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/TNF-
Synergism as the Final Effector in Autoimmune Diabetes: A Key Role for STAT1/IFN Regulatory Factor-1 Pathway in Pancreatic 
Cell Death1
*
Clinical Research Center, Samsung Biomedical Research Institute, and
Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea;
Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan;
Department of Medicine, Chungnam University, Taejun, Korea; and
¶ Graduate School of East-West Medical Science, Kyunghee University, Seoul, Korea
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1, and NO have been
considered as effector molecule(s) leading to 
cell death in
autoimmune diabetes. However, the real culprit(s) in cell
destruction have long been elusive, despite intense investigation. We
and others have demonstrated that FasL is not a major effector molecule
in autoimmune diabetes, and previous inability to transfer diabetes to
Fas-deficient nonobese diabetic (NOD)-lpr mice was due
to constitutive FasL expression on lymphocytes from these mice. Here,
we identified IFN-
/TNF- synergism as the final effector molecules
in autoimmune diabetes of NOD mice. A combination of IFN- and
TNF-, but neither cytokine alone, induced classical
caspase-dependent apoptosis in insulinoma and pancreatic islet cells.
IFN- treatment conferred susceptibility to TNF--induced apoptosis
on otherwise resistant insulinoma cells by STAT1 activation followed by
IFN regulatory factor (IRF)-1 induction. IRF-1 played a central role in
IFN-/TNF--induced cytotoxicity because inhibition of IRF-1
induction by antisense oligonucleotides blocked IFN-/TNF--induced
cytotoxicity, and transfection of IRF-1 rendered insulinoma cells
susceptible to TNF--induced cytotoxicity. STAT1 and IRF-1 were
expressed in pancreatic islets of diabetic NOD mice and colocalized
with apoptotic cells. Moreover, anti-TNF- Ab inhibited the
development of diabetes after adoptive transfer. Taken together, our
results indicate that IFN-/TNF- synergism is responsible for
autoimmune diabetes in vivo as well as cell apoptosis in vitro and
suggest a novel signal transduction in IFN-/TNF- synergism that
may have relevance in other autoimmune diseases and synergistic
anti-tumor effects of the two cytokines. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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islet cells (4, 5). Previous adoptive
experiments indicated that CD4+ T lymphocytes are
the final effector cells in cell destruction, whereas
CD8+ T lymphocytes play a role in the initiation
period of islet destruction (2, 6). The role of
macrophages as effector cells has also been suggested. Besides their
role as APCs, they may also be a source of oxygen radicals or other
soluble cytotoxic mediators (7).
Although effector cells and their roles are rather clearly defined, it
is far from clear which molecule(s) are the real effector(s) in
autoimmune diabetes. Perforin, Fas ligand
(FasL),4 TNF-,
IL-1, and NO have been implicated in cell destruction. Although the
role of perforin in autoimmune diabetes has been reported, perforin
alone accounts for only part of cell death by
CD8+ T lymphocytes (8). We and
others recently reported that FasL is not a major effector molecule in
autoimmune diabetes (5, 9, 10), and previous inability to
transfer diabetes to Fas-deficient nonobese diabetic
(NOD)-lpr mice (11) was due to constitutive
FasL expression on lymphocytes from these mice (12). The
role of TNF- as an effector has been extremely ambiguous. Its
diabetogenic role was suggested in some studies (13, 14, 15, 16, 17, 18),
whereas opposite effects were reported in other studies (19, 20). Neonatal islet-specific expression of TNF- promoted
diabetes by enhancing the presentation of islet Ags (16, 17). The period between 21 and 25 days following the initiation
of TNF- expression was characterized as the time point when the
decision to progress from nondestructive to destructive insulitis is
made, underscoring the temporal importance of TNF- expression in the
development of diabetes (18). Also, treatment of newborn
NOD mice with TNF- led to an earlier onset of diabetes, and the
administration of an anti-TNF- Ab before 3 wk of age resulted in
the complete prevention of diabetes (13). In contrast,
other transgenic mouse studies failed to demonstrate the role of
TNF- as an effector molecule (21, 22). In these
reports, transgenic expression of TNF- in pancreatic islets led to
insulitis, but not diabetes, or even prevented diabetes (21, 22). Thus, it appears that TNF- either inhibits or promotes
diabetes depending on the condition under which it acts. The effect of
IL-1 or NO on islet cell death was studied mostly in vitro (23, 24), and their in vivo effects were not demonstrated (14, 19). IFN- has been regarded as a sensitizing or
immunomodulatory cytokine rather than an effector molecule
(25).
