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Gene Expression Unit, Diabetes Research Center, Vrije Universiteit Brussel, B-1090 Brussels, Belgium
Address all correspondence and requests for reprints to: Dr. D. L. Eizirik, Gene Expression Unit, Diabetes Research Center, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium. E-mail: deizirik{at}mebo.vub.ac.be
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Abstract |
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(IFN-) have been suggested as potential mediators of
ß-cell death in early T1DM. We presently investigated whether the
viral replicative intermediate double stranded RNA [here used as
synthetic polyinosinic-polycytidylic acid (PIC)] modifies the effects
of IL-1ß and IFN- on gene expression and viability of rat
pancreatic ß-cells. For this purpose, fluorescence-activated cell
sorting-purified rat ß-cells were exposed for 6–16 h (study of gene
expression by RT-PCR) or 6–9 days (study of viability by nuclear dyes)
to PIC and/or IL-1ß and IFN-. PIC increased the expression of Fas
and Mn superoxide dismutase messenger RNAs by 5- to 10-fold. IL-1ß
and a combination of PIC and IFN- (but not PIC or IFN- alone)
induced expression of inducible nitric oxide (NO) synthase (iNOS) and
consequent NO production. Induction of iNOS expression by PIC and
IFN- requires nuclear factor-
B activation, as suggested by
transfection experiments with iNOS promoter-luciferase reporter
constructs into primary ß-cells. Combinations of IL-1ß plus
IFN-, PIC plus IFN-, or PIC plus IL-1ß induced a 2- to 3-fold
increase in the number of apoptotic ß-cells. Blocking of iNOS
activity significantly decreased PIC- plus IL-1ß-induced, but not
PIC- plus IFN--induced, apoptosis.
In conclusion, PIC alone or in combination with cytokines modifies the
expression of several genes in pancreatic ß-cells. Two of these
genes, Fas and iNOS, may contribute to ß-cell death. The
transcription factor nuclear factor-B is required for PIC-induced
iNOS expression. PIC has an additive effect on cytokine-induced
ß-cell death by both NO-dependent (in the case of IL-1ß) and
NO-independent (in the case of IFN-) mechanisms. These findings
suggest that viral intermediates in synergism with local cytokine
production may play an important role in ß-cell apoptosis in early
T1DM.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A common pathway for viral-induced cellular responses is via the
accumulation of double-stranded RNA (dsRNA) in the cytoplasm of
eukaryotic cells (6). dsRNA can trigger apoptotic cell
death (7), and several studies suggest that apoptosis is
the main form of ß-cell death in animal models of T1DM and possibly
also in human T1DM (reviewed in Refs. 8 and
9). Another potential mediator of ß-cell apoptosis is
cytokines, peptides that may accumulate at high concentrations at the
insulitis site. The cytokines interleukin-1ß (IL-1ß), tumor
necrosis factor-
, and interferon- (IFN-) have been shown to
induce rodent and human ß-cell death mostly by apoptosis (reviewed in
Refs. 8 and 10, 11, 12, 13). Furthermore, cytokines
and the inducible form of nitric oxide (NO) synthase (iNOS) are
expressed in the vicinity of ß-cells during the induction of diabetes
by the D variant of encephalomyocarditis virus in DBA/2 mice, and both
antibodies against IL-1ß and iNOS blockers prevent the outbreak of
diabetes in these mice (14). Finally, it has been shown
that dsRNA [in the form of poly(IC)] inhibits glucose-stimulated
insulin biosynthesis in mouse islets (15), and that
exposure of rat pancreatic islets to dsRNA in combination with IFN-
leads to ß-cell dysfunction and death, apparently by an NO-dependent
mechanism (16). No indication was provided in this later
study of whether the ß-cells die by necrosis or apoptosis after
exposure to dsRNA and IFN- (16). Together the
observations described above suggest that local induction of
cytokines, perhaps triggered by a viral infection, may interact with
viral products, such as dsRNA, leading to ß-cell death.
