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Endocrinology Vol. 138, No. 7 2747-2753
Copyright © 1997 by The Endocrine Society

ARTICLES

Interferon-{gamma}-Induced Interferon Regulatory Factor-1 (IRF-1) Expression in Rodent and Human Islet Cells Precedes Nitric Oxide Production1

Malin Flodström and Décio L. Eizirik

Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden (M.F., D.L.E.) and Department of Metabolism and Endocrinology, Diabetes Research Center, Vrije Universiteit Brussel, Brussels, Belgium (D.L.E.)

Address all correspondence and requests for reprints to: Malin Flodström, Department of Medical Cell Biology, Uppsala University, Box 571, S-751 23 Uppsala, Sweden. E-mail: malin.flodstrom{at}medcellbiol.uu.se


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The radical nitric oxide (NO) may be a mediator of ß-cell damage in IDDM. The cytokines IFN-{gamma} and IL-1ß are required for expression of the enzyme nitric oxide synthase (iNOS), and NO production by human pancreatic islets. In this study, possible mechanisms by which IFN-{gamma} participates in iNOS messenger RNA (mRNA) expression were evaluated in both rodent and human islets cells. Addition of IFN-{gamma}, before or after arrest of IL-1ß-induced iNOS gene transcription by actinomycin D, did not prolong iNOS mRNA half life in the rat insulin-producing cell line RINm5F (RIN cells). IFN-{gamma} also failed to modify IL-1ß-induced activation of the transcription factor {kappa}B (NF-{kappa}B) in RIN cells, as determined by electrophoretic mobility shift assay. However, IFN-{gamma} induced an early (30 min–1 h) increase in interferon regulatory factor-1 (IRF-1) mRNA expression and a later (2 h) 19-fold increase in RIN cell nuclear IRF-1 protein content, an effect further potentiated by IL-1ß. The total cellular content of IRF-1 protein increased by 30- to 50-fold in human islets exposed for 2–8h to IFN-{gamma} or IFN-{gamma} + IL-1ß. IL-1ß alone induced a marginal and transient increase in IRF-1. It has been previously reported that nicotinamide prevents IL-1ß-induced IRF-1 expression in rat pancreatic islets. However, nicotinamide (20 mM) presently failed to prevent IL-1ß + IFN-{gamma}-induced IRF-1 protein expression in human pancreatic islets. In conclusion, the effects of IFN-{gamma} on iNOS expression can neither be explained by iNOS mRNA stabilization nor increased NF-{kappa}B activation. However, IFN-{gamma} induces an early increase in cellular IRF-1 content, and this may contribute to increased iNOS mRNA expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN-DEPENDENT diabetes mellitus (IDDM) results from a selective destruction of the insulin-producing pancreatic ß-cells. A mononuclear infiltration, consisting mainly of macrophages and T-lymphocytes, is observed early in the disease process, reviewed in Ref. 1 . Inflammatory products of these invading cells, such as cytokines and free radicals, have been hypothesised to play a relevant role for ß-cell damage, reviewed in (2, 3). In vitro exposure to cytokines have detrimental effects on both rat and human islet function (3). Rodent islets exposed to IL-1ß express the inducible form of nitric oxide synthase (iNOS), and the deleterious effects of the cytokine is mostly mediated by nitric oxide (NO) formation, reviewed in Refs. 4, 5 . Islet iNOS activation is further increased when IL-1ß is combined with other cytokines, such as tumour necrosis factor-{alpha} (TNF-{alpha}) and interferon-{gamma} (IFN-{gamma}). In human islet preparations, it is necessary to use a combination of two to three cytokines (i.e. IL-1ß + IFN-{gamma} or IL-1ß + IFN-{gamma} + TNF-{alpha}) to induce NO formation (5) and the role for NO in cytokine-induced human ß-cell dysfunction still remains unclear. Thus, while one study indicates that NO mediates cytokine-induced human ß-cell dysfynction (6), three other studies showed dissociation between NO production and the deleterious effects of cytokines (7, 8, 9, 10). Even if the amount of NO produced by human islet endocrine cells may not be enough to harm these cells, there are other possible sources for NO production during insulitits, namely activated macrophages (11, 12) and islet capillary endothelial cells (13). The combined production of NO by mononuclear cells, endothelial cells, and the endocrine cells may generate enough NO to damage human ß-cells (14), emphasizing the need to further understand regulation of iNOS expression in endocrine and nonendocrine islet cells.