In our effort to identify the final effector molecule(s) in autoimmune
diabetes, we have found that IFN- and TNF- synergistically induce
cell apoptosis, and we demonstrated a critical role for STAT1/IFN
regulatory factor (IRF)-1 in the signal transduction of the cytokine
synergism, which was also found to be pertinent to the in vivo cell
death and diabetes in NOD mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MIN6N8 cells, SV40 T-transformed insulinoma cells derived from
NOD mice (kindly provided by J.-i. Miyagaki, Osaka University, Osaka,
Japan) (26), were grown in DMEM containing 15% FBS, 2 mM
glutamine, and penicillin-streptomycin (Life Technologies,
Gaithersburg, MD). Recombinant rat IFN- was generously provided by
P. H. van der Meide (Utrecht University, Utrecht, The Netherlands).
Recombinant mouse TNF- and recombinant human IL-1 were
purchased from R&D Systems (Minneapolis, MN). Recombinant human IL-1
has been shown to be active in rodent systems (27).
Agonistic anti-TNFRI Ab and antagonistic anti-TNFRII Ab were
obtained from R&D Systems. Caspase inhibitors (z-VAD-fmk, Ac-YVAD-cmk,
Ac-YVAD-fmk, Ac-YVAD-CHO, Ac-DEVD-fmk, and Ac-IETD-fmk) were purchased
from Enzyme Systems Products (Livermore, CA).
N-monomethyl-L-arginine (NMMA) was
obtained from Calbiochem (San Diego, CA). All other chemicals were
obtained from Sigma (St. Louis, MO) unless stated otherwise.
Isolation of mouse pancreatic islets
Islets were isolated from overnight-fasted ICR or NOD mice using the collagenase digestion technique. In brief, after 2.5 ml of collagenase P (0.8 mg/ml) was injected into the bile duct of an anesthetized mouse, the swollen pancreas was gently pulled out and other attached tissues were removed. The pancreas was then incubated with collagenase at 37°C for 15 min with gentle shaking. After incubation, cold HBSS was added to stop the digestion. The tissue was then passed through a 400-µm screen and centrifuged on 25, 23, 21.5, and 11.5% Ficoll gradients. Islets were collected from the interface. Collected islets were washed with M199 medium, and individual islets were hand-picked using micropipettes. They were treated with trypsin-EDTA for 5 min to yield single-islet cells.
Assessment of cytotoxicity by MTT assays
Cells (3 x 104/well for MIN6N8 cells
and 2 x 104/well for mouse single-islet
cells) were seeded in 96-well microtiter plates and treated with
various combinations of cytokines for the indicated time periods. The
optimal concentrations of cytokines for the cytotoxic action were 1000
U/ml for IFN-, 10 ng/ml for TNF-, and 17.5 ng/ml for IL-1. In
some experiments, cells were pretreated with caspase inhibitors or NMMA
for 1 h before cytokine treatment. After cytokine treatment, the
medium was removed and MTT (0.5 mg/ml) was added and followed by
incubation at 37°C for 2 h in a CO2
incubator. After a brief centrifugation, supernatants were carefully
removed and DMSO was added. After insoluble crystals were completely
dissolved, absorbance at 540 nm was measured using a Thermomax
microplate reader (Molecular Devices, Menlo Park, CA).
Morphological analysis of apoptotic cells
Morphological changes in the nuclear chromatin of cells undergoing apoptosis were detected by staining with 2.5 µg/ml DNA-binding bis-benzimide Hoechst 33258 fluorochrome (Calbiochem), followed by an examination on a fluorescence microscope. In some experiments, islet cells were stained with anti-insulin Ab (Dako, Carpinteria, CA) and then with FITC-conjugated anti-guinea pig IgG Ab (Zymed, San Francisco, CA), followed by Hoechst 33258 staining. For transmission electron microscopy, cells were fixed in 4% glutaraldehyde, 1% paraformaldehyde, and 0.2 M phosphate (pH 7.2) at 4°C for 2 h. After two washes in 0.2 M phosphate, the cell pellets were postfixed with 2% OsO4 in the same buffer for 30 min. The pellets were dehydrated in ethanol and then in 100% propylene oxide, followed by embedding at 37°C overnight and 60°C for another 3 days. Ultrafine sections were cut and examined on an electron microscope (Hitachi H7100, 75 kV; Hitachi, Tokyo, Japan).
Agarose gel electrophoresis of DNA
For the isolation of genomic DNA, MIN6N8 cells were lysed by incubation in the extraction buffer overnight at 55°C (10 mM Tris-HCl (pH 8.0), 0.1 M EDTA, 0.5% SDS, and 100 mM NaCl), followed by phenol/chloroform extraction and ethanol precipitation. The final pellet was dissolved in distilled water containing 0.1 mg/ml RNase A. Isolated genomic DNA was electrophoresed on 1.5% agarose gel and stained with ethidium bromide to detect internucleosomal cleavage.
DNA ploidy analysis
Cells were suspended in PBS-5 mM EDTA and fixed by adding 100% ethanol dropwise. RNase A (40 µg/ml) was added to the resuspended cells, and incubation was conducted at room temperature for 30 min. Propidium iodide (50 µg/ml) was then added for flow cytometric analyses (FACSVantage; Becton Dickinson, Mountain View, CA).
