Interferon-alpha and interferon-gamma differentially affect pancreatic beta -cell phenotype and function

Manuel E. Baldeón1, Taehoon Chun2, and H. Rex Gaskins1,2

1 Division of Nutritional Sciences and 2 Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

To better clarify individual roles of interferon (IFN)-alpha and IFN-gamma in beta -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 beta -cell lines. IFN-gamma but not IFN-alpha decreased secreted and intracellular insulin concentrations in beta TC6-F7 and beta TC3 cells. Likewise, IFN-gamma but not IFN-alpha treatment of beta -cells upregulated mRNA expression of MHC class IA antigen-processing genes and surface expression of class IA molecules. Alternatively, class IA MHC expression was upregulated by IFN-gamma and IFN-alpha in the P388D1 macrophage cell line. The observation of constitutive Ifn-alpha 6 mRNA expression by a differentiated beta -cell line substantiates previous indications that local expression of IFN-alpha in islets may trigger insulitis. Evidence that IFN-gamma , a product of infiltrating leukocytes, directly decreases beta -cell glucose sensitivity and increases MHC class IA cell surface expression supports the postulate that IFN-gamma magnifies the insulitic process.

type I diabetes; major histocompatibility complex class IA locus; insulitis

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

TYPE ONE DIABETES IS AN autoimmune disease characterized by the selective destruction of pancreatic beta -cells by autoreactive T lymphocytes (1, 4, 36). The cytokines interferon (IFN)-alpha and IFN-gamma 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-alpha mRNA expression and the presence of immunoreactive IFN-alpha in beta -cells of patients with recent-onset type 1 diabetes have been reported (12, 17, 37). In addition, IFN-alpha 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 diabetes-prone DP-BB rat and streptozotocin-treated mice, Ifn-alpha mRNA expression in islets precedes insulitis and diabetes (16). Also, transgenic mice harboring a hybrid human insulin promoter-Ifn-alpha construct develop hypoinsulinemic diabetes accompanied by insulitis (39). Those studies indicate a potential role for IFN-alpha in the pathogenesis of autoimmune diabetes but do not demonstrate mechanisms by which IFN-alpha contributes to beta -cell demise.

Pancreatic expression of IFN-gamma in animal models of autoimmune diabetes has also been reported (31, 35). Transgenic mice harboring the Ifn-gamma gene linked to the human insulin promoter develop insulitis and subsequently autoimmune diabetes (34). Alternatively, administration of anti-IFN-gamma antibody decreases diabetes incidence in the BB/Wor rat and in nonobese diabetic mice (9, 24). We have demonstrated that IFN-gamma concurrently decreases insulin production and upregulates cell surface expression of class IA MHC molecules on pancreatic beta -cell lines, mimicking two major alterations of the prediabetic beta -cell (2). To better clarify individual roles of IFN-alpha and IFN-gamma in beta -cell pathology, the present study compares the effects of these cytokines on glucose responsiveness, the mRNA expression of Ifn-alpha 6, the expression of class IA MHC antigen-processing and antigen presentation genes, and cell surface expression of class IA MHC molecules in the pancreatic beta TC3 and beta TC6-F7 cell lines.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cells. beta TC3 and beta TC6-F7 cells were established from beta -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 beta TC6-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-alpha and IFN-gamma on intracellular insulin content and secretion in response to glucose, beta TC6-F7 cells were seeded at a density of 2 × 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-gamma (6, 12, 25, and 50 U/ml; Genentech, South San Francisco, CA; sp act 9.8 × 106 U/ml) or recombinant human IFN-alpha -A/D (50, 100, and 200 U/ml; Hoffmann-La Roche, Nutley, NJ; sp act 1.4 × 108 U/ml). This recombinant IFN-alpha , 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 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. beta -Cell-conditioned 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 -20°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-alpha and IFN-gamma on cell viability, total cellular DNA concentrations were measured from beta TC6-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. beta TC6-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-alpha (100 U/ml) or IFN-gamma (50 U/ml) for 3 days. After exposure to IFN-alpha or IFN-gamma , total cellular RNA was isolated by a single-step 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. Sequence-specific primers were designed on the basis of respective mouse cDNA nucleotide sequences (GenBank, Bethesda, MD) to amplify specific regions of Ifn-alpha 6, class IA MHC antigen-processing 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.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Primer sequences, optimal PCR cycles, and product sizes of specific cDNA regions

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-alpha and IFN-gamma on H-2K alpha -chain and of beta 2-microglobulin (beta 2m) 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 alpha -chain cDNA clone (pH-2II a) was obtained from ATCC, and mouse beta 2m and GAPDH cDNA probes were obtained by RT-PCR as indicated. Purified cDNA fragments were labeled with [alpha -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 -70°C with intensifying screens. Laser densitometric analysis 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 alpha -chain and beta 2m mRNA expression.