In the present study we investigated the effects of dsRNA [tested here
as synthetic polyinosinic-polycytidylic acid (PIC)] alone or in
combination with the cytokines IL-1ß and/or IFN- on gene
expression and viability of fluorescence-activated cell sorting
(FACS)-purified rat ß-cells. The data obtained suggest that PIC
contributes to the expression of genes that may participate in the
induction of ß-cell death, such as Fas and iNOS. The main form of
ß-cell death induced by combinations of PIC and cytokines is
apoptosis, which occurs by both NO-dependent and -independent
mechanisms.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PIC and cytokine treatment and nitrite determination
The effects of cytokines and/or PIC were examined after 6
h, 6 days, and 9 days of culture in the presence of recombinant murine
IFN- (1000 U/ml, 10 U/ng; Holland Biotechnology, Leiden, The
Netherlands), recombinant human IL-1ß (50 U/ml, 38 U/ng; gift from
Dr. C. W. Reynolds from NCI, Bethesda, MD), and synthetic PIC (100
µg/ml; Sigma, St. Louis, MO). The concentrations of
cytokines were selected based on our previous studies with rodent
pancreatic islets and ß-cells (11, 20, 21, 22), whereas the
concentration of PIC was selected based on data from the literature
(16, 23, 24) and our own dose-response studies (data not
shown). Culture media were collected after 72 h for nitrite
determination (nitrite is a stable product of NO oxidation), which was
performed spectrophotometrically at 546 nm wavelength after colored
reaction with the Griess reagent (25). Nitrite was not
determined at subsequent time points because ß-cells exposed to PIC
and cytokines for 6–9 days demonstrate an important decrease in
viability (see Results). In some experiments ß-cells were
exposed to cytokines and/or PIC in the presence of the iNOS inhibitor
NG-monomethyl-L-arginine
(MA; 1 mM). We have previously shown that
0.5–1.0 mM MA prevents cytokine-induced nitrite
production by rat pancreatic ß-cells (19) (Pavlovic, D.,
and D. L. Eizirik, unpublished data).
mRNA isolation and RT-PCR
Polyadenylated RNA was isolated from ß-cells (0.5 x
105 cells) using
oligo(deoxythymidine)25-coated polystyrene
Dynabeads (DynAl, Oslo, Norway). The RT reaction was
performed at 42 C for 1 h and contained (per 10 µl) mRNA
equivalent to 6 x 103 cells, 1 x RT
buffer, 5 mM MgCl2, 1 mM
of each deoxy-NTP, 2.5 µM random hexamer primers, and 100
U Moloney murine leukemia virus reverse transcriptase
(Perkin-Elmer Corp., Norwalk, CT). The subsequent PCR
reaction contained (in 25 µl reaction solution): 2.5 µl
complementary DNA (cDNA), 0.4 µM forward and reverse
primers, 200 µM of each deoxy-NTP, 1 x PCR buffer,
2 mM MgCl2, and 0.625 U AmpliTaq Gold
DNA polymerase (Perkin-Elmer Corp.) (22, 26).
PCR specificity and efficiency were enhanced using hot start PCR with
12-min predenaturation at 95 C and then 29 cycles for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 28 cycles for iNOS,
25 cycles for MnSOD (manganese superoxide dismutase), 36 cycles
for Fas, 28 cycles for major histocompatibility complex (MHC) class I,
and 33 cycles for Spi-3 (serine protease inhibitor 3) at 94 C for
45 sec, 58 C for 45 sec, and 72 C for 80 sec. The number of cycles was
selected to allow linear amplification of the cDNA under study. The
primer sequences used for determination of rat cDNAs for GAPDH, iNOS
(20), MnSOD (27), and Spi-3 (28)
were as described in the indicated references. The primer sequences
used for determination of rat cDNAs for Fas were: forward,
5'-GAATGCAAGGGACTGATAGC-3'; and reverse, 5'-TGGTTCGTGTGCAAGGCTC-3'. The
primer sequences used for determination of rat cDNAs for MHC I were:
forward, 5'-GCTCACACTCGCTGCGGTAT-3'; and reverse,
5'-GCCATACATCTCCTGGATGG-3'.
The ethidium bromide-stained agarose gels were photographed under UV transillumination using Kodak Digital Science DC40 camera (Eastman Kodak Co., Rochester, NY). The PCR band intensities on the image were quantified by Biomax 1D Image analysis software (Kodak) and expressed in pixel intensities (OD). The target cDNAs present in each sample were corrected for the respective GAPDH values. Expression of the housekeeping gene GAPDH is not affected by exposure to cytokines (29).