RT-PCR and RNase protection assays
Total RNA was extracted from MIN6N8 cells by a sequential
addition of 4 M guanidinium thiocyanate, 2 M sodium acetate, and acid
phenol/chloroform. Reverse transcription was conducted using
Superscript (Life Technologies) and oligo(dT) primer. PCR amplification
using primer sets specific for each caspase was conducted at 60°C
annealing temperature for 30 cycles. Nucleotide sequences of the
primers were based on published cDNA sequences (caspase-1 forward, AAG
ATG GCA CAT TTC CAG GAC; caspase-1 reverse, GGG CAC TTC AAA GTG TTC
ATC; caspase-2 forward, ATG CTA ACT GTC CAA GTC TA; caspase-2 reverse,
GTC TCA TCT TCA TCA ACT CC; caspase-3 forward, CTA AGC CAT GGT GAT GAA
GGG; caspase-3 reverse, CTG CAA AGG GAC TGG ATG AAC; caspase-7 forward,
GAA GTA ACC GTC CAC AAT GAC; caspase-7 reverse, TGC CAT GCT CAT TCA GGA
TGG; caspase-8 forward, GCT CTT CTA CCT CTT GAT AAG; caspase-8 reverse,
GAC CCT GTA GGG AGA AAT CTG; caspase-9 forward, AGC TCT TCT TCA TCC
AGG; caspase-9 reverse, CCC CCA GCC TCA TGA AGT T; caspase-11 forward,
CTT CAC AGT GCG AAA GAA CT; caspase-11 reverse, GGT CCA CAC TGA AGA ATG
TCT GGA GAA GCA TTT CA). Nucleotide sequences of forward and reverse
primers for mouse -actin were ATC CTG AAA GAC CTC TAT GC and AAC GCA
GCT CAG TAA CAG TC, respectively. Isolated total RNA was also subjected
to RNase protection assay, which was conducted using the HybSpeed RPA
kit (Ambion, Austin, TX) and RiboQuant Multi-Probe Template set (BD
PharMingen, San Diego, CA) according to the manufacturer’s protocols,
except that hybridization of the probe with sample RNA was done for
2 h.
Western blot analyses
Cells were lysed in triple-detergent lysis buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.02% sodium azide, 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 1 mM PMSF). Protein concentration in cell lysates was determined using the Bio-Rad protein assay kit (Bio-Rad, Richmond, CA). An equal amount of protein for each sample was separated by 10 or 12% SDS-PAGE and transferred to Hybond ECL nitrocellulose membrane (Amersham Pharmacia Biotech, Piscataway, NJ). After blocking with 5% skim milk, the membranes were sequentially incubated with one of the primary Abs (rabbit anti-mouse IRF-1, obtained from Santa Cruz Biotechnology (Santa Cruz, CA); rabbit anti-STAT1 and anti-phospho-STAT1, obtained from New England Biolabs (Beverly, MA); and rabbit anti-mouse caspase-1, which was generously provided by P. Vandenabeele, (University of Ghent, Ghent, Belgium) and then HRP-conjugated secondary Abs (anti-rabbit IgG; Amersham Pharmacia Biotech), followed by ECL detection (Amersham Pharmacia Biotech).
Assessment of caspase activity
Caspase-3 or -8-like activity was measured using a commercial caspase assay kit (BD PharMingen) according to the supplier’s instruction. In brief, caspase-3 or -8 fluorogenic substrates (Ac-DEVD-AMC or Ac-IETD-AMC) were incubated with cytokine-treated cell lysate for 1 h at 37°C. AMC liberated from Ac-DEVD-AMC or Ac-IETD-AMC was measured using a fluorometric plate reader with an excitation wavelength of 380 nm and an emission wavelength of 420–460 nm.
Transient transfection of MIN6N8 cells
MIN6N8 cells in 6-well plates were cotransfected with 1 µg
human STAT1 cDNA, dominant negative STAT1 cDNA (provided by
T. Hirano, Osaka University) or mouse IRF-1 cDNA
(provided by T. Taniguchi, University of Tokyo, Tokyo, Japan) together
with 0.2 µg lacZ (pCH110; Amersham Pharmacia Biotech)
using lipofectamine reagent (Life Technologies), according to the
supplier’s instruction. At 48 h after transfection, cells were
treated with cytokines. After another 72 h, cells were washed with
PBS and fixed with 0.5% glutaraldehyde for 10 min at room temperature
and stained with 5-bromo-4-chloro-3-indolyl
-D-galactoside (1 mg/ml) in 4 mM potassium
ferricyanide, 4 mM potassium ferrocyanide, and 2 mM magnesium chloride
at 37°C for blue cell counting. Viable blue cells in 10 random high
power (x200) fields were counted for each sample, excluding dark blue
apoptotic cells. For the transfection of IRF-1 antisense
oligonucleotides, antisense and sense oligonucleotides with
phosphothioated-terminal 3 bases were designed. A pair of sense and
antisense oligonucleotides (S and AS1) were based on the previously
published work (S, GCA TCT CGG GCA TCT TTC; AS1, GAA AGA TGC CCG AGA
TGC) (28). An additional antisense oligonucleotide
(AS2) was designed to encompass the translational start codon of IRF-1
(AS2, AGT GAT TGG CAT GGT GGC; bold-typed sequences indicate
antisense start codon). The cells were similarly transfected with
either sense or antisense oligonucleotides in the presence of
lipofectAMINE reagent. At 24 h after the transfection, the cells
were exposed to the cytokines for 48 h, then viability was
assessed by MTT assays. For IRF-1 immunoblotting, transfected cells
were treated with IFN- for 2 h before the cells were lysed for
SDS-PAGE.