Cytofluorometric analysis. To study cell surface expression of MHC class IA molecules, beta TC3, beta TC6-F7, and P388D1 macrophages were seeded at a density of 2 × 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-alpha (100 U/ml) or IFN-gamma (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 × 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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effects of IFN-alpha or IFN-gamma on insulin secretion and intracellular content in glucose-stimulated beta TC6-F7 cells. Routine microscopic inspection of beta TC6-F7 cells did not reveal readily distinguishable morphological changes in cultures treated for up to 3 days with either IFN-alpha or IFN-gamma . Likewise, IFN treatment did not affect cell viability, as indicated by comparable protein and DNA concentrations in cultures treated without or with IFN-alpha or IFN-gamma (Table 2).

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Effect of IFN-alpha or IFN-gamma on soluble protein and total DNA in beta TC6-F7 cells

Insulin concentrations in the culture medium of IFN-gamma -treated beta TC6-F7 cells were significantly lower than insulin concentrations in untreated control cultures after 2 h of glucose challenge (Fig. 1A). The range of IFN-gamma 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 beta TC6-F7 cultures treated with increasing IFN-alpha concentrations were similar to untreated control cells (Fig. 1A).


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 1.   Effects of interferon (IFN)-alpha or IFN-gamma on glucose-stimulated insulin secretion (A) and content (B) in beta TC6-F7 cells. Cells were cultured in DMEM (25 mM glucose) for 3 days in presence or absence of IFN-alpha or IFN-gamma 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 ± SE) are representative of 3 independent experiments with similar results. * P < 0.05.

Similar to effects on secreted insulin, IFN-gamma significantly decreased intracellular insulin concentrations in beta TC6-F7 cell cultures compared with untreated controls (Fig. 1B). Decreases in intracellular insulin content provoked by IFN-gamma 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-gamma , intracellular insulin concentrations of IFN-alpha -treated beta TC6-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 beta TC3 cell line (not shown).

Effects of IFN-alpha or IFN-gamma on Ifn-alpha 6 mRNA expression by beta TC6-F7 cells and P388D1 macrophages. Ifn-alpha 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-alpha within islets nor its modes of regulation have been established (12). Accordingly, the expression of Ifn-alpha mRNA in beta TC6-F7 cells without or with IFN-alpha or IFN-gamma treatment was studied and compared with the control P388D1 macrophage cell line. Among possible Ifn-alpha mRNAs, the expression of Ifn-alpha 6 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-alpha 6 mRNA was expressed constitutively by both P388D1 macrophages and beta TC6-F7 cells (Fig. 2). Furthermore, steady-state Ifn-alpha 6 mRNA expression by P388D1 macrophages or beta TC6-F7 cells was not altered by IFN-alpha or IFN-gamma treatment (Fig. 2).


View larger version (35K):
[in this window]
[in a new window]
 
Fig. 2.   Effects of IFN-alpha or IFN-gamma on Ifn-alpha 6 mRNA expression by beta TC6-F7 cells and P388D1 macrophages. Cells were cultured for 3 days without (control; A) or with 100 U/ml IFN-alpha (B) or 50 U/ml IFN-gamma (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.