Cellular NO production may be regulated by transcriptional and posttranscriptional events, such as expression and stability of iNOS messenger RNA (mRNA) and protein (5), as well as by the availability of substrate and cofactors (15). It has been previously shown that activation of the transcription factor NF-{kappa}B is a necessary step for cytokine-induced iNOS mRNA expression and NO formation both in insulin producing cell lines rodent and human islets (16, 17, 18, 19). However, IL-1ß alone induces nuclear NF-{kappa}B binding but fails to stimulate NO production in human islets (19). Thus, while activation of NF-{kappa}B seems to be sufficient to induce iNOS expression in rodent islets, other factors are probably needed for iNOS expression in human islets. Because IFN-{gamma}, in combination with IL-1ß, is required for human iNOS induction, it is conceivable that some of these factors are induced by IFN-{gamma}.

Interferon regulatory factor-1 (IRF-1), a transcription factor activated in response to IFN-{gamma}, is indispensable for IFN-{gamma}- or LPS-induced expression of the iNOS gene in murine macrophages (20). Recently, it was shown that IL-1ß induces IRF-1 mRNA expression and NO production in rat islets, and that both phenomena were prevented by high concentrations of nicotinamide (20 mM) (21). Unlike NF-{kappa}B, which is present in the cytosol bound to an inhibitory subunit I{kappa}-B (22), IRF-1 has to be synthesized de novo (23). Considering that protein synthesis is necessary for iNOS mRNA expression in insulin producing cells (5), it is conceivable that IRF-1 is one of the newly synthesized proteins involved in iNOS expression. Besides the putative effects of IFN-{gamma} on iNOS mRNA expression discussed above, the cytokine may also increase iNOS mRNA stability, as described for murine macrophages (24).

In the present study, we examined IFN-{gamma} actions on NF-{kappa}B and IRF-1 activation and the potential posttranscriptional effects of the cytokine on iNOS mRNA stability in the insulin producing cell line RINm5F. Some of these experiments were also reproduced in rat and human pancreatic islets.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cytokines and chemicals
Human recombinant IL-1ß (bioactivity of 50 U/ng) was kindly provided by Dr. K. Bendtzen (Laboratory of Medical Immunology, Rigs-hospitalet, Copenhagen, Denmark). Recombinant human and murine IFN-{gamma} (bioactivities of 6.7 and 10 U/ng, respectively) were purchased from AMS Biotechnology (Täby, Sweden). The cytokine concentrations used in this study were derived from data previously obtained in RINm5F cells (25), rat (26), and human pancreatic islets (8). Human IL-1ß was used in both human and rat experiments.

Chemicals were purchased from the following sources: culture medium RPMI-1640 (containing 0 or 11 mM glucose) and FCS, Northumbria Biologicals (Irvine, UK); collagenase from Clostridium histolyticum, Boehringer-Mannheim (Mannheim, Germany); agarose, FMC Bioproducts (Rockland, ME); MagnaGraph Nylon Transfer Membrane, Micron Separation (Westboro, MA); nitrocellulose filters, Schleicher & Schuell (Dassel, Germany). Double-stranded oligomers for electrophoretic mobility shift assay (EMSA) were produced by Dr. J. Seibt (Department of Immunology, Uppsala University, Uppsala, Sweden) and labeled with [{alpha}-32P]-deoxy-CTP using the Megaprime DNA labeling kit, Amersham International (Aylesbury, UK). All other chemicals of analytical grade were obtained from E. Merck (Darmstadt, Germany) or Sigma.

