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Interferon-a and interferon-g differentially affect pancreatic b-cell phenotype and function MANUEL E. BALDEÓN,1 TAEHOON CHUN,2 AND H. REX GASKINS1,2 of Nutritional Sciences and 2Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 1Division Pancreatic expression of IFN-g in animal models of autoimmune diabetes has also been reported (31, 35). Transgenic mice harboring the Ifn-g gene linked to the human insulin promoter develop insulitis and subsequently autoimmune diabetes (34). Alternatively, administration of anti-IFN-g antibody decreases diabetes incidence in the BB/Wor rat and in nonobese diabetic mice (9, 24). We have demonstrated that IFN-g concurrently decreases insulin production and upregulates cell surface expression of class IA MHC molecules on pancreatic b-cell lines, mimicking two major alterations of the prediabetic b-cell (2). To better clarify individual roles of IFN-a and IFN-g in b-cell pathology, the present study compares the effects of these cytokines on glucose responsiveness, the mRNA expression of Ifn-a6, the expression of class IA MHC antigenprocessing and antigen presentation genes, and cell surface expression of class IA MHC molecules in the pancreatic bTC3 and bTC6-F7 cell lines. type I diabetes; major histocompatibility complex class IA locus; insulitis Cells. bTC3 and bTC6-F7 cells were established from b-cell adenomas derived from transgenic mice harboring a hybrid rat insulin promoter-simian virus 40 large T-antigen gene construct (10, 14, 20). The more differentiated bTC6-F7 cells were derived by soft agar cloning and maintain normal glucose sensitivity (20). The P388D1 murine macrophage cell line was obtained from the American Type Culture Collection (ATCC; Rockville, MD; ATTC TIB-63). Cells were serially passaged in 75-cm2 tissue culture flasks (Corning Glass, Corning, NY) and maintained in DMEM supplemented with a final glucose concentration of 25 mM and with Eagle’s minimum essential medium nonessential amino acid supplement (GIBCO, Grand Island, NY), 44 mM sodium bicarbonate, 15 mM HEPES, 10,000 U/ml penicillin plus 10,000 µg/ml streptomycin, 15% (vol/vol) horse serum (HS), and 2.5% (vol/vol) fetal clone II (FC; HyClone, Logan, UT). HS was heat inactivated at 56°C for 30 min. Cultures were maintained in a humidified atmosphere of 95% air and 5% CO2 at 37°C. Cell culture studies. To characterize the individual effects of IFN-a and IFN-g on intracellular insulin content and secretion in response to glucose, bTC6-F7 cells were seeded at a density of 2 3 105 cells/well into 24-well tissue culture plates (Corning). On reaching 70–90% confluence, culture medium was replaced with fresh DMEM supplemented with 5% FC without glucose for 24 h to minimize basal levels of insulin secretion. Cultures were then exposed for 3 days to treatment medium consisting of DMEM with 15% HS, 2.5% FC, and 25 mM glucose, without or with increasing doses of recombinant mouse IFN-g (6, 12, 25, and 50 U/ml; Genentech, South San Francisco, CA; sp act 9.8 3 106 U/ml) or recombinant human IFN-a-A/D (50, 100, and 200 U/ml; Hoffmann-La Roche, Nutley, NJ; sp act 1.4 3 108 U/ml). This recombinant IFN-a, a hybrid of human IFN-A and IFN-D proteins (A/D Bgl II), is biologically active on mouse cells (32, 39). After cytokine exposure, cultures were washed three times in DMEM without glucose. Cells were TYPE ONE DIABETES IS AN autoimmune disease characterized by the selective destruction of pancreatic b-cells by autoreactive T lymphocytes (1, 4, 36). The cytokines interferon (IFN)-a and IFN-g have been associated with type 1 diabetes pathogenesis both in humans and in animal models of autoimmune diabetes (3, 17, 33). For example, pancreatic Ifn-a mRNA expression and the presence of immunoreactive IFN-a in b-cells of patients with recentonset type 1 diabetes have been reported (12, 17, 37). In addition, IFN-a expression has been associated with hyperexpression of major histocompatibility complex (MHC) class IA antigens in human islets (12, 37). In two rodent models of autoimmune diabetes, the diabetesprone DP-BB rat and streptozotocin-treated mice, Ifn-a mRNA expression in islets precedes insulitis and diabetes (16). Also, transgenic mice harboring a hybrid human insulin promoter-Ifn-a construct develop hypoinsulinemic diabetes accompanied by insulitis (39). Those studies indicate a potential role for IFN-a in the pathogenesis of autoimmune diabetes but do not demonstrate mechanisms by which IFN-a contributes to b-cell demise. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. MATERIALS AND METHODS 0363-6143/98 $5.00 Copyright r 1998 the American Physiological Society C25 Downloaded from http://ajpcell.physiology.org/ by 10.220.33.5 on August 3, 2017 Baldeón, Manuel E., Taehoon Chun, and H. Rex Gaskins. Interferon-a and interferon-g differentially affect pancreatic b-cell phenotype and function. Am. J. Physiol. 275 (Cell Physiol. 44): C25–C32, 1998.—To better clarify individual roles of interferon (IFN)-a and IFN-g in b-cell pathology during the onset of type 1 diabetes mellitus, we compared the effects of these cytokines on insulin production and major histocompatibility complex (MHC) gene expression in pancreatic b-cell lines. IFN-g but not IFN-a decreased secreted and intracellular insulin concentrations in bTC6-F7 and bTC3 cells. Likewise, IFN-g but not IFN-a treatment of b-cells upregulated mRNA expression of MHC class IA antigenprocessing genes and surface expression of class IA molecules. Alternatively, class IA MHC expression was upregulated by IFN-g and IFN-a in the P388D1 macrophage cell line. The observation of constitutive Ifn-a6 mRNA expression by a differentiated b-cell line substantiates previous indications that local expression of IFN-a in islets may trigger insulitis. Evidence that IFN-g, a product of infiltrating leukocytes, directly decreases b-cell glucose sensitivity and increases MHC class IA cell surface expression supports the postulate that IFN-g magnifies the insulitic process. C26 PANCREATIC b-CELL RESPONSES TO IFN-a AND IFN-g Table 1. Primer sequences, optimal PCR cycles, and product sizes of specific cDNA regions Primer Sequences Ifn-a6 F: 58-AGCTACTGGCCAACCTGCTCTCTA R: 58-GTCCTCATTCAGTCTTGCCAGCAAC Lmp-2 F: 58-GCCGAGCCCCCGCTCTGCTGAGATG R: 58-CGGGGAGGACGCTTCCCTCCACACA Lmp-7 F: 58-CGAGGCCAAGTGGTCATGGCGTTAC R: 58-TTTATTGTTGAGCCCTTCT Tap-1 F: 58-CAGCAGGTTCCATCACATCTCG R: 58-ACACTCGTGGTCCAGACTTCAGC Tap-2 F: 58-CTGCGACTTGGAGTGCTGTGG R: 58-AGCGGCTCATCAGAGAGGTGTCA b2m (used for Northern blot analysis) F: 58-CCGAACATACTGAACTGCTACGTAACAC R: 58-GCCATACTGACATGCTTAACTCTGC GAPDH (also used for Northern blot analysis) F: 58-GGAAGCTTGTCATCAATGG R: 58-AGATCTCGTGGTTCACACC Product Cycles Size, bp 30 524 30 801 30 944 27 414 27 660 35 208 35 225 F, forward (sense) primer; R, reverse (antisense) primer; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. sis of autoradiographs was performed with a Personal Densitometer P.D. (Molecular Dynamics, Sunnyvale, CA), and data were analyzed with ImageQuant software from Molecular Dynamics. Arbitrary densitometric units (ADU) from interest transcript and control GAPDH were used to calculate a ratio to quantify H-2K a-chain and b2m mRNA expression. Cytofluorometric analysis. To study cell surface expression of MHC class IA molecules, bTC3, bTC6-F7, and P388D1 macrophages were seeded at a density of 2 3 105 in 35-mm tissue culture dishes (Corning) in DMEM (25 mM glucose) supplemented with 15% HS plus 2.5% FC. On reaching ,70% confluence, cultures were exposed to fresh DMEM without or with IFN-a (100 U/ml) or IFN-g (50 U/ml) for 3 days. After cytokine treatment, cells were collected with an enzyme-free cell dissociation buffer (GIBCO) for immunostaining and subsequent flow cytometric analysis. Briefly, 1 3 106 cells were incubated on ice in 50 µl of fluorescence-activated cell sorter (FACS) buffer (PBS and 1% BSA) with saturating concentrations of anti-pan MHC class I monoclonal antibody (M1-42; Ref. 38; The Jackson Laboratory, Bar Harbor, ME) for 30 min. Cells were then washed twice and incubated with 10 µg/ml of anti-rat IgG-phycoerythrin conjugate (Jackson ImmunoResearch Laboratories, West Grove, PA) in 50 µl of FACS buffer at 4°C for 30 min. After a final wash, cells were resuspended in 1 ml of FACS buffer. Fluorescence intensity was