

Address all correspondence and requests for reprints to: J. Seissler, M.D., German Diabetes Research Institute, University of Duesseldorf, Auf'm Hennekamp 65, D-40225 Duesseldorf, Germany. E-mail: .

	Abstract

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Cytokines released from activated antigen-presenting cells and T-lymphocytes are crucially involved in the pathogenesis of type 1 diabetes. Previous studies have shown that proinflammatory cytokines play an important role in the induction of autoimmunity and ß-cell damage. Inhibition of insulin expression has been described, but their effects on other major target autoantigens, such as the tyrosine phosphatase-like protein IA-2, is not known. In the present study, we established sensitive real-time RT-PCR to measure IA-2, insulin, and inducible nitric oxide (NO) synthase (iNOS) mRNA expression. Rat insulinoma INS-1 cells were stimulated with IL-1ß, TNF-, interferon (IFN)-, and IL-2 as well as with two combinations of these cytokines (C1: IL-1ß + TNF- + IFN-; C2: TNF- + IFN-). Treatment with IL-1ß, TNF-, or IFN- alone caused a significant down-regulation of IA-2 and insulin mRNA levels in a time and dose-dependent manner, whereas IL-2 had no effect. Exposure to cytokine combinations strongly potentiates the inhibitory effects. Incubation of cells with C1 and C2 for 24 h induces a significant inhibition of IA-2 mRNA levels by 78% and 58%, respectively. Under these conditions, an up to 5 x 10⁴-fold increase of iNOS gene expression was observed. The hypothesis that the formation of NO is involved in IA-2 regulation was confirmed by the finding that the coincubation of C1 with 4 mM L-N^G-monomethyL-L-arginine, an inhibitor of the iNOS, partly reversed the down-regulation of IA-2. Further, incubation with the synthetic NO-donor S-nitroso-N-acetyl-D-L-penicillamine significantly decreased IA-2 mRNA level to 51% of basal levels. In conclusion, we have demonstrated for the first time that IL-1ß, TNF-, and IFN- exert a strong inhibitory effect on expression of the diabetes autoantigen IA-2. The action of IL-1ß may be partly mediated by the activation of the NO pathway.

	Introduction

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TYPE 1 DIABETES IS caused by progressive destruction of the insulin-producing ß-cells in the pancreas mediated by a chronic autoimmune process (1). One member of the protein tyrosine phosphatase family, designated as IA-2 or ICA512, has been identified as a major target autoantigen in type 1 diabetes (2, 3). Autoantibodies to the cytoplasmic domain of IA-2 occur in 55–75% of newly diagnosed patients with type 1 diabetes (4, 5, 6). IA-2 is mainly expressed in neurons and in pancreatic islets as a membrane protein of the secretory vesicles (7) and is highly conserved in mammals (8). Neither human, murine, nor rat IA-2 has enzymatic activity using common substrates of protein tyrosine phosphatases (9). It was shown that IA-2 binds ß2-syntrophin and neuronal nitric oxide (NO) synthase (NOS), suggesting that IA-2 links the secretory granules with the actin cytoskeleton and the NO signaling pathways in ß-cells (10). Recently, we demonstrated that IA-2 mRNA is up-regulated in INS-1 cells by stimuli that increase cytoplasmic cAMP levels and by prolonged incubation with high glucose concentrations suggesting that IA-2 may be involved in the regulation of the ß- cell function (11).

Studies in animal models of type 1 diabetes have shown that the first cells infiltrating the pancreatic islets are activated macrophages and dendritic cells, followed by CD4⁺- and CD8⁺-positive T cells (12, 13). The infiltrating cells release a number of cytokines, mainly IL-1ß, TNF-, and interferon (IFN)-. These cytokines exert cytotoxic and inhibitory effects on pancreatic ß-cells and have been shown to represent major effector molecules involved in ß-cell destruction and the induction of ß-cell-specific autoimmunity (14, 15). These findings raise the question whether those cytokines could influence the expression of autoantigens, e.g. insulin and IA-2, and thereby promote the antigenicity of specific autoantigens.

