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• W2089100446 abstract "Viral infection is one environmental factor that may initiate β-cell damage during the development of autoimmune diabetes. Formed during viral replication, double-stranded RNA (dsRNA) activates the antiviral response in infected cells. In combination, synthetic dsRNA (polyinosinic-polycytidylic acid, poly(I-C)) and interferon (IFN)-γ stimulate inducible nitric-oxide synthase (iNOS) expression, inhibit insulin secretion, and induce islet degeneration. Interleukin-1 (IL-1) appears to mediate dsRNA + IFN-γ-induced islet damage in a nitric oxide-dependent manner, as the interleukin-1 receptor antagonist protein prevents dsRNA + IFN-γ-induced iNOS expression, inhibition of insulin secretion, and islet degeneration. IL-1β is synthesized as an inactive precursor protein that requires cleavage by the IL-1β-converting enzyme (ICE) for activation. dsRNA and IFN-γ stimulate IL-1β expression and ICE activation in primary β-cells, respectively. Selective ICE inhibition attenuates dsRNA + IFN-γ-induced iNOS expression by primary β-cells. In addition, poly(I-C) + IFN-γ-induced iNOS expression and nitric oxide production by human islets are prevented by interleukin-1 receptor antagonist protein, indicating that human islets respond to dsRNA and IFN-γ in a manner similar to rat islets. These studies provide biochemical evidence for a novel mechanism by which viral infection may initiate β-cell damage during the development of autoimmune diabetes. The viral replicative intermediate dsRNA stimulates β-cell production of pro-IL-1β, and following cleavage to its mature form by IFN-γ-activated ICE, IL-1 then initiates β-cell damage in a nitric oxide-dependent fashion. Viral infection is one environmental factor that may initiate β-cell damage during the development of autoimmune diabetes. Formed during viral replication, double-stranded RNA (dsRNA) activates the antiviral response in infected cells. In combination, synthetic dsRNA (polyinosinic-polycytidylic acid, poly(I-C)) and interferon (IFN)-γ stimulate inducible nitric-oxide synthase (iNOS) expression, inhibit insulin secretion, and induce islet degeneration. Interleukin-1 (IL-1) appears to mediate dsRNA + IFN-γ-induced islet damage in a nitric oxide-dependent manner, as the interleukin-1 receptor antagonist protein prevents dsRNA + IFN-γ-induced iNOS expression, inhibition of insulin secretion, and islet degeneration. IL-1β is synthesized as an inactive precursor protein that requires cleavage by the IL-1β-converting enzyme (ICE) for activation. dsRNA and IFN-γ stimulate IL-1β expression and ICE activation in primary β-cells, respectively. Selective ICE inhibition attenuates dsRNA + IFN-γ-induced iNOS expression by primary β-cells. In addition, poly(I-C) + IFN-γ-induced iNOS expression and nitric oxide production by human islets are prevented by interleukin-1 receptor antagonist protein, indicating that human islets respond to dsRNA and IFN-γ in a manner similar to rat islets. These studies provide biochemical evidence for a novel mechanism by which viral infection may initiate β-cell damage during the development of autoimmune diabetes. The viral replicative intermediate dsRNA stimulates β-cell production of pro-IL-1β, and following cleavage to its mature form by IFN-γ-activated ICE, IL-1 then initiates β-cell damage in a nitric oxide-dependent fashion. inducible nitric-oxide synthase interleukin interferon-γ double-stranded RNA tumor necrosis factor lipopolysaccharide fluorescein isothiocyanate reverse transcriptase-polymerase chain reaction interleukin-1 receptor antagonist protein IL-1β-converting enzyme fluorescence-activated cell sorting cycloheximide fluoromethyl ketone benzyloxycarbonyl-Tyr-Val-Ala-Asp(OMe)-fluoromethyl ketone glyceraldehyde-3-phosphate dehydrogenase Insulin-dependent diabetes mellitus is an autoimmune disease characterized by the selective destruction of insulin-secreting β-cells found in pancreatic islets of Langerhans (1Gepts W. Diabetes. 