Immunohistochemistry and TUNEL staining
Formalin-fixed sections of the mouse islet tissues were deparaffinized and briefly microwaved in 0.01 M sodium citrate buffer (pH 6.0). They were then incubated with the appropriate dilution of anti-STAT1 (New England Biolabs), anti-phospho-STAT1 (New England Biolabs), or anti-IRF-1 Ab (Santa Cruz Biotechnology) after goat serum blocking. Incubation with biotinylated anti-rabbit IgG Ab and then with avidin-biotin-peroxidase complex (Vector Laboratories, Burlingame, CA) followed. Diaminobenzidine was used as a color substrate. In some experiments, islet sections were double-stained for IRF-1 expression and apoptosis. For the double staining, tissue sections were first stained using TUNEL reagents (Roche Biomedical Laboratories, Burlington, NC) and diaminobenzidine. Immunostaining using primary Ab, biotinylated anti-rabbit IgG Ab, streptavidin-alkaline phosphatase, and then chromogenic substrate solutions containing Naphtol AS-MX salt (Sigma), Fast Blue (Sigma), and levamisole in TBS buffer followed.
Adoptive transfer of diabetes
The spleens from diabetic NOD mice were harvested, and 2 x 107 splenocytes were infused into the tail vein of each 9-wk-old NOD mouse. Recipient mice were irradiated (775 rad) 6 h before the transfer of splenocytes. The incidence of diabetes was >80% at 4 wk after the adoptive transfer in our previous experiments (5). From 2 wk after adoptive transfer, the mice were bled retro-orbitally every week, and nonfasting blood glucose levels were determined by the glucose oxidase method. Mice were considered diabetic if their blood glucose level was >300 mg/dl. To determine the severity of insulitis, >30 pancreatic islets from more than three parallel sections of different cut levels were analyzed per mouse unless the mice were devoid of pancreatic islets due to diabetes. The degree of insulitis was classified into four categories: 0, no insulitis; 1, peri-insulitis with minimal lymphocytic infiltration in islets; 2, invasive insulitis with islet destruction <50%; and 3, invasive isulitis with islet destruction >50%. Animal care was in accordance with institutional guidelines.
Anti-TNF- Ab treatment
A hybridoma producing anti-TNF- Ab, MP6-XT22.11, was
injected i.p. in nude mice pretreated with pristane. Ab was purified
using a protein G-agarose (Amersham Pharmacia Biotech) column. Bound
IgG was eluted with 50 mM glycine-HCl (pH 2.5). Collected fractions
were dialyzed against PBS and then sterilized by filtration. Ab (0.5
mg) was injected i.p. three times a week into NOD mice before and after
adoptive transfer. Control NOD mice were treated with PBS or control
IgG in the same manner.
Statistical analysis
The incidence of diabetes with or without anti-TNF- Ab
treatment was plotted according to the Kaplan-Meier method. The
incidences between the two groups were compared using the logrank test.
Other statistical analyses were performed using the Student’s
t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cell death by IFN- and TNF-
TNF-, IFN-, and IL-1 are the major proinflammatory
cytokines that have been implicated in the islet cell destruction in
vitro. To determine how these proinflammatory cytokines participate in
the cell destruction in vitro, we first tested the cytotoxic
effects of various combinations of these cytokines using MIN6N8
insulinoma cells. The combination of IFN- and TNF- significantly
reduced the viability of MIN6N8 cells as judged by the morphological
changes on light microscopy (data not shown) and MTT assays (Fig. 1
A). The addition of IL-1
to IFN-/TNF- combination or IL-1 alone had only a marginal
effect. The effect of IFN- and TNF- was synergistic, because
neither of the single cytokines alone had a significant cytotoxic
effect on insulinoma cells. NO production measured by Griess reaction
was negligible from the insulinoma cells treated with an
IFN-/TNF- combination (data not shown). NMMA, an inhibitor of
inducible NO synthase (iNOS), did not affect IFN-/TNF--induced
insulinoma cell death, indicating that NO had no significant effects on
the insulinoma/islet cell viability in this condition (data not shown).
TNF- signaling in IFN-/TNF- synergism was mediated through p55
TNFR (TNFRI) because agonistic Ab against TNFRI together with IFN-
induced apoptosis of cells, and antagonistic Ab against p75 TNFR
(TNFRII) did not interfere with cell apoptosis by IFN-/TNF-
synergism (data not shown).