Cell transfection and luciferase assay
Studies on iNOS promoter activity in single ß-cells (4 x
104 cells/condition) were performed by transient
transfection with the plasmid piNOS-1002luc containing nucleotides
-1002 to +132 of the rat iNOS promoter [wild-type (wt)] and mutants
from piNOS-1002luc in which either the distal or proximal nuclear
factor-B (NF-B)-binding sites, or the STAT (signal transducer and
activator of transcription)-binding (GAS) -interferon
activated site, were inactivated (30). We previously
observed that the promoter region containing nucleotides -1002 to +132
is required for maximal IL-1ß-induced iNOS activation in rat
insulin-producing cells (30). ß-Cells were transfected
with Lipofectamine (Life Technologies, Inc., Gaithersburg,
MD). After 4-h transfection they were exposed for 16 h to
cytokines and/or PIC. Luciferase activities were assayed with the dual
luciferase reporter assay (Promega Corp.) in a TD-20/20
Luminometer (Turner Designs, Sunnyvale, CA). The values of the test
plasmid were normalized for the luciferase activity value of the
cotransfected control plasmid, pRL-CMV (30). The different
conditions tested did not affect pRL-CMV activities, which for control,
IFN-- plus PIC-treated, and IL-1ß-treated ß-cells (means of
three experiments) were 521, 549, and 646, respectively.
Note that the molecular biology experiments described above require large numbers of cells. As ß-cells are difficult to obtain, these experiments are usually performed in insulin-producing cell lines (27, 30). Unfortunately, neither RINm5F cells nor MIN6 cells are responsive to PIC-induced modifications in gene expression (data not shown). Thus, all of our experiments were performed with primary ß-cells.
Assessment of ß-cell viability
The percentage of viable, apoptotic, and necrotic ß-cells was
determined after 6 or 9 days of exposure to cytokines and/or PIC, the
amount of time required to detect significant increases in cell death
in FACS-purified ß-cells (22, 31, 32) (data not shown).
For this purpose, ß-cells were incubated for 15 min with propidium
iodide (PI; 10 m/ml) and Hoechst (HO) 342 (20 mg/ml) (33).
PI is a highly polar dye that penetrates only cells with damaged
membranes, staining their nuclei red; HO 342 freely crosses the plasma
membrane, entering both cells with damaged and those with intact
membranes and staining the DNA blue (33). The cells were
examined in an inverted fluorescence microscope with UV excitation at
340–380 nm. Viable cells were identified by their intact nuclei with
blue fluorescence (HO 342), necrotic cells by their intact nuclei with
yellow-red fluorescence (HO 342 and PI), and apoptotic cells by their
fragmented nuclei, exhibiting either a blue (HO-342; early apoptosis)
or yellow-red fluorescence (HO 343 and PI; late apoptosis)
(33). Note that under the present experimental conditions
(culture in serum-free medium), nuclear remains from cells undergoing
apoptosis or necrosis are preserved, and the cells remain attached to
the culture dish (33). Thus, the values provided in Figs. 4
and 5 represent the cumulative number of apoptotic or necrotic cells
over a 6- to 9-day period of observation. This fluorescence assay for
single ß-cells is quantitative and has been validated by systematic
comparisons with electron microscopy observations (32, 33). The method has been successfully used to evaluate
apoptosis/necrosis in rat (32, 33) (present data), mouse
(22, 26), and human (31) ß-cells. The use
of purified ß-cells in these experiments provides a homogeneous and
well defined cell population (>95% ß-cells), decreasing the
detection of cell death in non-ß-cells, a problem inherent to studies
performed in whole isles. In each experimental condition, a minimum of
500 cells were counted by 2 observers, 1 of whom was unaware of the
sample identity. The necrosis and apoptosis indexes were calculated as
((% necrotic or apoptotic cells in experimental condition - %
necrotic or apoptotic cells in control)/(100 - % dead cells in
control)) x 100 (34). The mean values for necrosis
and apoptosis, respectively, in control single ß-cells (not exposed
to cytokines) after 6 days in culture were 14 ± 1% and 15
± 2% (n = 5). Similar values were observed after 9 days of
culture (data not shown).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, induced a clear increase in
Fas and MnSOD expression (Fig. 1). IL-1ß
plus PIC induced higher MnSOD expression than that induced by IL-1ß
or PIC alone (Fig. 1).
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alone (Fig. 1
)
did not express iNOS. Both IL-1ß alone and PIC plus IFN- induced
iNOS expression, but PIC failed to potentiate IL-1ß-induced iNOS
expression.
PIC did not consistently affect the expression of the mRNAs
encoding for MHC class I and serine protease inhibitor 3 (Spi-3;
Fig. 1). IFN- induced MHC class I expression, whereas IL-1 increased
the expression of Spi-3 mRNA. These effects of individual cytokines
were not potentiated by PIC or the other cytokines. None of the
above-described treatments modified the expression of the housekeeping
gene GAPDH (Fig. 1). The ODs for GAPDH expression were (mean ±
SEM of three experiments): control, 1.65 ±0.29; IL-1ß,
1.26 ± 0.14; IFN-, 1.34 ± 0.19; PIC, 1.41 ± 0.33;
IL-1ß plus PIC, 1.18 ± 0.23; IFN- plus PIC, 1.38 ±
0.25; and IL-1ß plus IFN-, 1.35 ± 0.28.