quantified by flow cytometry using an Epics 752 flow cytometer (Coulter, Hialeah, FL) equipped with an argon ion laser. Data were analyzed with ELITE software from Coulter. RESULTS Effects of IFN-a or IFN-g on insulin secretion and intracellular content in glucose-stimulated bTC6-F7 cells. Routine microscopic inspection of bTC6-F7 cells did not reveal readily distinguishable morphological changes in cultures treated for up to 3 days with either IFN-a or IFN-g. Likewise, IFN treatment did not affect cell viability, as indicated by comparable protein and DNA concentra- Downloaded from http://ajpcell.physiology.org/ by 10.220.33.5 on August 3, 2017 then preincubated in DMEM (0 mM glucose) for 1 h and subjected to an insulin secretion test for 2 h in DMEM supplemented with glucose (25 mM) and 5% FC. b-Cellconditioned medium and acid-ethanol culture extracts (1.5% HCl in 70% ethanol; overnight at 4°C) were collected at the end of the 2-h secretion tests and stored at 220°C until assayed for insulin. Insulin RIA and cellular protein determinations. Insulin concentrations in cell-conditioned medium and acid-ethanol cell extracts were determined by double-antibody RIA as described previously (22). Rat insulin was used as a standard. Standards, antibodies, and 125I-labeled insulin were obtained from Linco Research (St. Louis, MO). Inter- and intra-assay coefficients of variation were 9% and 3%, respectively. Cells were harvested and sonicated in 0.5 ml of PBS plus 0.1% Triton X-100 (Fisher Biotech, Fair Lawn, NJ) for later protein determination by the Bradford microassay method (Bio-Rad, Richmond, CA). Insulin concentrations are expressed as microunits per microgram of soluble cellular protein. Statistical analysis of treatment differences was made by paired t-tests and P values ,0.05 were considered significant. Cellular DNA content. To evaluate the individual effects of IFN-a and IFN-g on cell viability, total cellular DNA concentrations were measured from bTC6-F7 cultures treated with cytokines as described above for insulin production studies. Cells were then harvested and sonicated in 0.5 ml of DNA assay buffer (50 mM Na2HPO4, 2 M NaCl, and 2 mM EDTA, pH 7.4). Total DNA concentrations from crude homogenates were determined by fluorometry using bisbenzimide (Hoechst 33258; Molecular Probes, Eugene, OR) as described previously (21). Northern blot and RT-PCR analyses. bTC6-F7 cells and P388D1 macrophages were grown in 75-cm2 tissue culture flasks (Corning) in DMEM (25 mM glucose) supplemented with 15% HS plus 2.5% FC. On reaching ,70% confluence, cultures were exposed to fresh DMEM without or with IFN-a (100 U/ml) or IFN-g (50 U/ml) for 3 days. After exposure to IFN-a or IFN-g, total cellular RNA was isolated by a singlestep guanidinium thiocyanate method (7). One microgram of total RNA for each sample was reverse transcribed with avian myeloblastosis virus RT using an oligo(dT) primer. Sequencespecific primers were designed on the basis of respective mouse cDNA nucleotide sequences (GenBank, Bethesda, MD) to amplify specific regions of Ifn-a6, class IA MHC antigenprocessing and antigen presentation genes, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNAs. Reverse transcription reactions and PCR amplification were carried out in a thermal cycler (PTC-100, MJ Research, Watertown, MA). The temperature-time sequence of 95°C for 30 s, 58°C for 30 s, and 72°C for 1 min was carried out for each PCR cycle. Primer sequences, optimal PCR cycles, and product sizes of specific cDNA regions are shown in Table 1. Amplified cDNA products were size separated by electrophoresis in 1.5% agarose gels, stained with ethidium bromide, and visualized under ultraviolet light. To characterize the effects of IFN-a and IFN-g on H-2K a-chain and of b2-microglobulin (b2m) mRNA expression, 7 µg of total RNA for each sample were size-separated in 1.25% agarose-3% formaldehyde gels and blotted onto nylon membranes (Magna Graph, Westborough, MA). The mouse class IA a-chain cDNA clone (pH-2II a) was obtained from ATCC, and mouse b2m and GAPDH cDNA probes were obtained by RT-PCR as indicated. Purified cDNA fragments were labeled with [a-32P]dCTP (DuPont, Wilmington, DE) using