Tissue culture
Human islets were isolated from pancreata retrieved from 14 heart-beating organ donors sent to the Central Unit of the ß-Cell Transplant, Brussels as previously described (27). The age of the donors (mean ± SEM) was 32 ± 4 yr (range 8–61 yr). Aliquots of the preparations were examined by electron microscopy (n = 14), indicating less than 0.5% exocrine cells. The prevalence of insulin- and glucagon-positive cells was determined by light microscopical examination of immunohistochemically stained islets (28), showing 46.5 ± 3.8% insulin-positive cells and 11.4 ± 2.0% glucagon-positive cells. After isolation, islets were cultured in Brussels for 3–16 days (8 ± 1 days) (29) before being sent by air to Uppsala (27). Adult male Sprague Dawley rats (local colony, Biomedical Centre, Uppsala, Sweden) were housed, fed, and cared for according to the guidelines of the Swedish Regulations for Animal Care. All experiments involving rat tissue were approved by the Ethical Committee on Animal Experiments, Uppsala, Sweden. Rats were killed by cervical dislocation under ether anesthesia and islets isolated by collagenase digestion. Islets were cultured for to 2–4 days before experiments. In Uppsala, both human and rat islets were cultured free-floating in RPMI-1640 medium containing 10% (vol/vol) FCS, benzylpenicillin (100 U/ml), streptomycin (0.1 mg/ml), and either 5.6 (human islets) (8, 27) or 11 mM glucose (30). Medium was changed every second day.

The insulinoma cell line RINm5F, originally provided by Professor Å. Lernmark (then at the Hagerdorn Institute, Copenhagen, Denmark), was trypsinized and subcultured in medium RPMI-1640 supplemented with 10% (vol/vol) FCS, benzylpenicillin (100 U/ml), streptomycin (0.1 mg/ml) (25). Experiments were performed when cells reached 60–80% confluence.

Nitrite determination
The presence of nitrite, a stable product of NO reacting with molecular oxygen, in the culture mediums was determined with the Griess reagent (31). Thus, 10 µl of freshly prepared reagent, consisting of 0.5% naphtylethylenediamin dihydrochloride, 5% sulphanilamide and 25% concentrated H3PO4, was added to triplicate aliquots of culture medium (100 µl). After incubation for two min at 60 C, the absorbance of the reaction product was measured spectrophotometrically at 546 nm. A standard curve of sodium nitrite was used to calculate nitrite concentrations.

Northern blot analyses and studies of mRNA stability
For the Northern blotting, total RNA was extracted from 5 x 106 RINm5F cells using the RNeasy-kit (Qiagen, Hilden, Germany). Equal amounts of RNA (20–30 µg) were then electrophoretically separated on 1% agarose gels containing 2.2 M formaldehyde. After acridine orange staining of gels, to control for similar sample loadings, the RNA was transferred to a nylon membrane and the Northern blots hybridized to [32P]-labeled complementary DNAs (cDNAs) coding for mouse macrophage iNOS (a kind gift from Dr. J. M. Cunningham, Hematology-Oncology Division, Harvard Medical School, Boston, MA) or human IRF-1 (kindly provided by Dr. H. Ueda, Molecular and Cellular Biology Department; Nippon Boehringer Ingelheim Co. Ltd., Kawanishi, Japan). Membranes were subsequentially hybridized to cDNA encoding human glyceraldehyde 3-phosphate dehydrogenase (GAPDH, American Type Culture Collection, Rockville, MD), used as an internal control. GAPDH mRNA expression is unaffected by different stages of cell growth in distinct cell lines (32), or by acute IL-1ß exposure in insulin-producing and rat islets (33). The hybridization and autoradiography were performed as previously described (33, 34). The autoradiograms were subjected to densitometric scanning using the Quick Scan Jr. densitometer (Helena Laboratories, Beaumont, TX) and expressed in arbitrary units of OD. In all experiments, iNOS or IRF-1 ODs were corrected by values of GAPDH OD.