The aim of the present study was to investigate whether cytokines modulate the expression of the autoantigen IA-2. Results were compared with insulin that is known to be regulated by proinflammatory cytokines. To quantify mRNA levels, we established a real-time RT-PCR technique based on the LightCycler system (16) that allows a highly specific and sensitive measurement of amplified PCR products. We here demonstrate that IL-1ß, TNF-, and IFN- synergistically decrease IA-2 and insulin mRNA levels in rat insulinoma cells. Our data suggest that the activation of the inducible iNOS (iNOS)/NO pathway is involved in IA-2 regulation.

	Materials and Methods

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Cell culture and stimulation
Expression of IA-2 and insulin mRNA was investigated in INS-1 cells, a partial glucose sensitive rat insulinoma cell line, which were a kind gift from Prof. C. B. Wollheim (University of Geneva, Geneva, Switzerland) (17). INS-1 cells (passages 102–110) were cultured as described previously (11). Three days before stimulation, cells were cultured in RPMI 1640 medium with 7 mM glucose, 10% fetal calf serum, 2 mM glutamine, 50 µM 2-mercaptoethanol (basal medium). The influence of cytokines was studied by the treatment with rat IL-1ß (0.1–100 U/ml), TNF- (10–1000 U/ml), IFN- (1–1000 U/ml), and IL-2 (10–100 U/ml) for 6 and 24 h. In addition, two cytokine combinations were tested: C1, IL-1ß (10 U/ml) + IFN- (100 U/ml) + TNF- (500 U/ml) and C2, IFN- (100 U/ml) + TNF- (500 U/ml). All cytokines were supplied by Biosource Technologies, Inc. Europe (Nivelles, Belgium). The viability of the INS-1 cells was assessed by simultaneously staining with propidium iodide and acridine orange and analysis under a fluorescent microscope as described (18). At least 300 cells were counted in each field.

The role of NO was investigated by coincubation of the cytokine combination C1 with 4 mM of L-N^G-monomethyL-L-arginine (L-NMMA), a potent inhibitor of the iNOS), for 24 h and by incubation with the synthetic NO-donor S-nitroso-N-acetyl-D-L-penicillamine (SNAP, 250 µM) for 6 h. L-NMMA and SNAP were purchased from Alexis (Gruenberg, Germany).

LightCycler RT-PCR
Total RNA extraction and cDNA synthesis was performed as described (11). To generate control templates for the quantification we cloned rat ß-actin (bp 478–819; GenBank accession no. J00691), IA-2 (bp 1642–2463; D38222), insulin-1 (bp 4243–4538; J00747) and iNOS (bp 2560–2882; NM012611) cDNA fragments from INS-1 cells by RT-PCR into the pGEM-T Easy vector (Promega Corp., Madison, WI). Expression of mRNA was analyzed by quantitative real-time RT-PCR using the LightCycler system (Roche Diagnostics, Mannheim, Germany). PCRs were run in glass capillaries in a volume of 20 µl containing 2 µl FastStart DNA SybrGreen I mix, 3 mM MgCl₂, 0.5 µM of each primer, and 2 µl linearized control plasmids (standard) or cDNA. After each PCR, a melting curve analysis was performed to confirm the specificity of the amplified PCR product. Quantification was based on a set of four standards for each gene product which were run in parallel with the cDNA samples under identical PCR conditions. Standard curves were generated by serial dilution of the control templates ranging from 10⁷ to 10⁴ copies (ß-actin), 10⁶ to 10³ copies (IA-2), 10⁸ to 10⁵ copies (insulin), and 10⁸ to 10¹ copies (iNOS), respectively. To generate standard curves, the crossing cycle numbers of the logarithmic-linear PCR phase (crossing points) of the standards were plotted vs. the logarithm of their concentrations (Fit Points method). To exclude interassay variations cDNA samples from basal and stimulated cells were only compared with each other when they were run together in one PCR. The constitutively expressed housekeeping gene ß-actin was used to normalize the probes to equal mRNA/cDNA levels. The relative changes in levels of specific IA-2 mRNA were expressed in percent of basal values that were set equal to 100%.