a random hexamer primer oligolabeling kit (Pharmacia, Piscataway, NJ). Hybridization was carried out as described previously (13). Autoradiographic exposure of the membranes to Kodak X-Omat AR film was carried out at 270°C with intensifying screens. Laser densitometric analy- PANCREATIC b-CELL RESPONSES TO IFN-a AND IFN-g Table 2. Effect of IFN-a or IFN-g on soluble protein and total DNA in bTC6-F7 cells Total DNA, µg/ml Soluble protein, µg/ml Control IFN-a Control IFN-g 192 6 9 70 6 9 213 6 7 66 6 7 162 6 25 76 6 6 178 6 6 74 6 4 Values are combined means 6 SE from 3 independent experiments. Treatments were performed in triplicate in each experiment. Cells were cultured for 3 d without (Control) or with 100 U/ml interferon (IFN)-a or 50 U/ml IFN-g. ern blot analysis. Laser densitometric analysis indicated that IFN-g treatment increased H-2K a-chain and b2m mRNA expression by bTC6-F7 cells 5-fold and 10-fold compared with untreated controls (3.3 ADU without IFN-g vs. 16.3 ADU with IFN-g for H-2K a-chain; 1.4 ADU without IFN-g vs. 14 ADU with IFN-g for b2m; Fig. 3). Treatment of P388D1 macrophages with IFN-g upregulated H-2K a-chain and b2m mRNA expression eightfold compared with untreated controls (1 ADU without IFN-g vs. 8.3 ADU with IFN-g for H-2K a-chain; 1.5 ADU without IFN-g vs. 8 ADU with IFN-g for b2m; Fig. 3). Similar to the effects of IFN-g, steady-state expression of H-2K a-chain and b2m mRNA by P388D1 macrophages was increased after 3 days of IFN-a treatment (Fig. 3). In contrast, IFN-a treatment of bTC6-F7 cells did not significantly alter the steadystate level of H-2K a-chain or b2m mRNA expression after 3 days of cytokine exposure (2 ADU without IFN-a vs. 3 ADU with IFN-a for H-2K a-chain; 1.4 ADU without IFN-a vs. 1.4 ADU with IFN-a for b2m; Fig. 3). RT-PCR analysis demonstrated that low-molecularmass polypeptide 2 (Lmp-2) and Lmp-7 genes were expressed constitutively in P388D1 macrophages and bTC6-F7 cells (Fig. 4). An increase in steady-state Lmp-2 and Lmp-7 mRNA expression was observed after 3 days of exposure to IFN-g for both cell lines (Fig. 4). Similarly, IFN-a treatment of P388D1 macrophages and bTC6-F7 cells increased Lmp-2 and Lmp-7 mRNA basal expression, although the degree of induction was less than that observed for IFN-g treatment (Fig. 4). Basal expression of transporter associated with antigen processing 1 (Tap-1) and Tap-2 mRNA was observed for P388D1 macrophages, whereas basal expression of Tap-2 mRNA but not Tap-1 mRNA was observed for bTC6-F7 cells (Fig. 4). Increases in steady-state Tap-1 and Tap-2 mRNA expression by P388D1 macrophages were observed after 3 days of exposure to IFN-g. For bTC6-F7 cells, IFN-g treatment induced Tap-1 mRNA and enhanced Tap-2 mRNA expression (Fig. 4). As for Lmp-2 and Lmp-7, IFN-a treatment of P388D1 macrophage cultures increased Tap-1 and Tap-2 mRNA expression, with the level of induction also being lower than that observed for IFN-g treatment (Fig. 4). In contrast, basal Tap-2 mRNA expression by bTC6-F7 cells was not altered by IFN-a treatment nor did IFN-a induce Tap-1 mRNA expression by these b-cells (Fig. 4). Cell surface expression of MHC class IA molecules in response to IFN-a or IFN-g. Cell surface expression of MHC class IA molecules on P388D1 macrophages, bTC3, and bTC6-F7 cells in response to IFN-a or IFN-g was compared. Basal cell surface expression of MHC class IA was observed for P388D1 macrophages and for both b-cell lines in the absence of IFN treatment (Fig. 5, A–C). Basal MHC class IA cell surface expression was higher for P388D1 macrophages compared with either of the b-cell lines (Fig. 5, A–C). Treatment with IFN-g (50 U/ml) for 3 days approximately doubled MHC class IA cell surface expression on P388D1 macrophages and increased MHC class IA expression by two orders of magnitude on each of the b-cell lines compared with untreated control cultures (Fig. 5, G–I). After IFN-g Downloaded from http://ajpcell.physiology.org/ by 10.220.33.5 on August 3, 2017 tions in cultures treated without or with IFN-a or IFN-g (Table 2). Insulin concentrations in the culture medium of IFN-g-treated bTC6-F7 cells were significantly lower than