For iNOS mRNA stability studies, cells were initially exposed to IL-1ß (25 U/ml) or IL-1ß (25 U/ml) + IFN-{gamma} (1000 U/ml) for 6 h, to achieve maximal iNOS mRNA expression (25). Cells were then washed and fresh medium containing actinomycin D (act D, 5 mg/ml) added to arrest transcription. In some experiments, IFN-{gamma} was added together with act D. Cells were harvested after 0, 2, 4, and 6 h and iNOS mRNA contents analyzed by Northern blot (see above).

Nuclear protein extraction and EMSA
3–5 x 106 RINm5F cells were exposed to cytokines for 20–60 min (19). Nuclear protein was extracted and NF-{kappa}B binding activity in the protein fractions was determined by EMSA as previously described in (19). A double-stranded 26 mer oligonuclotide containing the {kappa}B binding site 5'-AGCTTCAGAGGGGACTTTCCGAGAGG was labeled with [32P] dCTP and used for the EMSA. A 100-fold excess of nonlabeled oligonucleotide was used as a negative control. The samples were then separated on 5% nondenaturing polyacrylamide gels, exposed to film, and the band intensities quantified by densitometric scanning with a Quick Scan Jr. densitometer. OD values were corrected for the amount of loaded protein, determined with Bradford reagent (35).

Western blot analysis
Groups of 60 rat or 100–150 human islets were exposed to cytokines and/or nicotinamide (20 mM) for 2–8 h, washed twice in cold PBS, pelleted by centrifugation, and sonicated for 10 sec in 100 µl cold TE (10 mM Tris, 1 mM EDTA) containing 0.5 mM phenylmethylsulfonylfluoride. One aliquot was taken for total protein content determinations (35), and the remaining lysate was precipitated with two volumes of cold acetone. Protein was pelleted by centrifugation (10 min at 12 000 rpm) and solubilized in SDS-gel sample buffer (2% SDS; 100 mM Tris, pH 6.8; 100 mM ß-mercaptoethanol; 0.01% bromophenol blue; 10% glycerol) by boiling for 4 min. Equal amounts of protein (10–20 µg) were then run on 9% SDS-polyacrylamide gels and transferred to nitrocellulose filters. The same method was used for immunoblotting of nuclear proteins extracted from RINm5F (for extraction of nuclear proteins see above). Filters were preblocked with 5% fat-free milk powder before incubation with antibodies against rat or human IRF-1 (Santa Cruz Biotechnology, CA) diluted 1:400 in PBS + 5% fat-free milk powder. Horse-radish peroxidase (HRP) linked goat antirabbit Ig was used as a secondary antibodies. Immunodetection was then performed using the ECL immunoblotting detection system (Amersham International, Aylesbury, UK). Band intensities were quantified from non saturated exposures using the Quick Scan Jr densitometer.

Statistical analysis
Results are presented as means ± SEM. Data were compared using Student’s unpaired or paired t test. When multiple comparisons were performed, the data were evaluated by ANOVA, followed by group comparisons with Student’s t test and correction of P values for multiple comparisons by the Bonferroni method (36). In the experiments with human pancreatic islets, results obtained from each donor were considered as one individual observation, even when experiments were performed in duplicate or in triplicate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In an initial series of experiments, we evaluated whether the potentiating effects of IFN-{gamma} on IL-1ß-induced iNOS expression were due to a synergistic effect on NF-{kappa}B activation (Fig. 1Go). Densitometric scanning of the autoradiographs showed an 55-fold increase in binding activity after exposure to IL-1ß alone (control, 82 ± 33; IL-1ß, 4531 ± 489; P < 0.001 vs. control; n = 3), but no increase after exposure to IFN-{gamma} (IFN-{gamma}, 101 ± 50; n = 3). Moreover, IFN-{gamma} did not potentiate IL-1ß effects on NF-{kappa}B binding (increase compared with control: IL-1ß alone, 70-fold; IL-1ß + IFN-{gamma}, 72-fold; n = 2). When exposure time was extended to 60 min, IFN-{gamma} again failed to activate or further increase IL-1ß induced NF-{kappa}B nuclear binding (data not shown). Thus, IFN-{gamma} neither activates NF-{kappa}B nor potentiates the stimulatory effect of IL-1ß on this transcription factor.