Each real-time PCR was performed with an initial denaturation step of 10 min at 95 C and an amplification for 40 cycles using the following conditions: ß-actin: 5'-ACCCACACTGTGCCC ATCTA-3', 5'-GCC ACA GGA TTC CAT ACC CA-3', 15 sec at 95 C, 5 sec at 58 C and 15 sec at 72 C; IA-2: 5'-TGCGCTCATTGCTGCTTACTCTG-3', 5'-GGCGCTCCTTATCCCGTTGT TT-3', 15 sec at 95 C, 5 sec at 63 C and 10 sec at 72 C; insulin: 5'-ACCCAAGTCCCGTCGTGA AGT-3', 5'-CCAGTTGGTAGAGGGAGCAGATG-3', 15 sec at 95 C, 5 sec at 61 C and 10 sec at 72 C; iNOS: 5'-GCCACGGAAGAGACGCACAGG-3', 5'-TGGGTCTTCGGGCTTCAG GTTATT-3', 15 sec at 95 C, 5 sec at 65 C, and 10 sec at 72 C.

	Northern blot analysis

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Northern blot analysis was performed with 5 µg total RNA of each sample as described previously (11). Briefly, RNA blotted and UV-cross-linked onto a nylon membrane were hybridized simultaneously with a digoxigenin (DIG)-labeled 757-bp fragment of rat ß-actin cDNA, a DIG-labeled 822-bp fragment of rat IA-2 cDNA, and a DIG-labeled 296-bp fragment of rat insulin-1 cDNA on the same blot for 18 h at 68 C. The membranes were stained with an anti-DIG alkaline phosphatase labeled antibody and disodium 3-(4-methoxyspiro(1,2-dioxetane-3,2'-(5-chloro)- tricyclodecan)-4yl) phenyl phosphate solution. Chemiluminescent signals were detected and quantified by the Lumi Imager system (Roche Diagnostics).

	Griess test for the measurement of nitrite concentration

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Nitrite production of the INS-1 cells was determined by mixing 50 µl of culture supernatants with 50 µl of Griess reagent (Sigma, Taufkirchen, Germany) in 96-well plates (19). The absorbance was measured at 550 nm in an ELISA plate reader (Emax; Molecular Devices Corp., Graefeling, Germany) and nitrite concentrations were calculated from a sodium nitrite standard curve (0.75–100 µM).

	Statistical analysis

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Results are presented as means ± SEM from at least three independent experiments. Significant differences between experimental groups were analyzed by one-way ANOVA with Bonferroni’s multiple comparisons test. P < 0.05 was considered to be significant.

	Results

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Establishment of the LightCycler RT-PCR
Quantification of the absolute copy numbers of cDNA samples by LightCycler RT-PCR is based on the real-time detection of amplification products in the log phase of the PCR. Figure 1 illustrates the amplification of IA-2 standards and a cDNA sample in a representative LightCycler run. The calculated error in the standard curves was always less than 0.1. The efficiency of all PCRs, given by the equation: efficiency = 10[-1/slope]*, was between 1.8 and 1.95, which is fairly consistent with the theoretical PCR efficiency of 2.0 corresponding to a doubling of the original copy number in each cycle during the exponential PCR phase. The intraassay coefficient of variation of the PCRs ranged from 9.7–11.7%. The respective cycle threshold crossing points resulted in an intraassay coefficient of variation between 0.8% and 1.4%.

View larger version (28K): [in this window] [in a new window]   Figure 1. LightCycler fluorescence curves from a typical IA-2 PCR run. Online fluorescence curves for the PCR amplification of IA-2 standards (106 to 103 copies), a cDNA obtained from unstimulated INS-1 cells and the no-template control (ntc) (A). Melting curves of all samples show a single melting maximum of 88 C for IA-2 indicating the specificity of the reaction (B).

Influence of cytokines on IA-2 and insulin mRNA levels
To determine the dose dependency, INS-1 cells were incubated for 24 h with different cytokine concentrations followed by analysis of mRNA expression and measurement of cell viability. We observed a dose-dependent decrease of IA-2 mRNA concentrations (up to 34%, 67%, and 62% of basal levels) after treatment with IL-1ß, TNF-, and IFN-, respectively. In contrast, stimulation with IL-2 did not significantly change IA-2 mRNA levels (Fig. 2). Because cell viability was significantly reduced at high cytokine concentrations, 50 U/ml IL-1ß, 500 U/ml TNF-, 100 U/ml IFN-, and 100 U/ml IL-2 were chosen for further experiments at which 77 ± 10%, 90 ± 4%, 67 ± 2%, and 96 ± 1% of INS-1 cells were viable after a 24-h incubation period (Fig. 3). At these concentrations, cytokine treatment did not significantly affect mRNA expression of ß-actin indicating that this gene can be used as housekeeping gene to normalize the cDNA content (Fig. 3A).