1965; 14: 619-633Crossref PubMed Scopus (966) Google Scholar). Triggering events that precipitate β-cell damage have remained elusive; however, evidence supports a role for viral infection in the initiation of autoimmune diabetes. Viruses have been isolated from pancreata, and virus-specific IgM antibodies have been identified in newly diagnosed diabetic patients (2Bach J.-F. Endocr. Rev. 1994; 15: 516-541Crossref PubMed Scopus (758) Google Scholar, 3Yoon J.-W. Diabetes Metab. Rev. 1995; 11: 83-107Crossref PubMed Scopus (69) Google Scholar). Diabetes can be induced in genetically susceptible strains of mice, rats, and primates by infection with encephalomyocarditis virus, Coxsackie B4 virus, Kilham's rat virus, rubella virus, and retrovirus (3Yoon J.-W. Diabetes Metab. Rev. 1995; 11: 83-107Crossref PubMed Scopus (69) Google Scholar, 4von Herrath M.G. Oldstone M.B.A. Curr. Opin. Immunol. 1996; 8: 878-885Crossref PubMed Scopus (109) Google Scholar). Encephalomyocarditis-induced diabetes in mice is associated with increased expression of iNOS1 and macrophage-derived cytokines IL-1β, IL-12, and TNF-α. Administration of neutralizing antisera for IL-1β or TNF or of the iNOS-selective inhibitor aminoguanidine attenuates diabetes development in this mouse model (5Hirasawa K. Jun H.S. Maeda K. Kawaguchi Y. Itagaki S. Mikami T. Baek H.S. Doi K. Yoon J.-W. J. Virol. 1997; 71: 4024-4031Crossref PubMed Google Scholar).In vitro studies have identified cytokine-stimulated iNOS expression and nitric oxide production by β-cells as one mechanism by which IL-1 inhibits insulin secretion and induces islet damage (6Corbett J.A. McDaniel M.L. Lancaster Jr., J.R. Nitric Oxide: Principles and Action. Academic Press, Inc., San Diego, CA1996: 177-217Crossref Google Scholar,7Mandrup-Poulsen T. Diabetologia. 1996; 39: 1005-1029Crossref PubMed Scopus (512) Google Scholar). Double-stranded RNA (dsRNA), formed during viral replication, is an active component of a viral infection that triggers antiviral responses in infected cells (8Jacobs B.L. Langland J.O. Virology. 1996; 219: 339-349Crossref PubMed Scopus (516) Google Scholar). The antiviral response includes the expression of type 1 interferons (9Kerr I.M. Stark G.R. J. Interferon Res. 1992; 12: 237-240Crossref PubMed Scopus (56) Google Scholar), nitric oxide production (10Karupiah G. Xie Q. Buller M.L. Nathan C. Duarte C. MacMicking J.D. Science. 1993; 261: 1445-1448Crossref PubMed Scopus (747) Google Scholar), macrophage IL-1 release (11Heitmeier M.R. Scarim A.L. Corbett J.A. J. Biol. Chem. 1998; 273: 15301-15307Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), and a general inhibition of protein translation (12Wu S. Kaufman R.J. J. Biol. Chem. 1997; 272: 1291-1296Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar,13de Haro C. Mendez R. Santoyo J. FASEB. J. 1996; 10: 1378-1387Crossref PubMed Scopus (234) Google Scholar). Similar to a viral infection, the synthetic dsRNA molecule polyinosinic-polycytidylic acid (poly(I-C)) also activates these antiviral responses (14Kreil T.R. Eibl M.M. Virology. 1996; 219: 304-306Crossref PubMed Scopus (109) Google Scholar, 15Melkova Z. Esteban M. J. Immunol. 1995; 155: 5711-5718PubMed Google Scholar, 16Clemens M.J. Elia A. J. Interferon Cytokine Res. 1997; 17: 503-524Crossref PubMed Scopus (512) Google Scholar). In vivo, administration of poly(I-C) to diabetes-resistant and -prone BioBreeding rats results in the induction and acceleration of diabetes, respectively (17Ewel C.H. Sobel D.O. Zeligs B.J. Bellanti J.A. Diabetes. 