As mentioned above, PIC cooperated with IFN- to induce iNOS mRNA
expression (Fig. 1). Sequence analysis and functional studies of the
5'-flanking region of the rat iNOS gene in insulin-producing cells have
previously revealed two NF-B sites and one GAS site that mediate the
stimulatory effects of IL-1ß and IFN- (30). To
delineate the PIC- plus IFN--responsive regions in the iNOS
promoter, transient transfections were performed with the plasmid
piNOS-1002luc containing nucleotides -1002 to +132 of the rat iNOS
promoter (wt), and mutants from piNOS-1002luc in which either the
distal or proximal NF-B binding site or the STAT-binding (GAS) site
was inactivated. The values for relative luciferase activity were
(mean ± SEM of three experiments): wt not exposed to
cytokines, 2 ± 1; wt exposed to PIC and IFN-, 156 ± 51;
and wt exposed to IL-1ß, 101 ± 10. Thus, PIC plus IFN-
induced a nearly 70-fold increase in iNOS promoter activity, similar to
the induction observed in wt constructs exposed to IL-1ß (used as a
positive control) (30). Site mutations of the proximal or
distal NF-B binding sites decreased PIC- plus IFN--induced iNOS
promoter activity by 80% (Fig. 2),
whereas inactivation of GAS lead to a minor and nonsignificant
reduction in promoter activity (Fig. 2). These data indicate that
NF-B is a crucial transcription factor for PIC-induced iNOS
expression.
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), ß-cells exposed for
72 h to IL-1ß presented a 4-fold increase in nitrite production,
whereas neither PIC nor IFN- alone increased medium nitrite
accumulation above basal levels (Fig. 3). A combination of PIC and
IFN- increased nitrite production similarly to that observed with
IL-1ß. These levels were significantly higher than those observed in
the control condition or after exposure to PIC or IFN- alone
(P < 0.05). On the other hand, PIC failed to further
increase the nitrite production induced by IL-1ß or IL-1ß plus
IFN-.
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and data not shown). Combinations
containing IL-1ß and IFN-, IL-1ß and PIC, IFN- and PIC, or
PIC, IL-1ß, and IFN- induced a significant increase in the
percentage of apoptotic cells after 6 and 9 days when compared against
controls (P < 0.001; data not shown) and when compared
with individual cytokines or PIC alone (Fig. 4). None of the treatments
used induced a significant increase in the fraction of necrotic cells
(data not shown). These data indicate that PIC synergizes with IL-1ß
and/or IFN- to induce ß-cell death and that the main form of cell
death under the present experimental conditions was apoptosis.
As PIC in combination with cytokines induced iNOS expression
(Fig. 1), NO production (Fig. 3), and ß-cell apoptosis (Fig. 4), we
next evaluated whether NO production is required for PIC- plus
cytokine-induced apoptosis. For this purpose, ß-cells were exposed to
different proapoptotic conditions in the presence or absence of the
iNOS blocker MA (Fig. 5). MA, cytokines,
or PIC alone did not modify the percentage of apoptotic cells, and none
of the different treatments lead to an increased number of necrotic
cells (data not shown). On the other hand, as observed in Fig. 4, there
was an increase in the apoptotic index after exposure to IL-1ß plus
IFN-, IL-1ß plus PIC, or IFN- plus PIC on both days 6 and
9. MA abolished the apoptosis induced by IL-1ß and PIC and
partially protected against apoptosis induced by IL-1ß and IFN-,
but it did not prevent apoptosis induced by IFN- and PIC. As
previously described (19) (Pavlovic, D., and D. L.
Eizirik, unpublished data), MA prevented the increased NO formation
induced by IL-1ß, IL-1ß plus IFN-, IL-1ß plus PIC, or IFN-
plus PIC (data not shown). As a whole, these observations suggest that
PIC contributes to cytokine-induced ß-cell apoptosis by both
NO-dependent and NO-independent mechanisms.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(10, 11, 12, 14).
To further investigate the mechanisms of ß-cell dysfunction/death
after exposure to these agents, we presently characterized the effects
of PIC alone or in combination with IL-1ß and/or IFN- on the gene
expression and viability of FACS-purified rat ß-cells. The use of
purified ß-cells in the present experiments allows discrimination
between necrosis and apoptosis (9, 31, 32, 33) and removes
potential biases due to effects of PIC and/or cytokines on
non-ß-cells that are usually present in whole islet preparations
(41, 42).