Effects of IFN-alpha or IFN-gamma on expression of class IA MHC antigen processing and antigen presentation genes by beta TC6-F7 cells and P388D1 macrophages. The ability of IFN-alpha (100 U/ml) and IFN-gamma (50 U/ml) to individually modulate the expression of the class IA MHC antigen-processing and antigen presentation genes in beta TC6-F7 cells vs. P388D1 macrophages was compared by Northern blot analysis. Laser densitometric analysis indicated that IFN-gamma treatment increased H-2K alpha -chain and beta 2m mRNA expression by beta TC6-F7 cells 5-fold and 10-fold compared with untreated controls (3.3 ADU without IFN-gamma vs. 16.3 ADU with IFN-gamma for H-2K alpha -chain; 1.4 ADU without IFN-gamma vs. 14 ADU with IFN-gamma for beta 2m; Fig. 3). Treatment of P388D1 macrophages with IFN-gamma upregulated H-2K alpha -chain and beta 2m mRNA expression eightfold compared with untreated controls (1 ADU without IFN-gamma vs. 8.3 ADU with IFN-gamma for H-2K alpha -chain; 1.5 ADU without IFN-gamma vs. 8 ADU with IFN-gamma for beta 2m; Fig. 3). Similar to the effects of IFN-gamma , steady-state expression of H-2K alpha -chain and beta 2m mRNA by P388D1 macrophages was increased after 3 days of IFN-alpha treatment (Fig. 3). In contrast, IFN-alpha treatment of beta TC6-F7 cells did not significantly alter the steady-state level of H-2K alpha -chain or beta 2m mRNA expression after 3 days of cytokine exposure (2 ADU without IFN-alpha vs. 3 ADU with IFN-alpha for H-2K alpha -chain; 1.4 ADU without IFN-alpha vs. 1.4 ADU with IFN-alpha for beta 2m; Fig. 3).


View larger version (57K):
[in this window]
[in a new window]
 
Fig. 3.   Effects of IFN-alpha or IFN-gamma on H-2K alpha -chain and beta 2m mRNA expression by beta TC6-F7 cells and P388D1 macrophages. Cells were cultured for 3 days without (control; A) or with 100 U/ml IFN-alpha (B) or 50 U/ml IFN-gamma (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.

RT-PCR analysis demonstrated that low-molecular-mass polypeptide 2 (Lmp-2) and Lmp-7 genes were expressed constitutively in P388D1 macrophages and beta TC6-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-gamma for both cell lines (Fig. 4). Similarly, IFN-alpha treatment of P388D1 macrophages and beta TC6-F7 cells increased Lmp-2 and Lmp-7 mRNA basal expression, although the degree of induction was less than that observed for IFN-gamma treatment (Fig. 4).


View larger version (56K):
[in this window]
[in a new window]
 
Fig. 4.   Effects of IFN-alpha or IFN-gamma on Lmp-2, Lmp-7, Tap-1, and Tap-2 mRNA expression by beta TC6-F7 cells and P388D1 macrophages. Cells were cultured for 3 days without (control; A) or with 100 U/ml IFN-alpha (B) or 50 U/ml IFN-gamma (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.

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 beta TC6-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-gamma . For beta TC6-F7 cells, IFN-gamma treatment induced Tap-1 mRNA and enhanced Tap-2 mRNA expression (Fig. 4). As for Lmp-2 and Lmp-7, IFN-alpha 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-gamma treatment (Fig. 4). In contrast, basal Tap-2 mRNA expression by beta TC6-F7 cells was not altered by IFN-alpha treatment nor did IFN-alpha induce Tap-1 mRNA expression by these beta -cells (Fig. 4).

Cell surface expression of MHC class IA molecules in response to IFN-alpha or IFN-gamma . Cell surface expression of MHC class IA molecules on P388D1 macrophages, beta TC3, and beta TC6-F7 cells in response to IFN-alpha or IFN-gamma was compared. Basal cell surface expression of MHC class IA was observed for P388D1 macrophages and for both beta -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 beta -cell lines (Fig. 5, A-C). Treatment with IFN-gamma (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 beta -cell lines compared with untreated control cultures (Fig. 5, G-I). After IFN-gamma treatment, a similar level of fluorescence intensity for surface MHC class IA staining was observed for P388D1 macrophages and the two beta -cell lines (Fig. 5, G-I).


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 5.   Comparison of effects of IFN-alpha or IFN-gamma on cell surface major histocompatibility complex (MHC) class IA expression on beta TC3 (B, E, and H) and beta TC6-F7 (C, F, and I) pancreatic beta -cells and on P388D1 macrophages (A, D, and G). Cells were cultured in DMEM without (control; A-C) or with 100 U/ml IFN-alpha (D-F) or 50 U/ml IFN-gamma (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.