1992; 41: 1016-1021Crossref PubMed Google Scholar, 18Sobel D.O. Newsome J. Ewel C.H. Bellanti J.A. Abbassi V. Creswell K. Blair O. Diabetes. 1992; 41: 515-520Crossref PubMed Scopus (73) Google Scholar). Although viral infection has been implicated in the development of autoimmune diabetes, the response of islets and specifically β-cells to a viral insult has been poorly defined. We have shown that dsRNA, in combination with IFN-γ, inhibits glucose-stimulated insulin secretion and induces islet degeneration in a nitric oxide-dependent manner (19Heitmeier M.R. Scarim A.L. Corbett J.A. J. Biol. Chem. 1999; 274: 12531-12536Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Alone, neither poly(I-C) nor IFN-γ stimulates iNOS expression or inhibits insulin secretion by rat islets (19Heitmeier M.R. Scarim A.L. Corbett J.A. J. Biol. Chem. 1999; 274: 12531-12536Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). In combination with IFN-γ, dsRNA also activates macrophages, stimulating iNOS expression, nitric oxide formation, and IL-1 release (11Heitmeier M.R. Scarim A.L. Corbett J.A. J. Biol. Chem. 1998; 273: 15301-15307Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Islets contain 5–10 resident macrophages that express and release IL-1 when activated in response to TNF + LPS. Local accumulation of IL-1 in islets stimulates the expression of iNOS and production of nitric oxide by β-cells resulting in a potent inhibition of insulin secretion and islet degeneration (20Arnush M.A. Scarim A.L. Heitmeier M.R. Kelly C.B. Corbett J.A. J. Immunol. 1998; 160: 2684-2691PubMed Google Scholar). In this report, we show that the inhibitory and destructive effects of dsRNA + IFN-γ on insulin secretion and islet viability are mediated by the intra-islet production of IL-1. Furthermore, we show that β-cells themselves are a source of IL-1 in response to dsRNA and that β-cell production of IL-1 leads to IL-1- and nitric oxide-dependent inhibition of β-cell function. CMRL-1066 tissue culture medium,l-glutamine, penicillin, streptomycin, and rat recombinant IFN-γ were from Life Technologies, Inc. Fetal calf serum was obtained from HyClone (Logan, UT). Male Harlan Sprague-Dawley rats (250–300 g) were purchased from Harlan Breeders (Indianapolis, IN). Poly(I-C) and collagenase type XI were from Sigma. [α-32P]dCTP and enhanced chemiluminescence (ECL) reagents were purchased from Amersham Pharmacia Biotech. Human recombinant IL-1β was from Cistron Biotechnology (Pine Brook, NJ). Horseradish peroxidase-conjugated donkey anti-rabbit IgG, FITC-conjugated donkey anti-guinea pig, and CY3-conjugated donkey anti-rabbit secondary antibodies were obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Rabbit antiserum specific for the C-terminal 27 amino acids of mouse macrophage iNOS was a gift from Dr. Thomas Misko (G. D. Searle, St. Louis, MO). iNOS and cyclophilin cDNAs were gifts from Dr. Charles Rodi (Monsanto Corporate Research, St. Louis, MO) and Dr. Steve Carroll (Department of Pathology, University of Alabama, Birmingham, AL), respectively. Guinea pig anti-human insulin antibody was from Linco Research, Inc. (St. Louis, MO), and goat anti-rat IL-1β antibody was from R & D Systems (Minneapolis, MN). All other reagents were from commercially available sources. Islets were isolated from male Harlan Sprague-Dawley rats by collagenase digestion as described previously (21McDaniel M.L. Colca J.R. Kotagal N. Lacy P.E. Methods Enzymol. 