Download Interferon- and interferon- differentially affect pancreatic

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
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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.
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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-
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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
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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-
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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
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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
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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).
We suggest that, in susceptible individuals, early expression of IFN-a by the b-cell may contribute to insulitis,
whereas IFN-g, 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 b-cell, thereby magnifying the insulitic process.
Received 21 January 1998; accepted in final form 23 March 1998.
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This work was supported by National Institute of Diabetes and
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Mouse recombinant IFN-g was provided by Genentech (South San
Francisco, CA), and human recombinant IFN-a-A/D was provided by
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