The mean fluorescence intensity of MHC class IA expression on the cell surface of P388D1 macrophages was approximately doubled in response to IFN-alpha 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-alpha or IFN-gamma (Fig. 5, D and G). In contrast, basal MHC class IA expression on the surface of beta TC3 and beta TC6-F7 cells was not altered significantly by 3 days of IFN-alpha treatment (Fig. 5, E and F).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

This investigation demonstrates that IFN-gamma but not IFN-alpha directly diminishes insulin production and induces expression of the MHC class IA antigen presentation pathway in pancreatic beta -cells. The demonstration of comparable IFN-gamma and IFN-alpha 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-alpha on beta -cell phenotype and function. Rhodes and Taylor (33) demonstrated that treatment of isolated human islets with high doses of IFN-alpha (1,000 U/ml) inhibits the synthesis of proinsulin. However, those authors indicate that the high dose of IFN-alpha 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-gamma decrease insulin production by pancreatic beta -cell lines without altering total protein biosynthesis or compromising cell viability. The more differentiated beta TC6-F7 cells are more susceptible than beta TC3 cells to the inhibitory effects of IFN-gamma , indicating that beta -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-gamma compromises beta -cell insulin production. To date, those mechanisms remain undefined. Available evidence indicates that IFN-gamma inhibits processes that occur after preproinsulin gene transcription but before insulin secretory granule exocytosis (Ref. 2; unpublished observations).

Consistent with previous observations, IFN-gamma treatment of beta -cells upregulated expression of the MHC class IA pathway (2, 13). In contrast, IFN-alpha treatment did not affect the basal level of cell surface class I MHC expression or mRNA expression of the H-2K alpha -chain, beta 2m, or the endoplasmic reticulum peptide transporter genes (Tap-1 and Tap-2). That IFN-alpha increased Lmp-2 and Lmp-7 mRNA expression by beta TC6-F7 cells and markedly upregulated MHC class IA expression by P388D1 macrophages indicates that an effective dose of a biologically active IFN-alpha was used in the present study. The differential response of the beta -cell lines to IFN-gamma vs. IFN-alpha 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-alpha or IFN-gamma response pathways in beta -cells.

Treatment of "purified" islet cell preparations from human islets (30) with either IFN-gamma or IFN-alpha increased cell surface MHC class IA expression (30), with IFN-gamma having a greater stimulatory effect than IFN-alpha . The IFN-alpha reagent used in those studies was derived from medium conditioned by Namalwa cells, and quantitative data is not provided for IFN-alpha stimulation of MHC class I expression, making it difficult to relate those results to the current study, which evaluated responses specifically in beta -cells and used lower doses of a recombinant IFN-alpha product. In a more recent study with isolated human islets, both IFN-gamma and IFN-alpha 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-gamma was shown to be a more potent stimulator of both TAP-1 and HLA class I expression than IFN-alpha when individual effects of those cytokines were compared, even with the use of a relatively large dose (500 U/ml) of a recombinant IFN-alpha product. Nonetheless, apparently on the basis of those results and the clear evidence for IFN-alpha expression in pancreases from newly diagnosed patients with type 1 diabetes (12, 17, 37), those authors predict that IFN-alpha is "most probably" the important cytokine among those capable of inducing cell surface HLA class I expression on beta -cells (41). The present results bring into question that postulate, although differences in cytokine responsiveness between mouse and human islet cells may well exist that would invalidate a direct comparison between species.

Despite clear evidence for IFN-alpha expression in islets from newly diagnosed patients with type 1 diabetes (12, 17, 37), the cellular origin of IFN-alpha within islets has not been conclusively defined. Foulis et al. (12) localized IFN-alpha in insulin-containing cells via immunocytochemistry; however, that observation does not prove beta -cell expression. Accordingly, our study provides the additional contribution of demonstrating constitutive Ifn-alpha 6 mRNA expression by a differentiated beta -cell line. Neither IFN-alpha nor IFN-gamma modulated basal Ifn-alpha 6 mRNA expression in either beta TC6-F7 cells or P388D1 macrophages. Further studies are required to determine the potential of exogenous cytokines to modulate IFN-alpha secretion from beta -cells.