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  		Figure 1. Nuclear NF-{kappa}B binding in RINm5F cells exposed to cytokines. RINm5F cells were exposed to IL-1ß (25 U/ml) and/or IFN-{gamma} (1000 U/ml) for 20 min and nuclear protein fractions extracted as described in Materials and Methods. NF-{kappa}B binding activities were evaluated by EMSA. Lanes: 1, control; 2, IFN-{gamma}; 3, IL-1ß; 4, IL-1ß + IFN-{gamma}; 5, negative control using a 100-fold excess of nonlabeled oligonucleotide. The autoradiograph is representative for two to three separate experiments.

 
In parallel to these experiments, RIN cells were exposed to cytokines for 12 h for medium nitrite determination. Cells exposed to IL-1ß produced 25–30 times more nitrite than control cells (control, 2 ± 1 pmol NO2-/µg DNA x h, n = 6; IL-1ß-exposed cells, 55 ± 20 pmol NO2-/µg DNA x h; n = 5; P < 0.01, ANOVA). IFN-{gamma} alone failed to increase nitrite levels (1.0 ± 0.3 pmol NO2-/µg DNA x h, n = 6), but it potentiated IL-1ß effects (148 ± 29, n = 6; P < 0.01 vs. both control and cells exposed to IL-1ß alone; ANOVA).

To test whether IFN-{gamma} alters the stability of iNOS mRNA, RINm5F cells were treated either with IL-1ß alone or IL-1ß + IFN-{gamma} for 6 h, and transcription then arrested by actinomycin D (act D, Fig. 2Go). A similar 40–70% decline in iNOS mRNA expression was observed 2–6 h after act D addition in both IL-1ß and IL-1ß + IFN-{gamma}-treated cells. In a second series of experiments, cells were exposed to IL-1ß alone for 6 h, and then act D added with or without IFN-{gamma}. Again, there was a rapid decline in iNOS mRNA levels already after 2 h (IL-1ß, 73%; IL-1ß + IFN-{gamma}, 51% of control; n = 2; experimental conditions as in Fig. 2Go), followed by further decrease after 4 and 6 h (4 h, 56 and 40%; 6 h, 33 and 28% of control, respectively for IL-1ß and IL-1ß + IFN-{gamma}). These observations suggest that IFN-{gamma} does not potentiate IL-1ß-induced iNOS expression by increasing mRNA stability.



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  		Figure 2. Time course study of iNOS mRNA stability in RINm5F cells exposed to cytokines. RINm5F cells were treated with IL-1ß (25 U/ml) alone or in combination with IFN-{gamma} (1000 U/ml). After 6 h (time 0), the cells were rinsed with RPMI, and fresh medium containing actinomycin D (5 mg/ml), but without cytokines, was added. Total RNA was extracted at time 0 and at 2, 4, and 6 h after blockage of gene transcription. iNOS mRNA expression was determined as described in Materials and Methods and presented as percentage of iNOS mRNA at time 0. The results are means ± SEM of three separate experiments.

 
We next investigated the expression of the transcription factor IRF-1 in RINm5F cells. As evaluated by Northern blot analysis (Fig. 3Go), cytokines induced a clear increase in IRF-1 mRNA expression already after 30 min, with a peak after 1 h. To further evaluate this phenomenon, we performed additional experiments where RINm5F cells were exposed for 1 h to cytokines (methods as in Fig. 3Go, data as OD corrected for GAPDH expression). IL-1ß and IFN-{gamma} induced respectively a 6- and 21-fold increase in IRF-1 mRNA expression (n = 8; respectively P < 0.05 and P < 0.01 vs. control; ANOVA), whereas the combination of IL-1ß + IFN-{gamma} induced a 68-fold increase in IRF-1 expression vs. control and an 11-fold increase compared with cells treated with IL-1ß alone (n = 8; P < 0.01 both vs. control and cells treated with IL-1ß alone; ANOVA).