1983; 98: 182-200Crossref PubMed Scopus (117) Google Scholar). Following isolation, islets were cultured overnight in complete CMRL-1066 (CMRL-1066 containing 2 mml-glutamine, 10% heat-inactivated fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin) under an atmosphere of 95% air and 5% CO2 at 37 °C. Prior to each experiment, islets were washed 3 times in complete CMRL-1066, counted, and then cultured for an additional 3 h at 37 °C. Experiments were initiated by the addition of poly(I-C) and IFN-γ followed by culture for the indicated times. Where indicated, islets were pretreated for 30 min with IRAP prior to the addition of poly(I-C) and IFN-γ. The Islet Isolation Core Facility at Washington University School of Medicine and the Diabetes Research Institute at the University of Miami provided human islets. Isolated human islets were cultured for 3 days at 37 °C in complete CMRL-1066 prior to experimentation. Where indicated, human islets were pretreated for 30 min with IRAP prior to incubation with cytokines and poly(I-C). Islets isolated from 12 rats were cultured overnight (∼1200 islets/3 ml) in complete CMRL-1066 media under an atmosphere of 95% air and 5% CO2 at 37 °C. Islets were then dispersed into individual cells by treatment with trypsin (1.0 mg/ml) in Ca2+- and Mg2+-free Hanks solution at 37 °C for 3 min as stated previously (21McDaniel M.L. Colca J.R. Kotagal N. Lacy P.E. Methods Enzymol. 1983; 98: 182-200Crossref PubMed Scopus (117) Google Scholar). Dispersed islet cells were incubated for 60 min at 37 °C in complete CMRL-1066 prior to cell sorting. Islet cells were purified as described previously (22Pipeleers D.G. Int Veld P.A. Van De Winkel M. Maes E. Schuit F.C. Gepts W. Endocrinology. 1985; 117: 806-816Crossref PubMed Scopus (322) Google Scholar) using a FACSTAR + flow cytometer (Becton Dickinson, San Jose, CA). The cells were illuminated at 488 nm, and emission was monitored at 515–535 nm. This procedure results in β- and α-cell purity of 90–95 and 80–85%, respectively. For RT-PCR analysis of IL-1α and IL-1β mRNA expression, narrow gated windows were used to enhance β-cell purity, which was greater than 98% based on post-sort FACS analysis and immunohistochemical analysis of insulin-containing cells (data not shown). Islets (220/ml of complete CMRL-1066) were cultured for 40 h with the indicated concentrations of poly(I-C), rat IFN-γ, and IRAP. The islets were isolated and washed three times in Krebs-Ringer bicarbonate buffer (KRB: 25 mmHepes, 115 mm NaCl, 24 mm NaHCO3, 5 mm KCl, 1 mm MgCl2, 2.5 mm CaCl2, and 0.1% bovine serum albumin, pH 7.4) containing 3 mmd-glucose, and insulin secretion was performed as described (23Heitmeier M.R. Scarim A.L. Corbett J.A. J. Biol. Chem. 1997; 272: 13697-13704Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Medium insulin content was determined by radioimmunoassay (24Wright P.H. Makulu D.R. Vichick D. Sussman K.E. Diabetes. 1971; 20: 33-45Crossref PubMed Scopus (73) Google Scholar). Islets (25/500 μl of complete CMRL-1066) were cultured for 96 h in 24-well microtiter plates with the indicated concentrations of poly(I-C), IFN-γ, and IRAP. Islet degeneration was determined in a double-blind manner by phase-contrast microscopic analysis. Islet degeneration is characterized by the loss of islet integrity, disintegration, and partial dispersion of islets as described previously (23Heitmeier M.R. Scarim A.L. Corbett J.A. J. Biol. Chem. 1997; 272: 13697-13704Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 25Lacy P.E. Finke E.H. Am. J. Pathol. 