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/TNF--induced cell death is a typical apoptosis
Next, we studied whether cell death by IFN-/TNF- synergism
was the classical apoptosis. The cytotoxic effects of the cytokines on
MIN6N8 cells were effectively blocked by z-VAD-fmk, indicating that the
cytokine combination induced cytotoxicity in a caspase-dependent manner
(Fig. 1
B). The electrophoresis of genomic DNA, nuclear
staining with Hoechst 33258, electron microscopy, and DNA ploidy
analysis all indicated that the two cytokines synergistically induced
apoptosis of MIN6N8 cells (Fig. 1, C–F). DNA ploidy assays
indicated that the effect of the IFN-/TNF- combination was not
due to the growth arrest, as was shown by the absence of a decrease in
the S phase cell percentage (Fig. 1F).
IFN- induces phosphorylation of STAT1 and the expression of
IRF-1: a key role for STAT1/IRF-1 in IFN-/TNF- synergism
To study the mechanism of IFN-/TNF- synergism on cell
apoptosis, MIN6N8 cells were sequentially treated with the two
cytokines. Upon pretreatment with IFN-, TNF- alone was sufficient
to induce cytotoxic effects comparable to those by the combination of
cytokines (Table I). However,
pretreatment with TNF- followed by IFN- treatment did not induce
significant insulinoma cell death, suggesting that IFN- pretreatment
sensitizes MIN6N8 cells to TNF--mediated cytotoxicity by inducing
IFN--responsive genes (Table I). We studied the possible involvement
of STAT protein, an important element of IFN- signal transduction,
in the induction of susceptibility to TNF-. Phosphorylated STAT1 was
detected as early as 30 min after IFN- treatment (STAT1 activation)
and rapidly disappeared (Fig. 2A,
left). Interestingly, the level of unphosphorylated form of STAT1
was also increased 24–48 h after IFN- treatment (STAT1 induction;
Fig. 2A, right). TNF- treatment alone did not activate or
induce STAT1 protein. We first thought STAT1 induction might be related
to the sensitization of islet cells to TNF--mediated apoptosis
because it has been reported that transfection of wild-type or
phosphorylation-defective STAT1 rendered STAT1-deficient U3A cells
susceptible to TNF--mediated apoptosis (29). However,
transfection of STAT1 failed to induce susceptibility to
TNF--mediated apoptosis in insulinoma cells (Fig. 2B,
left), reflecting the differences in signal transduction between
the two cell types. In contrast, IFN-/TNF--mediated insulinoma
cell death was inhibited by transfection with dominant-negative
phosphorylation-defective STAT1 (Fig. 2B, right),
suggesting that STAT1 activation, rather than STAT1 induction, plays a
critical role in inducing susceptibility to TNF--mediated apoptosis.
We further traced the signal mediators downstream of STAT1 activation.
Among the genes acting downstream of STAT1 activation, we found that
IRF-1 was induced at 24–48 h after IFN- treatment but not by
TNF- (Fig. 3A). The
inhibition of IFN--mediated IRF-1 induction by antisense
oligonucleotides abolished the cytotoxic effects of IFN-/TNF-,
strongly suggesting that IRF-1 is the key player in IFN-/TNF-
synergism (Fig. 3B). Decreased expression of IRF-1 protein
in antisense oligonucleotide-transfected cells was confirmed by Western
blot analyses (Fig. 3C). Furthermore, IRF-1
transfection alone rendered insulinoma cells susceptible to TNF-
(Fig. 3D), suggesting that IRF-1 was both necessary and
sufficient for the induction of TNF- sensitivity in insulinoma
cells.
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Because those papers linking IRF-1 to apoptosis demonstrated the
induction of caspases by IRF-1 (30, 31), we next studied
the possibility of caspase induction as a downstream event following
IRF-1 induction. When the expression of various caspases was assessed
by RT-PCR, the expression of caspase-1 and -11 was induced by IFN-,
whereas constitutive expression of other caspases (-2, -3, -7, -8, and
-9) was not affected by the cytokine treatment (Fig. 4A). TNF- alone appeared to
have induced the expression of caspase-11, but not caspase-1. Western
blot analysis also showed that caspase-1 was induced at the protein
level by IFN- but not by TNF- (Fig. 4C). Although
caspase-1 has been regarded as a proinflammatory caspase, its
involvement in apoptosis has also been described in a number of
instances, particularly in relation to IRF-1 (30, 32). The
involvement of caspase-1 in TNF--induced apoptosis also has been
reported (33). Caspase-11 is essential for the activation
of caspase-1 by physically interacting with procaspase-1
(34). Induction of caspase-11 by IFN- alone or
IFN-/TNF-, as observed in this study, could be essential for
apoptosis mediated by caspase-1, which itself was induced by IFN-.