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  		Figure 3. Time course of IRF-1 mRNA expression in RINm5F cells. Total RNA (20–30 µg) was obtained from RINm5F cells treated with IL-1ß, IFN-{gamma} or IL-1ß + IFN-{gamma} for 0.5, 1.0, or 2.0 h. Sequential hybridizations were performed with cDNAs coding for IRF-1 and GAPDH. Top, Northern blot representative of three separate experiments. Bottom, Northern blots were evaluated by densitometric scanning and, after correction for GAPDH content, expressed as arbitrary units of optical density. Results are means ± SEM of three separate experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with respective controls (i.e. no cytokines added; unpaired t test).

 
To examine whether IRF-1 is also expressed in primary cells, isolated rat and human islets of Langerhans were exposed to the cytokines for 2 h and IRF-1 protein expression evaluated by Western blotting of total proteins (Fig. 4Go, A and B). A low basal expression of IRF-1 was detected both in rat and human islets. In rat islets, IFN-{gamma} induced a 4- to 5-fold increase in IRF-1 expression, an effect not further increased by IL-1ß (4). In human islets (4), IFN-{gamma} alone or in conjunction with IL-1ß induced a marked increase in IRF-1 (32- to 40-fold above control levels). As observed for rat islets (4), IL-1ß alone induced a small (4- to 6-fold) but nonsignificant increase of IRF-1 content in human islets (4). In two experiments, human islet IRF-1 contents were determined after longer exposure periods to cytokines. IRF-1 expression was still slightly increased (6- to 7-fold above control) after 4-h exposure to IL-1ß, with a return to basal levels (1–2-fold above control) after 8 h, whereas in islets treated with IFN-{gamma} and IFN-{gamma} + IL-1ß, IRF-1 levels were still markedly elevated both after 4 h (52-to 53-fold above control) and 8 h (25- to 40-fold above control).



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  		Figure 4. Fig. 4 A and B. IRF-1 protein contents in control or cytokine-exposed rat (A) and human islets (B). Rat and human islets were exposed to IL-1ß (rat, 25 U/ml); human, 50 U/ml) and/or IFN-{gamma} (1000 U/ml) for 2 h. Protein was extracted and Western blotting performed as described in Materials and Methods. The blots were evaluated by densitometric scanning and expressed as arbitrary units of OD. All results are means ± SEM of four (rat) or five (human) experiments. *, P < 0.05; **, P < 0.01 compared with respective controls, ANOVA.

 
In parallel to these experiments, human islet nitrite production was evaluated after 14 h exposure to cytokines. Neither IL-1ß nor IFN-{gamma} alone increased basal medium nitrite contents (pmol/µg DNA x h; control, 1.4 ± 0.7, n = 5; IL-1ß, 0.4 ± 0.2, n = 5; IFN-{gamma}, 0.4 ± 0.4, n = 4). On the other hand, islets exposed to IL-1ß + IFN-{gamma} produced 6- to 7-fold more nitrite than control islets (9.0 ± 2.3 pmol/µg DNA x h, n = 5; P < 0.05 vs. control, ANOVA).

Nuclear migration is a crucial event for IRF-1-induced gene transcription. Thus, we next examined the appearance of IRF-1 in the nuclear protein fractions of RINm5F cells exposed to IL-1ß and/or IFN-{gamma} for 2 h (Fig. 5Go). IL-1ß and IFN-{gamma} alone increased nuclear IRF-1 contents by respectively 3-fold and 19-fold (compared with control), whereas IRF-1 contents in islets exposed to a combination of IL-1ß + IFN-{gamma} were 37-fold higher than in control cells.