1991; 138: 1183-1190PubMed Google Scholar, 26Corbett J.A. McDaniel M.L. Biochem. J. 1994; 299: 719-724Crossref PubMed Scopus (81) Google Scholar). Rat or human islets (120/400 μl of complete CMRL-1066), cultured for the indicated times with poly(I-C), rat or human IFN-γ, and IRAP were isolated, lysed, and protein separated by SDS-gel electrophoresis as described (23Heitmeier M.R. Scarim A.L. Corbett J.A. J. Biol. Chem. 1997; 272: 13697-13704Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Detection of iNOS was by ECL according to the manufacturer's specifications (Amersham Pharmacia Biotech) and as described previously (23Heitmeier M.R. Scarim A.L. Corbett J.A. J. Biol. Chem. 1997; 272: 13697-13704Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Rat islets (900/3 ml complete CMRL-1066) were cultured for 18 h at 37 °C with poly(I-C), rat IFN-γ, IRAP, and cycloheximide (CHX) as indicated. After culture, the islets were washed 3 times with 0.1 m phosphate-buffered saline, pH 7.4, and total RNA was isolated using the RNeasy kit (Qiagen, Inc., Chatsworth, CA). RNA (5–10 μg) was denatured, fractionated, and transferred to Duralon UV nylon membranes (Stratagene, La Jolla, CA) as described (23Heitmeier M.R. Scarim A.L. Corbett J.A. J. Biol. Chem. 1997; 272: 13697-13704Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Membranes were hybridized to a 32P-labeled probe specific for rat iNOS or cyclophilin (27Brown R. Mackey K. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struh L.K. Current Protocols in Molecular Biology. 2. Green Publishing Associates and Wiley-Interscience, New York1997: 4.9.1-4.9.13Google Scholar). The cDNA probe was radiolabeled with [α-32P]dCTP by random priming using the Prime-a-Gene nick translation system from Promega (Madison, WI). iNOS cDNA probe corresponds to bases 509–1415 of the rat iNOS coding region. Cyclophilin was used as an internal control for RNA loading. Hybridization and autoradiography were performed as described previously (28Burd P.R. Rogers H.W. Gordon J.R. Martin C.A. Jayaraman S. Wilson S.D. Dvorak A.M. Galli S.J. Dorf M.E. J. Exp. Med. 1989; 170: 245-257Crossref PubMed Scopus (473) Google Scholar). Nitrite production was determined by mixing 50 μl of culture medium with 50 μl of Griess reagent (29Green L.C. Wagner D.A. Glogowski J. Skipper P.L. Wishnok J.S. Tannenbaum S.R. Anal. Biochem. 1982; 126: 131-138Crossref PubMed Scopus (10586) Google Scholar). The absorbance at 540 nm was measured, and nitrite concentrations were calculated from a sodium nitrite standard curve. RT-PCR analysis of IL-1α and IL-1β mRNA accumulation by rat and human islets (100/condition) and FACS-purified β- and α-cells (100,000 cells/condition) was performed as described previously (20Arnush M.A. Scarim A.L. Heitmeier M.R. Kelly C.B. Corbett J.A. J. Immunol. 1998; 160: 2684-2691PubMed Google Scholar, 30Arnush M. Heitmeier M.R. Scarim A.L. Marino M.A. Manning P.A. Corbett J.A. J. Clin. Invest. 1998; 102: 516-526Crossref PubMed Scopus (214) Google Scholar). GAPDH mRNA accumulation was used as a control for PCRs, and total RNA isolated from rat islets treated for 4 h with TNF + LPS was used as a positive control for islet expression of IL-1α and IL-1β. We have previously shown that resident macrophages are the source of IL-1 in response to TNF + LPS (20Arnush M.A. Scarim A.L. Heitmeier M.R. Kelly C.B. Corbett J.A. J. Immunol. 1998; 160: 2684-2691PubMed Google Scholar). For immunoprecipitations, rat islets (500 islets/ml of methionine-deficient minimum Eagle's medium) were treated for 18 h with poly(I-C) + IFN-γ. [35S]Methionine (500 μCi) was added, and the islets were cultured for 6 additional h. The islets were isolated, and lysed, and IL-1 was immunoprecipitated using hamster anti-IL-1α- and hamster anti-IL-1β-specific antisera as described (20Arnush M.A. Scarim A.L. Heitmeier M.R. Kelly C.B. Corbett J.A. J. Immunol. 1998; 160: 2684-2691PubMed Google Scholar). Immunohistochemistry was performed as described previously (20Arnush M.A. Scarim A.L. Heitmeier M.R. Kelly C.B. Corbett J.A. J. Immunol. 