Thus, IFN- may induce susceptibility to TNF- by inducing both
caspase-1 and caspase-11 that form a complex. To confirm caspase-1
involvement in the cytokine synergism, we studied the effect of
Ac-YVAD-cmk, an inhibitor of caspase-1. Only high concentrations of
Ac-YVAD-cmk (600–1000 µM) were able to block the cytokine-induced
cytotoxicity in MIN6N8 cells (Fig. 4B, left). Similar
results were obtained using another caspase-1 inhibitor, Ac-YVAD-CHO
(data not shown). Because Ac-YVAD of this high concentration may
nonspecifically inhibit other caspases, we could not at present
definitively determine whether caspase-1 is involved in cell
apoptosis by IFN-/TNF- synergism. In contrast to Ac-YVAD,
Ac-IETD-fmk and Ac-DEVD-fmk at 50 µM significantly inhibited
cytotoxic activity of IFN-/TNF- (Fig. 4B, right),
suggesting the involvement of other caspases such as caspase-8 and -3
in the cytokine-induced apoptosis. Caspase assays using fluorogenic
substrates also indicated that caspase-3 and caspase-8 were activated
after IFN-/TNF- treatment of MIN6N8 cells (Fig. 4D),
further supporting the involvement of these caspases in the apoptosis
of insulinoma cells. Besides caspases, IFN- is known to modulate the
expression of other apoptosis-related genes including TNFRI
(35). However, the expression of TNFRI, which mediated
apoptotic signaling of TNF- in MIN6N8 insulinoma cells, was not
significantly affected by IFN- as demonstrated by RNase protection
assays (data not shown). Similarly, the expression of Bcl-2
and other important apoptosis-related genes such as TRADD, RIP,
FADD, FAF, and Fas was not significantly influenced by
IFN- treatment of insulinoma cells as demonstrated by Western blot
analyses or RNase protection assays (data not shown).
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in mouse primary
islets
We next studied whether this scheme of cytotoxic synergism between
IFN- and TNF- found in insulinoma cells could be applicable to
primary islet cells. A similar synergistic cytotoxic effect of IFN-
and TNF- was observed in single-islet cells isolated from ICR mice
(Fig. 5A) as well as humans
(data not shown). The addition of IL-1 again had only negligible
effects on the islet cell viability. Moreover, the cytokine combination
also induced classical apoptosis on pancreatic islet cells. Typical
apoptotic morphology, such as nuclear condensation and fragmentation,
was observed in the cytokine-treated mouse islet cells as indicated by
Hoechst 33258 staining (Fig. 5B). STAT1 phosphorylation and
IRF-1 induction by IFN- were also observed in murine single-islet
cells (Fig. 5C), indicating that a similar cytokine
synergism is operative in MIN6N8 cells and primary islet cells. To
confirm the death of islet cells by IFN-/TNF-, immunostaining
of insulin was conducted simultaneously with Hoechst 33258 nuclear
staining. About 90% of islet cells were insulin-positive, indicating
that the majority of islet cells were cells. The percentage of
apoptotic cells with nuclear condensation increased from 16.6%
(incubation for 3 days without cytokines) to 62.7% upon
IFN-/TNF- treatment for 3 days, indicating that insulin-positive
cells were killed by IFN-/TNF- (>200 insulin-positive
cells were counted). We next asked whether a similar cytokine-induced
death of islet cells could be observed in studies using islet cells
isolated from insulitis-free NOD mice at 7 wk of age. IFN-/TNF-
treatment led to 49.3% viability compared with untreated control
(viability set to 100%), indicating that IFN-/TNF- combination
induced the death of islets from NOD mice as well.
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Having shown that IFN--mediated up-regulation of IRF-1 plays a
central role in cytokine-induced islet destruction in vitro, we
speculated that a similar cytokine synergism between IFN- and
TNF- may be responsible for cell death in natural diabetes of
NOD mice. IFN- secreted by islet-infiltrating T lymphocytes may
activate or induce STAT1 and/or IRF-1 in islet cells, conferring
susceptibility to apoptosis by TNF- derived mainly from macrophages.
To test our hypothesis, we evaluated by immunohistochemical staining
the expression of STAT1 and IRF-1 in pancreatic islets of NOD mice. In
pancreatic islets of nondiabetic ICR mice or young (5- to 8-wk-old)
female NOD mice, minimal or no expression of STAT1/IRF-1 was observed
(Fig. 6, C and F).
However, in adoptive transferred-NOD mice (Fig. 6, A and
D) or 20-wk-old female prediabetic NOD mice with
(peri)insulitis (Fig. 6, B and E), a strong
expression of STAT1 and IRF-1 was observed in several islet cells (Fig. 6, A, B, D, and E). Moreover, some IRF-1-positive
cells were colocalized with apoptotic nuclei in pancreatic islets of
diabetic NOD mice (Fig. 6G), further supporting the role of
IRF-1 in the cytokine-induced apoptosis of pancreatic cells. The
expression of phosphorylated STAT1 was not detected in pancreatic
islets of NOD mice regardless of age, probably because its expression
in vivo was transient as was in vitro (data not shown).