insulin concentrations in untreated control cultures after 2 h of glucose challenge (Fig. 1A). The range of IFN-g inhibition of insulin release varied between 71 and 88%, for the lowest and highest cytokine doses, respectively (Fig. 1A). In contrast, insulin concentrations in medium from bTC6-F7 cultures treated with increasing IFN-a concentrations were similar to untreated control cells (Fig. 1A). Similar to effects on secreted insulin, IFN-g significantly decreased intracellular insulin concentrations in bTC6-F7 cell cultures compared with untreated controls (Fig. 1B). Decreases in intracellular insulin content provoked by IFN-g ranged from 53% for the 6 U/ml dose to 82% for the 50 U/ml dose (Fig. 1B). In contrast to the effects of IFN-g, intracellular insulin concentrations of IFN-a-treated bTC6-F7 cells were similar to untreated controls (Fig. 1B). Similar results have been observed for both secreted and intracellular insulin with the less differentiated pancreatic bTC3 cell line (not shown). Effects of IFN-a or IFN-g on Ifn-a6 mRNA expression by bTC6-F7 cells and P388D1 macrophages. Ifn-a mRNA expression in islets is an early pathological feature of autoimmune diabetes in humans and in rodent models of type 1 diabetes (16, 17, 37). However, neither the cellular origin of IFN-a within islets nor its modes of regulation have been established (12). Accordingly, the expression of Ifn-a mRNA in bTC6-F7 cells without or with IFN-a or IFN-g treatment was studied and compared with the control P388D1 macrophage cell line. Among possible Ifn-a mRNAs, the expression of Ifn-a6 mRNA was chosen because this mouse gene locus has been conclusively demonstrated to encode a biologically active protein (15, 19, 44). RT-PCR analysis demonstrated that Ifn-a6 mRNA was expressed constitutively by both P388D1 macrophages and bTC6-F7 cells (Fig. 2). Furthermore, steady-state Ifn-a6 mRNA expression by P388D1 macrophages or bTC6-F7 cells was not altered by IFN-a or IFN-g treatment (Fig. 2). Effects of IFN-a or IFN-g on expression of class IA MHC antigen processing and antigen presentation genes by bTC6-F7 cells and P388D1 macrophages. The ability of IFN-a (100 U/ml) and IFN-g (50 U/ml) to individually modulate the expression of the class IA MHC antigenprocessing and antigen presentation genes in bTC6-F7 cells vs. P388D1 macrophages was compared by North- C27 C28 PANCREATIC b-CELL RESPONSES TO IFN-a AND IFN-g treatment, a similar level of fluorescence intensity for surface MHC class IA staining was observed for P388D1 macrophages and the two b-cell lines (Fig. 5, G–I). The mean fluorescence intensity of MHC class IA expression on the cell surface of P388D1 macrophages was approximately doubled in response to IFN-a relative to untreated control macrophages (Fig. 5, A vs. D). Thus MHC class IA expression on the surface of P388D1 macrophages was increased equally by IFN-a or IFN-g (Fig. 5, D and G). In contrast, basal MHC class IA expression on the surface of bTC3 and bTC6-F7 cells was not altered significantly by 3 days of IFN-a treatment (Fig. 5, E and F). DISCUSSION This investigation demonstrates that IFN-g but not IFN-a directly diminishes insulin production and induces expression of the MHC class IA antigen presentation pathway in pancreatic b-cells. The demonstration of comparable IFN-g and IFN-a upregulation of MHC class IA expression in macrophages further indicates that these two IFNs may play distinct roles in the insulitic process. A limited number of studies have analyzed the direct effects of IFN-a on b-cell phenotype and function. Rhodes and Taylor (33) demonstrated that treatment of isolated human islets with high doses of IFN-a (1,000 U/ml) inhibits the synthesis of proinsulin. However, those authors indicate that the high dose of IFN-a used may have reduced total protein biosynthesis, as demonstrated with other cell types (33). The present data and our previous report (2) conclusively demonstrate that low doses of IFN-g decrease insulin production by pancreatic b-cell lines without altering total protein biosynthesis or compromising cell viability. The more differentiated bTC6-F7 cells are more susceptible than bTC3 cells to the inhibitory