Although it is demonstrated that IFN-alpha does not directly induce major phenotypic or functional changes in beta -cells, those results do not necessarily argue against an important role for this cytokine in insulitis. Indeed, the demonstration of IFN-alpha expression by beta -cells enables possible clarification of the contributions of both IFN-alpha and IFN-gamma during prediabetes. From clear evidence for IFN-alpha expression in islets of patients with type 1 diabetes, it has been suggested that local expression of IFN-alpha 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 beta -cell IFN-alpha expression. In support of its role as an initiating agent, IFN-alpha 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-alpha facilitates T cell stimulation by the induction of the costimulatory molecules ICAM-1 and B7.2 on antigen-presenting cells in islets (5). IFN-alpha 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-alpha expression by beta -cells may be a critical event in the initiation of autoimmune diabetes (5, 16, 17).

The observation that IFN-gamma , but not IFN-alpha , directly affects the phenotype and function of pancreatic beta -cells agrees with the notion that IFN-gamma plays a direct pathogenic role in autoimmune diabetes (23, 40, 42). We suggest that, in susceptible individuals, early expression of IFN-alpha by the beta -cell may contribute to insulitis, whereas IFN-gamma , a product of islet-infiltrating leukocytes, may mediate characteristic decreases in glucose sensitivity and increased cell surface expression of MHC class IA in the prediabetic beta -cell, thereby magnifying the insulitic process.

    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases grant DK-49192 (to H. R. Gaskins). Mouse recombinant IFN-gamma was provided by Genentech (South San Francisco, CA), and human recombinant IFN-alpha -A/D was provided by Hoffmann-La Roche (Nutley, NJ).

    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests: H. R. Gaskins, University of Illinois at Urbana-Champaign, 1207 W. Gregory Dr., Urbana, IL 61801.

Received 21 January 1998; accepted in final form 23 March 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Bach, J. F. Insulin-dependent diabetes mellitus as an autoimmune disease. Endocr. Rev. 15: 516-542, 1994[Abstract].

2.   Baldeón, M. E., D. J. Neece, D. Nandi, J. J. Monaco, and H. R. Gaskins. Interferon-gamma independently activates the MHC class I antigen processing pathway and diminishes glucose responsiveness in pancreatic beta -cell lines. Diabetes 46: 770-778, 1997[Abstract].

3.   Campbell, I. L., T. W. H. Kay, L. Oxbrow, and L. C. Harrison. Essential role for interferon-gamma and interleukin-6 in autoimmune insulin-dependent diabetes in NOD/Wehi mice. J. Clin. Invest. 87: 739-742, 1991[Medline].

4.   Castano, L., and G. S. Eisenbarth. Type-I diabetes: a chronic autoimmune disease of human, mouse and rat. Annu. Rev. Immunol. 8: 647-679, 1990[Medline].

5.   Chakrabarti, D., X. Huang, J. Beck, J. Henrich, N. McFarland, R. F. L. James, and T. A. Stewart. Control of islet intercellular adhesion molecule-1 expression by interferon-alpha and hypoxia. Diabetes 45: 1336-1343, 1996[Abstract].

6.   Chakrabarti, D., B. Hultgren, and T. A. Stewart. IFN-alpha induces autoimmune T cells through the induction of intracellular adhesion molecule-1 and B7.2. J. Immunol. 157: 522-528, 1996[Abstract].

7.   Chomczynski, P., and N. Sacchi. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159, 1987[Medline].

8.   Darnell, J. E., I. M. Kerr, and G. R. Stark. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 264: 1415-1421, 1994[Medline].

9.   Debray-Sachs, M., C. Carnaud, C. Boitard, H. Cohen, I. Gresser, P. Bedossa, and J.-F. Bach. Prevention of diabetes in NOD mice treated with antibody to murine IFN-gamma . J. Autoimmun. 4: 237-248, 1991[Medline].

10.   Efrat, S., S. Linde, H. Kofod, D. Spector, M. Delannoy, S. Grant, D. Hanahan, and S. Baekkeskov. Beta cell lines derived from transgenic mice expressing a hybrid insulin gene-oncogene. Proc. Natl. Acad. Sci. USA 85: 9037-9041, 1988[Abstract].

11.   Farrar, M. A., and R. D. Schreiber. The molecular cell biology of interferon-gamma and its receptor. Annu. Rev. Immunol. 11: 571-611, 1993[Medline].

12.   Foulis, A. K., M. A. Farquharson, and A. Meager. Immunoreactive alpha -interferon in insulin-secreting beta  cells in type 1 diabetes mellitus. Lancet 2: 1423-1427, 1987[Medline].