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Ab blocks diabetes development
Because the expression of STAT1 and IRF-1 in pancreatic islets of
NOD mice suggested the possibility that IFN- induces susceptibility
to TNF- on islet cells through IRF-1 induction, we investigated
whether the blockade of TNF- could abrogate diabetes development
after adoptive transfer of lymphocytes from diabetic NOD mice. As we
hypothesized, administration of anti-TNF- Ab during adoptive
transfer blocked the development of diabetes (Fig. 7) (p < 0.05),
supporting the role of TNF- as the final effector molecule in the
cell death and diabetes. The administration of anti-TNF- Ab
also significantly decreased the insulitis score from 2.13 ± 0.45
(n = 6) to 1.44 ± 0.22 (n = 7)
(p < 0.01).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNF- is
responsible for cell apoptosis in vitro. In previous papers that
reported the significant effects of IL-1 on the viability of islet
cells, the effects of IL-1 varied widely among different species
from which islets were isolated. Although IL-1 alone appears to be a
major effector in the destruction of rat islets, the combinations of
proinflammatory cytokines such as TNF- or IFN- were necessary for
the induction of mouse or human islet cell death (24). In
vitro effects of NO have also been reported, mostly using islet cells
from rats (23, 36). NO production by cytokine-stimulated
islet cells seems to be negligible in species other than rats. Thus,
the effects of IL-1 and NO observed in the studies using rat islets
cannot be generalized to other species. A previous paper described the
cytotoxicity of pancreatic islets by IFN- and TNF- combination;
however, the nature of cell death was not investigated
(37). Also, although a few reports suggested the
involvement of DNA strand break or DNA fragmentation in
cytokine-induced cell death (38, 39), detailed
morphological changes associated with apoptosis or caspase involvement
were not demonstrated. Moreover, in other in vitro studies in which
apoptosis of cells have been reported, apoptosis was induced by
nonphysiological agents, such as thapsigargin or serum deprivation,
that were irrelevant to natural autoimmune diabetes (40, 41). Thus, we have established a physiological in vitro cell
apoptosis model, which could be used not only for further investigation
on the mechanism of cell destruction, but also for the screening of
anti-apoptotic agents that could be used for the treatment of
autoimmune diabetes.
We also demonstrated that IFN- induced IRF-1 expression following
STAT1 activation and that the transfection of IRF-1 could be
substituted for IFN- treatment to induce apoptosis of cells.
These results suggest a collaborative signal transduction mechanism for
the cytokine synergism in the destruction of pancreatic cells.
IFN- induces susceptibility to TNF--mediated apoptosis by
sequentially activating or inducing STAT1 and IRF-1. Previous studies
have indicated the role of IRF-1 in apoptosis induced by DNA damage or
IFN- (30, 31, 42), which supported the proapoptotic
action of IRF-1. However, the role of IRF-1 in conferring
susceptibility to TNF--induced apoptosis has not been reported.
Caspase induction has been suggested as a possible downstream event
following IRF-1 induction in IFN--induced apoptosis
(31). Our results showing inhibitory effect of Ac-YVAD
only at concentrations above 500 µM suggests that caspase-1, although
induced by IFN-, might not be directly involved in
IFN-/TNF--induced cell apoptosis. Caspase-11 has been
reported to be crucial for the activation of caspase-1
(34); however, recent papers demonstrated that caspase-11
also activated caspase-3 as well (43). Furthermore,
caspase-11-deficient mice are partly resistant to the induction of
experimental allergic encephalomyelitis (44) and to the
development of stroke after middle cerebral artery occlusion
(43). Thus, IFN--induced caspase-11, as noted in our
experiment, may play a role in the activation of caspase-3 during
apoptosis of cells. Besides caspases, IRF-1 has also been shown to
induce iNOS (45) that catalyzes the production of NO.
However, our results argued against the possible involvement of NO in
IRF-1-mediated apoptosis. A significant amount of NO was not generated
by MIN6N8 cells treated with IFN-/TNF-, and iNOS inhibitor did
not affect the cytokine-induced cytotoxicity (data not shown).