effects of IFN-g, indicating that b-cells that maintain normal glucose responsiveness may be more sensitive to this cytokine (not shown). That possibility may provide an important clue in the search for mechanisms by which IFN-g compromises b-cell insulin production. To date, those mechanisms remain undefined. Available evidence indicates that IFN-g inhibits processes that occur after preproinsulin gene transcription but before insulin secretory granule exocytosis (Ref. 2; unpublished observations). Consistent with previous observations, IFN-g treatment of b-cells upregulated expression of the MHC class IA pathway (2, 13). In contrast, IFN-a treatment did not affect the basal level of cell surface class I MHC expression or mRNA expression of the H-2K a-chain, b2m, or the endoplasmic reticulum peptide transporter Downloaded from http://ajpcell.physiology.org/ by 10.220.33.5 on August 3, 2017 Fig. 1. Effects of interferon (IFN)-a or IFN-g on glucose-stimulated insulin secretion (A) and content (B) in bTC6-F7 cells. Cells were cultured in DMEM (25 mM glucose) for 3 days in presence or absence of IFN-a or IFN-g and subjected to a 2-h insulin secretion test. Insulin concentrations in conditioned medium and acid-ethanol cellular extracts were determined by RIA and are expressed as µU/µg of cellular protein. Experimental treatments were performed in triplicate. Data (means 6 SE) are representative of 3 independent experiments with similar results. * P , 0.05. PANCREATIC b-CELL RESPONSES TO IFN-a AND IFN-g C29 genes (Tap-1 and Tap-2). That IFN-a increased Lmp-2 and Lmp-7 mRNA expression by bTC6-F7 cells and markedly upregulated MHC class IA expression by P388D1 macrophages indicates that an effective dose of a biologically active IFN-a was used in the present study. The differential response of the b-cell lines to IFN-g vs. IFN-a is consistent with evidence that these two cytokines bind to distinct cell surface receptors and activate different DNA-binding proteins (8, 11, 25, 27–29). Despite their candidacy as key insulitic cytokines, little is know about IFN-a or IFN-g response pathways in b-cells. Treatment of ‘‘purified’’ islet cell preparations from human islets (30) with either IFN-g or IFN-a increased cell surface MHC class IA expression (30), with IFN-g having a greater stimulatory effect than IFN-a. The Fig. 3. Effects of IFN-a or IFN-g on H-2K a-chain and b2m mRNA expression by bTC6-F7 cells and P388D1 macrophages. Cells were cultured for 3 days without (control; A) or with 100 U/ml IFN-a (B) or 50 U/ml IFN-g (C). Total RNA was extracted, and Northern blot analysis was performed as described in MATERIALS AND METHODS. GAPDH mRNA expression was assayed as a constitutive control. Fig. 4. Effects of IFN-a or IFN-g on Lmp-2, Lmp-7, Tap-1, and Tap-2 mRNA expression by bTC6-F7 cells and P388D1 macrophages. Cells were cultured for 3 days without (control; A) or with 100 U/ml IFN-a (B) or 50 U/ml IFN-g (C). Total RNA was extracted and analyzed by RT-PCR as described in MATERIALS AND METHODS. GAPDH mRNA expression was assayed as a constitutive control. Lane at left contains a 1-kb DNA marker. IFN-a reagent used in those studies was derived from medium conditioned by Namalwa cells, and quantitative data is not provided for IFN-a stimulation of MHC class I expression, making it difficult to relate those results to the current study, which evaluated responses specifically in b-cells and used lower doses of a recombinant IFN-a product. In a more recent study with isolated human islets, both IFN-g and IFN-a induced TAP-1 protein and mRNA expression (41). Furthermore, a correlation was observed for cytokine enhancement of TAP-1 and human leukocyte antigen (HLA) class I expression in both isolated islets and the human HP62 pancreatic endocrine cell line (41). In agreement with the earlier study (41) and partially in agreement with the present results, IFN-g was shown to be a more potent stimulator of both TAP-1 and HLA class I expression than IFN-a when individual effects of those cytokines were compared, even with the use of a relatively large dose (500 U/ml) of a recombinant IFN-a product. Nonetheless, apparently on the basis of those