1998; 160: 2684-2691PubMed Google Scholar). In brief, islets were isolated, dispersed into individual cells by trypsin treatment as stated above, and centrifuged onto slides. The cells were fixed in 4% paraformaldehyde containing 0.1% Triton X-100 for 30 min and then blocked for 1 h with 5% bovine serum albumin (in 0.1m phosphate-buffered saline). ICE was identified using rabbit anti-mouse ICE (1:40 dilution) antiserum specific for the p10 active form of ICE, IL-1β was identified using goat anti-rat IL-1β antiserum (1:20 dilution), and insulin was identified using guinea pig anti-human insulin (1:200 dilution). Secondary antibodies included FITC- or CY3-conjugated donkey anti-rat, donkey anti-guinea pig, and donkey anti-mouse antisera (1:200 dilution). All figures for immunohistochemistry were at a × 40 magnification. To determine whether the endogenous production of IL-1 is required for dsRNA + IFN-γ-induced damage, islets were incubated for 40 h with dsRNA and IFN-γ in the presence or absence of the interleukin-1 receptor antagonist protein (IRAP). IRAP competes with IL-1 for receptor binding and thereby prevents IL-1-induced signaling events (31Arend W.P. J. Clin. Invest. 1991; 88: 1445-1451Crossref PubMed Scopus (548) Google Scholar). Treatment of rat islets with dsRNA + IFN-γ results in a 2.5-fold increase in nitrite production (poly(I-C) + IFN-γ treated, 30 pmol/islet; untreated, 12 pmol/islet). In a concentration-dependent manner, IRAP prevents poly(I-C) + IFN-γ-induced nitrite formation with maximal ∼70% inhibition at 1–10 μg/ml (Fig.1 a). We have recently shown that dsRNA + IFN-γ-induced iNOS mRNA accumulation and protein expression are maximal following 18- and 40-h incubations, respectively, and that concentrations of 50 μg/ml poly(I-C) + 150 units/ml IFN-γ stimulate maximal iNOS expression and nitrite formation by rat islets (19Heitmeier M.R. Scarim A.L. Corbett J.A. J. Biol. Chem. 1999; 274: 12531-12536Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). As shown in Fig. 1 b, IRAP prevents dsRNA + IFN-γ-induced iNOS mRNA accumulation following an 18-h incubation. IRAP also prevents dsRNA + IFN-γ-induced iNOS protein expression following a 40-h incubation (Fig. 1 c). Alone, neither IFN-γ nor dsRNA stimulates iNOS expression or nitrite formation by rat islets (Fig. 1 b and Ref. 19Heitmeier M.R. Scarim A.L. Corbett J.A. J. Biol. Chem. 1999; 274: 12531-12536Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Consistent with a requirement for IL-1 production, dsRNA + IFN-γ-induced iNOS mRNA accumulation requires de novo protein synthesis. CHX, at a concentration (10 μm) that inhibits islet total protein synthesis by greater than 95% (32Hughes J.H. Colca J.R. Easom R.A. Turk J. McDaniel M.L. J. Clin. Invest. 1990; 86: 856-863Crossref PubMed Scopus (84) Google Scholar), prevents dsRNA + IFN-γ-induced iNOS mRNA accumulation by rat islets (Fig.1 b). Importantly, CHX (at 10 μm) does not alter the levels of iNOS mRNA that accumulate in response to 1 unit/ml IL-1, nor does CHX inhibit glucose-stimulated insulin secretion by rat islets (6Corbett J.A. McDaniel M.L. Lancaster Jr., J.R. Nitric Oxide: Principles and Action. Academic Press, Inc., San Diego, CA1996: 177-217Crossref Google Scholar, 32Hughes J.H. Colca J.R. Easom R.A. Turk J. McDaniel M.L. J. Clin. Invest. 