13.   Gaskins, H. R., J. J. Monaco, and E. H. Leiter. Expression of intra-MHC transporter (Ham) genes in diabetes-susceptible NOD mice. Science 256: 1826-1828, 1992[Medline].

14.   Hanahan, D. Heritable formation of pancreatic beta  cell tumors in transgenic mice expressing recombinant insulin/simian virus 40 oncogenes. Nature 315: 115-122, 1985[Medline].

15.   Hiscott, J., K. Cantell, and C. Weissmann. Differential expression of human interferon genes. Nucleic Acids Res. 12: 3727-3746, 1984[Abstract].

16.   Huang, X., H. Hultgren, and T. A. Stewart. Islet expression of interferon-alpha precedes diabetes in both the BB rat and streptozotocin-treated mice. Immunity 1: 469-478, 1994[Medline].

17.   Huang, X., J. Yuan, A. Goddard, A. Foulis, R. F. L. James, A. Lernmark, R. Pujol-Borrel, A. Rabinovitch, N. Somoza, and T. A. Stewart. Interferon expression in the pancreases of patients with type I diabetes. Diabetes 44: 658-664, 1995[Abstract].

18.   Kasaian, M. T., and C. A. Biron. Cyclosporin A inhibition of interleukin 2 gene expression, but not natural killer cell proliferation, after interferon induction in vivo. J. Exp. Med. 171: 745-762, 1990[Abstract].

19.   Kelley, K. A., and P. M. Pitha. Characterization of a mouse interferon gene locus. I. Isolation of a cluster of four alpha  interferon genes. Nucleic Acids Res. 13: 805-823, 1985[Abstract].

20.   Knaack, D., M. D. Fiore, M. Surana, M. Leiser, M. Laurence, D. Fusco-DeMane, O. D. Hegre, N. Fleischer, and S. Efrat. Clonal insulinoma cell line that stably maintains correct glucose responsiveness. Diabetes 43: 1413-1417, 1994[Abstract].

21.   Labarca, C., and K. Paigen. A simple, rapid, and sensitive DNA assay procedure. Anal. Biochem. 102: 344-352, 1980[Medline].

22.   Morgan, C., and A. Lazarow. Immunoassay of insulin: two antibody system. Plasma insulin levels in normal, subdiabetic and diabetic rats. Diabetes 12: 115-122, 1963.

23.   Muir, A., A. Peck, M. Clare-Salzier, Y. H. Song, J. Cornelius, R. Luchetta, J. Krischer, and N. Maclaren. Insulin immunization of nonobese diabetic mice induces a protective insulitis characterized by diminished intraislet interferon-gamma transcription. J. Clin. Invest. 95: 628-634, 1995[Medline].

24.   Nicoletti, F., P. Meroni, S. Landolfo, M. Gariglio, S. Guzzardi, W. Barcellini, M. Lunetta, L. Mughini, and C. Zanussi. Prevention of diabetes in BB/Wor rats treated with monoclonal antibodies to interferon-gamma (Abstract). Lancet 336: 319, 1990[Medline].

25.   Pace, J. L., S. W. Russell, P. A. LeBlanc, and D. M. Murasco. Comparative effects of various classes of mouse interferons on macrophage activation for tumor cell killing. J. Immunol. 134: 977-981, 1985[Abstract/Free Full Text].

26.   Parronchi, P., M. de Carli, R. Manetti, C. Simonelli, S. Sampognaro, M.-P. Piccinni, D. Macchia, E. Maggi, G. del Prete, and S. Romagnani. IL-4 and IFN (alpha  and gamma ) exert opposite regulatory effects on the development of cytolytic potential by Th1 or Th2 human T cell clones. J. Immunol. 149: 2977-2983, 1992[Abstract/Free Full Text].

27.  Pestka, S. The human interferon alpha species and hybrid proteins. Semin. Oncol. 24, Suppl. 9: S4-S17, 1997.

28.  Pestka, S. The interferon receptors. Semin. Oncol. 24, Suppl. 9: S18-S40, 1997.

29.   Pestka, S., J. A. Langer, K. C. Zoon, and C. E. Samuel. Interferons and their actions. Annu. Rev. Biochem. 56: 727-777, 1987[Medline].

30.   Pujol-Borrel, R., I. Todd, M. Doshi, D. Gray, M. Feldmann, and G. F. Bottazzo. Differential expression and regulation of MHC products in the endocrine and exocrine cells of the human pancreas. Clin. Exp. Immunol. 65: 128-139, 1986[Medline].