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  		Figure 5. IRF-1 protein contents in nuclear protein fractions from RINm5F cells exposed to cytokines. RINm5F cells were exposed to IL-1ß (25 U/ml) and/or IFN-{gamma} (1000 U/ml) for 2 h. Nuclear protein fractions were extracted and Western blotting performed as described in Materials and Methods. The blots were evaluated by densitometric scanning and expressed as arbitrary units of OD. Results are expressed as the mean ± SEM of four separate experiments. *, P < 0.01 compared with control, ANOVA.

 
To evaluate whether nicotinamide prevents cytokine-induced IRF-1 expression, human pancreatic islets were pretreated with nicotinamide (20 mM) for 1 h, and then exposed for 2 h to IL-1ß + IFN-{gamma} (Fig. 6Go). Nicotinamide neither effected basal IRF-1 protein expression nor prevented IL-1ß + IFN-{gamma}-induced increase in IRF-1 expression.



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  		Figure 6. Nicotinamide effects on IL-1ß- and IFN-{gamma}-induced IRF-1 protein expression. Human islets were precultured during 1 h with nicotinamide (20 mM) before exposure to IL-1ß (50 U/ml) plus IFN-{gamma} (1000 U/ml) for 2 h. Nicotinamide was also present during cytokine exposure. Protein was extracted and Western blotting performed as described in Materials and Methods. The blots were evaluated by densitometric scanning and expressed as arbitrary units of OD. Results are expressed as the mean ± SEM of four separate experiments. *, P < 0.01 compared with control, ANOVA.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
iNOS mRNA expression in insulin producing cells depends on gene transcription, protein synthesis and activation of the transcription factor NF-{kappa}B (5). It has been previously shown that, whereas IL-1ß is a sufficient stimulus for induction of iNOS mRNA expression and NO production in the insulinoma cell line RINm5F and rat islets of Langerhans, a combination of at least IL-1ß + IFN-{gamma} is required to induce NO formation by human islets ((5), and present data). It is noteworthy that IL-1ß alone activates NF-{kappa}B in human islets without inducing iNOS activity (19). Thus, it is likely that additional signal(s) provided by IFN-{gamma} are required to increase expression of the human iNOS gene.

Data obtained in other cell types suggest that putative sites for IFN-{gamma} action include stabilization of iNOS mRNA (24), potentiation of IL-1ß-induced NF-{kappa}B activation (37) and induction of other nuclear transcription factors, such as IRF-1 (38). In the present study, we observed that IFN-{gamma} alone or in combination with IL-1ß and added before or after transcriptional arrest by actinomycin D, did not increase the stability of iNOS mRNA in RINm5F cells. Moreover, IFN-{gamma} did not potentiate IL-1ß-induced activation of the transcription factor NF-{kappa}B. However, IFN-{gamma} or IFN-{gamma} + IL-1ß induced an early expression of the transcription factor IRF-1 in RINm5F cells, rat and human islets. Moreover, this was followed by appearance of IRF-1 protein in the nucleus, an important step for IRF-1 action (23). To our knowledge, this is the first demonstration that IFN-{gamma} induces IRF-1 expression in human and rodent islet cells. Binding sequences for IRF-1 are present in the rat and human iNOS promoters (39, 40), and deletion analysis in the rodent macrophage cell line RAW 264.7 suggests that this factor regulates iNOS expression by binding to an interferon response element (IRE) in the murine iNOS promoter (41). It is thereby possible that IFN-{gamma}-induced IRF-1 expression plays a role for iNOS mRNA expression in human pancreatic islets.