1990; 86: 856-863Crossref PubMed Scopus (84) Google Scholar). These results show that de novoprotein synthesis is required for dsRNA + IFN-γ-induced iNOS expression by rat islets and suggest that dsRNA + IFN-γ-induced iNOS expression and nitrite formation is mediated by the intra-islet release of IL-1. We have previously shown that dsRNA + IFN-γ inhibits glucose-stimulated insulin secretion and induces islet degeneration in a nitric oxide-dependent manner (19Heitmeier M.R. Scarim A.L. Corbett J.A. J. Biol. Chem. 1999; 274: 12531-12536Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). To determine whether the inhibitory and destructive effects of dsRNA + IFN-γ are mediated by the intra-islet release of IL-1, rat islets were incubated for 40 (insulin secretion) or 96 h (islet viability) with 50 μg/ml poly(I-C) + 150 units/ml IFN-γ in the presence or absence of IRAP. As shown in Fig. 2, dsRNA + IFN-γ inhibits glucose-stimulated insulin secretion and induces the degeneration of 89% of islets. IRAP prevents the inhibitory effects of dsRNA + IFN-γ on glucose-stimulated insulin secretion and attenuates islet degeneration by ∼75%. These results indicate that dsRNA + IFN-γ-induced inhibition of insulin secretion, and induction of islet degeneration is mediated by IL-1. To examine whether β-cells are a source of IL-1 in response to dsRNA + IFN-γ, primary β-cells purified by fluorescence-activated cell sorting (FACS) were incubated with poly(I-C) and IFN-γ in the presence or absence of IRAP. Following a 40-h incubation, β-cells express high levels of iNOS in response to dsRNA + IFN-γ (Fig. 3). IRAP prevents dsRNA + IFN-γ-induced iNOS expression by FACS-purified β-cells, indicating that β-cells may be one islet cellular source of IL-1. Alone, neither dsRNA nor IFN-γ induces iNOS expression by FACS-purified β-cells, and dsRNA and IFN-γ, alone or in combination, fail to induce iNOS expression by FACS-purified α-cells (data not shown). Two isoforms of IL-1 have been identified, IL-1α and IL-1β. To determine the isoform(s) of IL-1 expressed by β-cells, the effects of dsRNA and IFN-γ on IL-1α and IL-1β mRNA accumulation were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoprecipitation. Treatment of FACS-purified β-cells with poly(I-C) or poly(I-C) + IFN-γ results in IL-1β mRNA accumulation following an 18-h incubation (Fig.4 a). FACS-purified α-cells fail to express either IL-1α or IL-1β (data not shown), and FACS-purified β-cells fail to express IL-1α in response to either poly(I-C), IFN-γ, or poly(I-C) + IFN-γ (Fig. 4 a). In rat islets, poly(I-C) and poly(I-C) + IFN-γ stimulate both IL-1α and IL-1β mRNA accumulation that is first apparent following a 12-h incubation and that persists for up to 24 h (Fig. 4 b). Resident macrophages appear to be the islet cellular source of IL-1α, as dsRNA + IFN-γ fails to stimulate IL-1α mRNA accumulation in islets depleted of resident macrophages (data not shown) and primary β-cells purified by FACS (Fig. 4 a). These results indicate that dsRNA alone stimulates IL-1β expression by primary β-cells. To confirm that IL-1 is expressed at the protein level in response to dsRNA + IFN-γ, IL-1α and IL-1β were sequentially immunoprecipitated from [35S]methionine-labeled rat islets using hamster monoclonal antisera previously used to immunoprecipitate both the pro-forms and mature forms of IL-1α and IL-1β from activated macrophages (33Hill J.R. Corbett J.A. Kwon G. Marshall C.A. McDaniel M.L. J. Biol. Chem. 1996; 271: 22672-22678Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Poly(I-C) + IFN-γ stimulates the expression of immunoprecipitable pro-IL-1β from isolated rat islets following a 24-h incubation (Fig. 4 c). We were unable to immunoprecipitate IL-1α from rat islets, a finding that is consistent with IL-1α expression by the limited number of macrophages found in rat islets (∼10/islet). Also, we were unable to immunoprecipitate the mature form of IL-1β from islets, consistent with its release following proteolytic processing (Fig. 4 c(34Wilson K.P. Black J.A. Thomson J.A. Kim E.E. Griffith J.P. Navia M.A. Murcko M.A. Chambers S.P. Aldape R.A. Raybuck S.A. Livingston D.J. Nature. 