31.   Rabinovitch, A., W. Suarez-Pinzon, A. El-Sheikh, O. Sorenson, and R. F. Power. Cytokine gene expression in pancreatic islet-infiltrating leukocytes of BB rats: expression of Th1 cytokines correlates with beta -cell destructive insulitis and IDDM. Diabetes 45: 749-754, 1996[Abstract].

32.   Rehberg, E., B. Kelder, E. G. Hoal, and S. Pestka. Specific molecular activities of recombinant and hybrid leukocyte interferons. J. Biol. Chem. 257: 11497-11502, 1982[Abstract/Free Full Text].

33.   Rhodes, C. J., and K. W. Taylor. Effect of human lymphoblastoid interferon on insulin synthesis and secretion in isolated human pancreatic islets. Diabetologia 27: 601-603, 1984[Medline].

34.   Sarvetnick, N., D. Liggitt, S. L. Pitts, S. E. Hansen, and T. A. Stewart. Insulin-dependent diabetes mellitus induced in transgenic mice by ectopic expression of class II MHC and interferon-gamma. Cell 52: 773-778, 1988[Medline].

35.   Sarvetnick, N., J. Shizuru, D. Liggitt, L. Martin, B. McIntyre, A. Gregory, T. Parslow, and T. A. Stewart. Loss of pancreatic islet tolerance induced by beta -cell expression of IFN-gamma . Nature 346: 844-847, 1990[Medline].

36.   Shehadeh, N. N., and K. J. Lafferty. The role of T-cells in the development of autoimmune diabetes. Diabetes Rev. 1: 141-151, 1993.

37.   Somoza, N., F. Vargas, C. Roura-Mir, M. Vives-Pi, M. T. Fernández-Figueras, A. Ariza, R. Gomis, R. Bragado, M. Martí, D. Jaraquemada, and R. Pujol-Borrel. Pancreas in recent onset insulin-dependent diabetes mellitus changes in HLA, adhesion molecules and autoantigens, restricted T cell receptor Vbeta usage, and cytokine profile. J. Immunol. 153: 1360-1377, 1994[Abstract/Free Full Text].

38.   Springer, T., G. Galfre, D. Secher, and C. Milstein. Monoclonal xenogenic antibodies to mouse leukocyte antigens: identification of macrophage-specific and other differentiation antigens. Curr. Top. Microbiol. Immunol. 81: 45-50, 1978[Medline].

39.   Stewart, T. A., B. Hultgren, X. Huang, S. Pitts-Meek, J. Hully, and N. J. MacLachlan. Induction of type I diabetes by interferon-alpha in transgenic mice. Science 260: 1942-1946, 1993[Medline].

40.   Suarez-Pinzon, W., R. V. Rajotte, T. R. Mosmann, and A. Ravinovitch. Both CD4+ and CD8+ T cells in syngeneic islets grafts in NOD mice produce interferon-gamma during beta -cell destruction. Diabetes 45: 1350-1357, 1996[Abstract].

41.   Vives-Pi, M., M. P. Armengol, L. Alcalde, M. Costa, N. Somoza, F. Vargas, D. Jaraquemada, and R. Pujol-Borrel. Expression of transporter associated with antigen processing-1 in the endocrine cells of human pancreatic islets: effects of cytokines and evidence of hyperexpression in IDDM. Diabetes 45: 779-788, 1996[Abstract].

42.   Von Herrath, M. G., and M. B. A. Oldstone. Interferon-gamma is essential for destruction of beta  cells and development of insulin-dependent diabetes mellitus. J. Exp. Med. 185: 531-539, 1997[Abstract/Free Full Text].

43.   Yoshida, R., H. W. Murray, and C. F. Nathan. Agonist and antagonist effects of interferon alpha and beta on activation of human macrophages: two classes of interferon gamma receptors and blockade of the high-affinity sites by interferon alpha or beta. J. Exp. Med. 167: 1171-1185, 1988[Abstract].

44.   Zwarthoff, E. C., A. T. A. Mooren, and J. Trapman. Organization, structure and expression of murine interferon alpha genes. Nucleic Acids Res. 13: 791-804, 1985[Abstract].


Am J Physiol Cell Physiol 275(1):C25-C32
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society