In addition to our in vitro findings, we also present evidence that
IFN-/TNF- synergism involving STAT1/IRF-1 pathways plays a
critical role as the final effector in the development of diabetes in
vivo: 1) IFN- activated and induced STAT1/IRF-1 in primary islet
cells; 2) STAT1/IRF-1 was expressed in pancreatic islets of diabetic
mice and colocalized with apoptotic cells; and 3) anti-TNF- Ab
inhibited diabetes development. The role of TNF- in NOD mice has
been extensively studied. However, the in vivo role of the cytokine in
diabetes development has not yet been fully elucidated. Its
diabetogenic role was suggested in some studies (13, 14, 15, 16, 17, 18),
whereas opposite effects were reported in other studies (19, 20). Our IFN-/TNF- synergism model nicely explains why
transgenic expression of TNF- alone could not induce diabetes in
some of the previous studies in which pancreatic expression of TNF-
alone did not accelerate diabetes development (21). It is
also possible that the effects of systemic TNF- administration in
previous studies were mostly due to its immunomodulatory effects rather
than to the direct effects on target (islet) cell viability (13, 19). There is another previous report that demonstrated the
inability of anti-TNF- Ab treatment to inhibit diabetes in NOD
mice (46). However, in that report, the dose and duration
of Ab administration might not have been sufficient. In the current
study, we used anti-TNF- Ab whose neutralizing activity was
confirmed, and the dose was sufficient to block TNF- action in vivo
(47). We have also shown that the administration of
anti-TNF- Ab significantly decreased the insulitis score in
adoptive transfer experiments. Inhibition of cell apoptosis by
anti-TNF- Ab could decrease the release of islet Ags from dead
islet cells and further insulitis. In addition to its direct effect on
islet cell death, anti-TNF- Ab treatment may affect Ag
presentation or chemotaxis potentially mediated by TNF-. The
inhibition of diabetes by anti-TNF- Ab was not complete in our
current study. This result might be explained by the residual TNF-
activity, lymphotoxin, or perforin. Targeted disruption of perforin
significantly decreased diabetes incidence in NOD mice
(8); however, such results do not necessarily mean that
perforin is the major effector in cell death. Perforin is
predominantly used by CD8+ T lymphocytes that
initially infiltrate pancreatic islets, and immunostaining disclosed
that perforin was expressed on CD8+ but not on
CD4+ T lymphocytes in pancreatic islets of NOD
mice (48). Thus, the findings observed in
perforin-deficient NOD mice seem to be due to abrogation of the initial
islet cell injury by CD8+ T lymphocytes that is
critical for the sensitization of the majority of islet-specific
CD4+ T lymphocytes. Pakala et al.
(14) established a valuable model to test the effect of
single-gene disruption on the cell apoptosis and diabetes. In their
model, diabetogenic BDC2.5 T cells were transferred into
streptozotocin-treated NOD.scid/scid mice grafted with
islets deficient in the single gene of interest. They demonstrated that
p55 TNFRI is essential for cell apoptosis in vivo, consistent with
our result showing significant inhibition of diabetes transfer by
anti-TNF- Ab. However, IFN-R was not essential for the
development of diabetes in their model, in contrast to our results.
IFN that can induce STAT1 activation and IRF-1 induction might
compensate for the absence of IFN- action on those islets. It is
also possible that apoptosis by BDC2.5 T cells does not represent
naturally occurring cell apoptosis in toto by diabetogenic
lymphocytes.
According to our model, blockade of IFN- is also expected to inhibit
cell death and diabetes, although IFN- itself does not exert
apoptosis on islet cells. Ablation of the IFN- gene in NOD mice did
not completely prevent diabetes; however, the development of diabetes
was delayed (49). Anti-IFN- Ab (50) or
targeted disruption of IFN-R (51) abrogated diabetes in
NOD mice. On the contrary, it has been reported that cells are not
the direct targets of IFN- in autoimmune diabetes (52).
Thus, the role of IFN- in diabetes development is still
controversial. Our preliminary experiments showed that anti-IFN-
Ab partially inhibited adoptive transfer of diabetes (S. Kim, K. Suk
and M.-S. Lee, unpublished data). Incomplete abrogation of
diabetes in IFN--deficient NOD mice or IFN- Ab-treated mice could
be due to the overlapping effects of other cytokines, such as IFN,
which might compensate for the absence of IFN-. IRF-1, a key
mediator of islet cell apoptosis, can be also induced by type I IFN
(53). Importantly, IRF-1-deficient NOD mice did not have
insulitis or diabetes (54), consistent with the
proposed key role of IRF-1 in islet cell death. In our model, IFN-
plays a priming role for the cytotoxic action of TNF- in pancreatic
islet cells, and this priming role of IFN- is mediated by IRF-1
induction. Although previous reports showed that IFN- alone could
induce apoptosis on various types of cells, such as primary hepatocytes
(31) or HeLa, (55) through IRF-1 induction,
the cytokine seems to require another cytotoxic signaling event to kill
islet cells.
Based on the results presented here, we propose that
CD4+ T lymphocytes (as a major source of IFN-)
act in collaboration with macrophages (as a major source of TNF-) to
induce cell death through delayed-type hypersensitivity-like
reaction. This type of cooperative immune response between innate and
adaptive immune responses may be also responsible for organ-specific
autoimmune diseases other than autoimmune diabetes. Moreover,
IFN-/TNF- synergism has been reported in numerous tumor cell
death models (56). The signal transduction of
IFN-/TNF- synergism we postulated may be relevant to the
pathophysiology of other autoimmune diseases and cytokine-induced tumor
cell apoptosis.
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Footnotes |
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2 K.S. and S.K. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Myung-Shik Lee, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-dong, Kangnam-ku, Seoul, 135-710, Korea.
4 Abbreviations used in this paper: FasL, Fas ligand; NOD, nonobese diabetic; IRF, IFN regulatory factor; NMMA, N-monomethyl-L-arginine; iNOS, inducible NO synthase.
Received for publication September 5, 2000. Accepted for publication January 30, 2001.
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).
Phosphorylated STAT1 and unphosphorylated STAT1 were detected 30 min
and 24–48 h, respectively, after treatment with IFN-
, n = 9),
anti-TNF-