results and the clear evidence for IFN-a expression in pancreases from newly diagnosed patients with type 1 diabetes (12, 17, 37), those authors predict that IFN-a is ‘‘most probably’’ the important cytokine among those capable of inducing cell surface HLA class I expression on b-cells (41). The present results bring into question that postulate, although differences in cytokine responsiveness between mouse and human islet cells may well Downloaded from http://ajpcell.physiology.org/ by 10.220.33.5 on August 3, 2017 Fig. 2. Effects of IFN-a or IFN-g on Ifn-a6 mRNA expression by bTC6-F7 cells and P388D1 macrophages. Cells were cultured for 3 days without (control; A) or with 100 U/ml IFN-a (B) or 50 U/ml IFN-g (C). Total RNA was extracted and analyzed by RT-PCR as described in MATERIALS AND METHODS. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression was assayed as a constitutive control. Lane at left contains a 1-kb DNA marker. C30 PANCREATIC b-CELL RESPONSES TO IFN-a AND IFN-g exist that would invalidate a direct comparison between species. Despite clear evidence for IFN-a expression in islets from newly diagnosed patients with type 1 diabetes (12, 17, 37), the cellular origin of IFN-a within islets has not been conclusively defined. Foulis et al. (12) localized IFN-a in insulin-containing cells via immunocytochemistry; however, that observation does not prove b-cell expression. Accordingly, our study provides the additional contribution of demonstrating constitutive Ifn-a6 mRNA expression by a differentiated b-cell line. Neither IFN-a nor IFN-g modulated basal Ifn-a6 mRNA expression in either bTC6-F7 cells or P388D1 macrophages. Further studies are required to determine the potential of exogenous cytokines to modulate IFN-a secretion from b-cells. Although it is demonstrated that IFN-a does not directly induce major phenotypic or functional changes in b-cells, those results do not necessarily argue against an important role for this cytokine in insulitis. Indeed, the demonstration of IFN-a expression by b-cells enables possible clarification of the contributions of both IFN-a and IFN-g during prediabetes. From clear evidence for IFN-a expression in islets of patients with type 1 diabetes, it has been suggested that local expression of IFN-a in response to potential diabetogenic stimuli such as viruses may trigger the insulitic process (12, 16, 17, 37). In that regard it will now be important to identify exogenous stimuli capable of modulating b-cell IFN-a expression. In support of its role as an initiating agent, IFN-a has been shown to induce intercellular adhesion molecule 1 (ICAM-1) and HLA class IA on endothelial cells from human islets (6). Increased expression of ICAM-1 and HLA class IA by endothelial cells may contribute to leukocyte infiltration during insulitis. Furthermore, IFN-a facilitates T cell stimulation by the induction of the costimulatory molecules ICAM-1 and B7.2 on antigen-presenting cells in islets (5). IFN-a also stimulates natural killer cells and Th1 lymphocyte responses (18, 26, 43). Together with previous data, the present results support the possibility that early IFN-a expression by b-cells may be a critical event in the initiation of autoimmune diabetes (5, 16, 17). Downloaded from http://ajpcell.physiology.org/ by 10.220.33.5 on August 3, 2017 Fig. 5. Comparison of effects of IFN-a or IFN-g on cell surface major histocompatibility complex (MHC) class IA expression on bTC3 (B, E, and H) and bTC6-F7 (C, F, and I) pancreatic b-cells and on P388D1 macrophages (A, D, and G). Cells were cultured in DMEM without (control; A–C) or with 100 U/ml IFN-a (D–F) or 50 U/ml IFN-g (G–I) for 3 days. Cells were stained with saturating concentrations of anti-pan MHC class IA monoclonal antibody M1-42 as described in MATERIALS AND METHODS. Fluorescence intensity was quantified by flow cytometry. PANCREATIC b-CELL RESPONSES TO IFN-a AND IFN-g The observation that IFN-g, but not IFN-a, directly affects the phenotype and function of pancreatic b-cells agrees with the notion that IFN-g plays a direct pathogenic role in autoimmune diabetes (23, 40, 42). 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