1994; 370: 270-274Crossref PubMed Scopus (751) Google Scholar)). To confirm directly that β-cells are a source of IL-1β, islets were treated for 18 h with poly(I-C) or poly(I-C) + IRAP, isolated, and dispersed into individual cells, and IL-1β expressing cells were identified by immunocytochemistry. As shown in Fig. 5 a, treatment of rat islets with poly(I-C) results in IL-1β expression (redfluorescence), and IL-1β immunoreactivity localizes with insulin-containing cells (green fluorescence), as indicated by the yellow fluorescence following double exposure. To control for IL-1β binding to cell membranes, rat islets were treated for 18 h with poly(I-C) + IRAP. Under these conditions, IRAP should antagonize the interactions of IL-1β with its surface receptors on β-cells. As shown in Fig. 5 b, IL-1β expression (green fluorescence) localizes to insulin-containing cells (red fluorescence), as indicated by the yellow following double exposure. Similar results were obtained from islets treated for 18 h with poly(I-C) + IFN-γ and poly(I-C) + IFN-γ + IRAP (data not shown). In addition, IL-1β was not expressed in untreated islets nor was it expressed in islets treated for 18 h with IFN-γ (data not shown). Whereas β-cells express high levels of IL-1β, less than 1% of islet cells express this cytokine in response to poly(I-C). As expected, poly(I-C) also stimulates IL-1β expression by a second population of islet cells that appear to be resident macrophages. As shown in Fig. 5 b, IL-1β immunoreactivity localizes with a large highly vacuolized cell containing a high degree of macrophage morphology. To confirm that these cells are macrophages, islets were treated for 5 h with TNF + LPS. We have previously shown that macrophages are the sole islet cellular source of IL-1β under these conditions (20Arnush M.A. Scarim A.L. Heitmeier M.R. Kelly C.B. Corbett J.A. J. Immunol. 1998; 160: 2684-2691PubMed Google Scholar). As shown in Fig. 5 c, a 5-h incubation of islets with TNF + LPS results in the expression of IL-1β (red fluorescence) by resident macrophages. The morphology of the IL-1β expressing macrophages in Fig. 5, b andc, are nearly identical. Similar numbers of macrophages express IL-1β in response poly(I-C) as compared with TNF + LPS. These findings provide direct support for β-cell expression of IL-1β and that resident islet macrophages also produce this cytokine in response to poly(I-C). The RINm5F cell IL-1 bioassay, which is specific for active IL-1 (14Kreil T.R. Eibl M.M. Virology. 1996; 219: 304-306Crossref PubMed Scopus (109) Google Scholar,33Hill J.R. Corbett J.A. Kwon G. Marshall C.A. McDaniel M.L. J. Biol. Chem. 1996; 271: 22672-22678Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), was used to quantitate the levels of IL-1 released by islets in response to poly(I-C) and IFN-γ. In this experiment, islets were treated for 40 h with 50 μg/ml poly(I-C), 150 units/ml IFN-γ, or both poly(I-C) and IFN-γ; the culture supernatant was isolated and IL-1 levels quantitated. Alone, neither IFN-γ nor poly(I-C) stimulate IL-1 release by islets; however, poly(I-C) + IFN-γ stimulates the accumulation of 32.6 ± 1.2 pg/ml of IL-1. We have previously shown that this level of IL-1 is sufficient to stimulate iNOS expression by β-cells in the presence of IFN-γ (23Heitmeier M.R. Scarim A.L. Corbett J.A. J. Biol. Chem. 1997; 272: 13697-13704Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). The interleukin-1β-converting enzyme (" @default.
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• W2089100446 title "Pancreatic β-Cell Damage Mediated by β-Cell Production of Interleukin-1" @default.
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