View larger version (17K): [in this window] [in a new window]   Figure 2. Dose-dependence of IA-2 and insulin mRNA levels in response to various cytokines. INS-1 cells were grown in basal medium and stimulated for 24 h with IL-1ß (0.1–100 U/ml), TNF- (10–1000 U/ml), IFN- (1–1000 U/ml), and IL-2 (10–100 U/ml). Quantification of IA-2 () and insulin () mRNA copy numbers was performed by LightCycler-PCR. Data are expressed as percent of IA-2 and insulin mRNA expression obtained under basal conditions and are from two to three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. basal.

View larger version (64K): [in this window] [in a new window]   Figure 3. Cytokines decrease cell viability but do not affect levels of ß-actin mRNA expression. INS-1 cells were stimulated with 50 U/ml IL-1ß, 500 U/ml TNF-, 100 U/ml IFN-, C1 or C2 for 24 h. Then equal amounts of total RNA were reverse transcribed to cDNA and amplified for 25 cycles (log phase) using the ß-actin primers in the LightCycler system. Products were separated on a 2% agarose gel and visualized by ethidium bromide staining (A). In parallel, cell viability was measured by simultaneous propidium iodide/acridine orange staining (B).

View larger version (25K): [in this window] [in a new window]   Figure 4. Time-dependent effects of cytokines on IA-2 and insulin mRNA expression. INS-1 cells were grown in basal medium and incubated with IL-1ß (50 U/ml), TNF- (500 U/ml), IFN- (100 U/ml), IL-2 (100 U/ml), and with cytokine combinations C1 (10 U/ml IL-1ß + 100 U/ml IFN- + 500 U/ml TNF-) or C2 (500 U/ml TNF- + 100 U/ml IFN-) for 6 and 24 h. Incubation with forskolin (10 µM) served as positive control. Quantification of IA-2 and insulin mRNA copy numbers was performed by LightCycler PCR. Data are expressed as percent of IA-2 and insulin mRNA expression obtained under basal conditions and are from three to four independent experiments. *, P < 0.05; **P < 0.01; ***, P < 0.001 vs. basal.

Effect of NO
To study the role of NO as a possible mediator of IA-2 gene expression iNOS mRNA levels were measured and studies with the iNOS inhibitor L-NMMA were performed. Exposure of INS-1 cells to IL-1ß, C1 and C2 resulted in highly significant increase of iNOS mRNA levels from low level basal expression (24.0 ± 10.0 cDNA copies, no template control: 0–9 copies) to 3.4 ± 3.2 x 10⁵, 12.6 ± 1.1 x 10⁵ and 1.6 ± 1.4 x 10⁵ cDNA copies per 1 x 10⁵ ß-actin molecules, respectively (P < 0.0001) (Fig. 5B). The 24-h accumulated nitrite concentration in response to IL-1ß, C1, and C2 was 22.8 ± 2.3 µmol/liter, 24.0 ± 1.1 µmol/liter, and 15.6 ± 0.8 µmol/liter, respectively. In contrast, only low level iNOS expression and NO production was observed by incubation with TNF- (1.2 ± 1.1 x 10⁴ copies; 2.1 ± 1.2 µmol/liter nitrite) or IFN- (71 ± 64 copies; 0.07 ± 0.03 µmol/liter nitrite) (Fig. 5A). Coincubation of C1 with 4 mM L-NMMA significantly reduced nitrite levels to 1.3 ± 0.4 µmol/liter indicating that L-NMMA indeed decreased NO production. Interestingly, in the presence of 4 mM L-NMMA cytokine induced suppression of IA-2 expression was partly reversed from 22 ± 2% to 64 ± 4% of basal levels (P < 0.05). In parallel, insulin gene expression increased from 6 ± 2% to 31 ± 5% (P < 0.01) (Fig. 4C). To compare the results obtained by LightCycler PCR with a conventional method, we performed a Northern blot of several RNA samples. Northern blot analysis confirmed the data obtained by the LightCycler (Fig. 6). In a second set of experiments, we investigated whether chemically derived NO can also inhibit IA-2 and insulin gene expression. Treatment of INS-1 cells with the synthetic NO-donor SNAP for 6 h was found to have a low effect on cell viability (90 ± 4% viable), but induces the formation of nitrite in a similar amount (21 ± 0.6 µmol/liter) as observed after incubation with the cytokine combination 1 for 24 h (Fig. 5A). This was accompanied by a significant down-regulation of IA-2 mRNA (51 ± 6%, P < 0.01) and insulin mRNA (30 ± 6%, P < 0.001) (Fig. 5B).