IRF-1 competes with IRF-2 for the binding to sequences found in the promoters from IFN-{gamma}-responsive genes and is presently known to be a key factor in regulation of cell growth and apoptosis (23). IFN-{gamma}-induced IRF-1 gene expression involves activation of STAT-1{alpha} (Signal Transducers and Activators of Transcription-1{alpha}) dimers and their subsequent binding to GAS (gamma-activated sequence) elements in the IRF-1 promoter. NF-{kappa}B binding sequences are present both in the human and rodent IRF-1 promoters, and NF-{kappa}B may regulate IRF-1 transcription in response to other stimuli then IFN-{gamma},(42). Because IL-1ß induces NF-{kappa}B activation in both human and rodent islet cells (17, 18), this may explain the presently observed marginal increase in IRF-1 expression in cells exposed to IL-1ß alone. Interestingly, IFN-{gamma}-induced IRF-1 mRNA levels in RINm5F cells are still increasing after 2 h exposure, whereas there is already a decreased IRF-1 expression in cells stimulated with IL-1ß alone or in combination with IFN-{gamma} (Fig. 3Go). In human islets, IFN-{gamma} also evokes a stronger and longer lasting effect on IRF-1 expression than IL-1ß. Moreover, human islet cells present a higher increase in IRF-1 protein content after exposure to IFN-{gamma}, as compared with rat islet cells (Fig. 4Go). Thus, if IRF-1 is indeed necessary for iNOS expression in human islets, it is conceivable that the modest IRF-1 induction by IL-1ß (Fig. 4BGo) is not sufficient to synergize with NF-{kappa}B and trigger iNOS expression. On the other hand, the marked increase in IRF-1 induced by IFN-{gamma} may be enough for, together with IL-1ß-induced NF-{kappa}B activation, the induction of iNOS mRNA. Clearly, future studies blocking either IRF-1 expression or nuclear binding are required to clarify this issue.

Nicotinamide, an inhibitor of poly (ADP-ribose) polymerase and, at high concentrations, a free radical scavenger, partially blocks cytotoxic effects of cytokines on rodent and human pancreatic islets (7, 10, 43, 44). It was recently shown that nicotinamide (20 mM) inhibits IL-1ß-induced IRF-1 mRNA expression and decreases NO production in rat pancreatic islets (21). Based on these data, it was concluded that these beneficial effects of nicotinamide are mediated via IRF-1 inhibition. However, our present data suggest that this is not the case for human pancreatic islets. Thus, 20 mM nicotinamide did not modify cytokine-induced IRF-1 in these cells.

Antibodies against IFN-{gamma} prevents diabetes development in the NOD mice, and the cytokine has been found in the insulitis lesion of NOD mice and BB rats, reviewed in Ref. 2 . Besides the above described effects of IFN-{gamma} on iNOS expression, the cytokine affect the expression of other proteins of potential relevance for ß-cell destruction. Thus, IFN-{gamma} alone or in combination with IL-1 up-regulate expression of MHC class I (45) and Fas (CD95 or Apo-1) (46) in cultured rodent islets and anti-IFN-{gamma}-antibodies prevent overexpression of MHC class I molecules in NOD mice (2). In other cell types, expression of MHC class I and ß2-microglobulin is coregulated by NF-{kappa}B and members of the IRF-family (47). Thus, it may be of interest to characterize the role of IRF-1 in the expression of these genes in rodent and human pancreatic islets.


   Acknowledgments
 
The skillful technical assistance of M. Engkvist, I.-B. Hallgren, and E. Törnelius is gratefully acknowledged. Authors thank Dr. T. Karlsson, Department of Medical Cell Biology, for valuable discussions and Professor D. Pipeleers, Coordinator of the ß-Cell Transplant, for providing the human islet preparations and information on human islet cell composition.


   Footnotes
 
1 This study made use of human islets prepared by the Central Unit of the ß-cell Transplant, supported by a Shared Costs Action of the European Community. It was also supported by Grants from the Juvenile Diabetes Foundation International, Swedish Medical Research Council (12X-9237; 12X-109; 12X-9886; 12X-6538; 12P-9287; and 12P-8982), the Swedish Diabetes Association, the Novo-Nordisk Insulin Fund, the Family Ernfors Fund and the Göran Gustafsson Foundation. Back

Received February 11, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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