View larger version (31K): [in this window] [in a new window]   Figure 5. NO is a mediator of cytokine-induced down-regulation of IA-2 and insulin mRNA. INS-1 cells were grown in basal medium and incubated with cytokines (50 U/ml IL-1ß, 500 U/ml TNF-, 100 U/ml IFN-, and C1 for 24 h), the cytokine combination C1 plus 4 mM L-NMMA (24 h) or with SNAP (250 µM) for 6 h. The accumulation of nitrite in cell culture supernatants was measured by Griess test (A). In parallel, probes were analyzed for iNOS (B) and IA-2/insulin (C) mRNA expression by LightCycler PCR. Copy numbers were expressed in percent of basal conditions (B). *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. basal; , P < 0.001 vs. C1.

View larger version (67K): [in this window] [in a new window]   Figure 6. Northern blot analyses of NO effects on IA-2 and insulin gene expression. Total RNA was extracted from INS-1 cells cultured under basal conditions (lanes 1 and 4) or incubated with the cytokine combination C1 for 24 h (lane 2), C1 and 4 mM L-NMMA for 24 h (lane 3) or 250 µM SNAP for 6 h (lane 5), blotted onto nylon membrane, and hybridized simultaneously with DIG-labeled ß-actin, IA-2 and insulin probes, respectively.

	Discussion

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To detect changes in mRNA levels, we here established a real-time RT-PCR that overcomes current limitations of mRNA quantification such as Northern blots and most forms of endpoint RT-PCR techniques (20, 21). This study demonstrates that the LightCycler-assisted PCR provides accurate values for target molecule number with high sensitivity (threshold of 10 copies per probe for IA-2, insulin and iNOS transcripts) and a high reproducibility and specificity indicated by the low intraassay CVs and the melting curve analysis. To our knowledge, this is the first study reporting on the real-time, quantitative measurement of not only IA-2 but also insulin mRNA levels in ß-cells. We show that diabetogenic cytokines exert a strong dose- and time-dependent inhibitory effect on the gene expression of the diabetes-associated autoantigen IA-2 in INS-1 cells. INS-1 cells were chosen because they represent a well-established ß-cell line that avoids interference with stimulatory and inhibitory hormonal factors from - and -cells (11). The regulation of IA-2 expression was compared with insulin, a ß-cell-specific protein and well defined autoantigen in type 1 diabetes, whose expression is known to be suppressed by the cytokines IL-1ß, IFN-, and TNF- (14, 22, 23, 24). In line with studies on the regulation of insulin expression in INS-1 cells or isolated islet cells, treatment with individual cytokines results in a 53–92% inhibition of insulin mRNA levels. Under these conditions, a significant down-regulation of IA-2 mRNA was observed decreasing to 55–72% compared with basal levels. Because we demonstrate that cytokine treatment did not affect ß-actin mRNA levels and we also detect significant effects after short-term incubation of 6 h, at a time point when the viability was apparently not yet impaired, our data suggest that the modulation of IA-2 and insulin gene expression may result from specific cytokine-mediated signals. Our findings are in agreement with a recent study on the identification of cytokine-regulated genes in which a decrease of IA-2 mRNA was described after exposure of rat islet cells to IL-1ß (25).

The strongest inhibition of both IA-2 and insulin expression was observed after exposure to combinations of proinflammatory cytokines. Previous studies have shown that the cytokines IFN- and TNF- potentiate the effect of IL-1ß. Incubation of isolated rodent islets with combinations of IL-1ß with IFN- and/or TNF- strongly inhibit ß-cell function and induce dose-dependent ß-cell damage (14, 26, 27). In vitro studies also indicate that IL-1ß alone or in combination with TNF- and/or IFN- influences the expression pattern of a number of ß-cell proteins (22, 25, 28, 29, 30, 31). These changes may occur on the translational as well as on the transcriptional level independently of the cytotoxic effects of the cytokines. It is important to note that another study has demonstrated that stimulation of isolated rat islets with IL-1ß, TNF-, or IFN- results in the down-regulation of protein and mRNA expression of glutamic acid decarboxylase (32). Thus, it is obvious that the expression the three major target antigens in type 1 diabetes, insulin, IA-2, and glutamic acid decarboxylase, are down-regulated after exposure to cytokines released by infiltrating mononuclear cells during the development of type 1 diabetes. Our data indicate that diabetogenic cytokines may not boost antigenicity by increasing autoantigen expression. Their major effects on antigenicity may be mediated by the release of antigens from damaged ß-cells and the activation of effector T cells. Other studies have shown that IL-1ß exerts inhibitory actions on ß- cell-specific proteins such as the transcription factor PDX-1, the glucose transporter Glut-2, and the proinsulin convertase PC2, whereas the expression of hemoxygenase HO-1, heat shock protein HSP70, and superoxide dismutase MnSOD was induced (22, 25, 29, 30, 31). These observations may be explained by a shift of the ß-cells toward a phenotype with increased expression of stress proteins involved in cell defense and repair and reduced expression of ß-cell-specific proteins.

We here show that the inhibition of IA-2 gene expression may be induced by the cytokine-mediated increase in NO formation. There is compelling evidence that NO is an important messenger molecule in the regulation of insulin expression and ß-cell function. Several studies conclusively demonstrate that IL-1ß induces the expression of iNOS and the production of the free radical NO from L-arginine after a lag period of 3–6 h (14, 26, 27, 28). NO inhibits the activity of mitochondrial enzymes such as aconitase, suppresses insulin secretion, and causes nuclear DNA damage (33, 34, 35). We demonstrate that stimulation of INS-1 cells with a combination of IL-1ß, TNF-, and IFN- modulates the expression of IA-2 and insulin by the cytokine-induced stimulation of iNOS expression and NO production. Suppression of iNOS by coincubation with L-NMMA attenuates the inhibitory cytokine effects. Furthermore, incubation of the INS-1 cells with the synthetic NO-donor SNAP results in a 50% inhibition of IA-2 expression. These findings clearly indicate that NO may activate factors that are required for down-regulating IA-2 mRNA and insulin mRNA. Interestingly, Ort et al. (10) recently reported on the presence of a NOS binding site in the cytoplasmic domain of IA-2, suggesting a link between IA-2 and the NO-related metabolic pathways. Because the iNOS inhibitor L-NMMA did not completely suppress the cytokine effects, other mechanisms such as the formation of oxygen reactive species may participate in the inhibition of IA-2 expression (14, 15). One alternative factor might be prostaglandin E₂, which is produced by cyclooxygenase-2 and proposed to be involved in IL-1ß-induced inhibition of insulin secretion (36). The reported reduction in cAMP content might contribute to the decreased IA-2 mRNA levels because we have shown recently that cAMP strongly stimulates IA-2 expression (11). The absence of NO formation after treatment with IFN- suggests that IFN- signaling involves alternative pathways such as the activation of the transcription factors STAT1 and IFN regulatory factor-1 (37, 38). The molecular mechanism of IFN--induced inhibition of IA-2 expression remained to be determined in further studies.

In conclusion, we here report on the influence of proinflammatory cytokines on IA-2 and insulin mRNA expression. By real-time RT-PCR, we demonstrate for the first time that IL-1ß, IFN-, and TNF- synergistically inhibit IA-2 gene expression and provide evidence that the formation of NO is involved in the signal transduction pathway. These findings support the concept that proinflammatory cytokines have an important impact on the regulation of autoantigen expression that could play a role in the induction and natural course of ß-cell autoimmunity.

	Footnotes

 
This work was supported by grants from the Eli Lilly Foundation International (to J.S.) and the Deutsche Diabetes-Gesellschaft (to J.S.). This study was a part of a Ph.D. thesis at the University of Duesseldorf (by H.S.).

Abbreviations: CV, Coefficient of variation; DIG, digoxigenin; IFN-, interferon-; L-NMMA, L-N^G-monomethyl-L-arginine; NO, nitric oxide; iNOS, inducible NOS; NOS, nitric oxide synthase; SNAP, S-nitroso- N-acetyl-D-L-penicillamine.

Received June 4, 2002.

Accepted for publication June 26, 2002.

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