PIC induced expression of Fas, MnSOD, and, to a minor extent, MHC class
I mRNA. Similarly, IL-1ß induced Fas and MnSOD expression, but it
also led to increased expression of two mRNAs not affected by PIC,
namely iNOS and Spi-3. On the other hand, IFN- did not induce the
expression of Fas, MnSOD, iNOS, or Spi3, but this cytokine was the most
potent inducer of MHC class I expression. This diverse pattern of mRNA
expression triggered by PIC, IL-1ß, and IFN- suggests that these
agents act on ß-cells at least to some extent by distinct signal
transduction pathways. It is noteworthy that the effects of PIC on mRNA
expression were more similar to the effects of IL-1ß than to those of
IFN-. It has been previously shown that IL-1ß triggers the
expression of iNOS (30, 43, 44), MnSOD (27),
and Fas (Darville, M., and D. L. Eizirik, manuscript in
preparation) via NF-B activation. IFN-, on the other hand,
potentiates the effects of IL-1ß on iNOS expression via STAT-1
nuclear binding (30), without affecting IL-1ß-induced
NF-B activation (45). We presently observed, by
site-directed mutagenesis, that two separate NF-B-binding sites in
the iNOS promoter are required for PIC- and IFN--induced iNOS
expression in primary ß-cells. Site mutation of STAT-1 also leads to
a decrease, of minor proportion, in iNOS expression. This inhibitory
effect of STAT-1 inactivation is probably due to blocking the IFN-
signal transduction, whereas blocking of NF-B probably acts by
preventing PIC signaling. Indeed, as mentioned above, IFN- does not
activate NF-B in ß-cells, whereas PIC has been shown in other
tissues to affect cellular function via NF-B activation (46, 47). The fact that both IL-1ß (30, 43, 44) and
PIC (present data) use NF-B for signal transduction in ß-cells may
explain the similarities between the effects of these agents on mRNA
expression in ß-cells.
PIC, in the range of 10–100 µg, has been shown to induce apoptosis
in diverse cell types (23, 48, 49). This was not the case
for pancreatic ß-cells, where the viral product alone failed to
induce cell death. When PIC was combined with either IL-1ß or
IFN-, however, it led to a significant increase in ß-cell death,
mostly by apoptosis. The present finding that apoptosis is the main
form of cell death induced by PIC and cytokines in rat ß-cells is in
line with previous studies showing that exposure of human, mouse, or
rat ß-cells to IL-1ß plus IFN- or IL-1ß, IFN-, plus tumor
necrosis factor- induces cell death mostly by apoptosis (22, 26, 31, 32).
As discussed above, a combination of IFN- and PIC induced iNOS
expression, NO production, and ß-cell death by apoptosis, suggesting
a potential link between NO synthesis and ß-cell apoptosis. Indeed,
it has been previously suggested that blockage of iNOS activity
prevents IFN-- and PIC-induced cell death in whole islets
(16). We presently observed that the iNOS blocker
L-NMMA prevents PIC- plus IFN--induced NO
formation, but does not prevent ß-cell death, suggesting that NO is
not a major mediator in ß-cell apoptosis induced by PIC and IFN-.
A possible explanation for the differences between our findings and
data reported by Heitmeier et al. (16) is the
use of different experimental models, namely whole islets
(16) vs. pure ß-cells (present data). NO
apparently has a more relevant role for cytokine-induced cell death
(mostly by necrosis) in whole islets than in purified ß-cells
(22, 32). Moreover, cell death seems to occur earlier in
cytokine-exposed whole islets than in pure ß-cells (22, 26). This may be due to the disappearance of intercellular
capillary spaces in whole islets maintained in culture, causing central
ischemia and allowing local accumulation of high amounts of NO. On the
other hand, it cannot be excluded that FACS purification of ß-cells
and the consequent loss of cell to cell contacts increases ß-cell
susceptibility to proapoptotic stimuli. Moreover, some of the effects
of PIC and/or cytokines on ß-cells in whole islets (in
vivo and in vitro) may be mediated via islet
non-ß-cells, absent in our FACS-purified preparations. Another reason
for the difference findings of our report and that by Heitmeier
et al. (16) is the lack of quantitative (number
of dead cells) and qualitative (apoptosis or necrosis) evaluation of
ß-cell death in the study by Heitmeier et al.
(16) compared with the present quantitative assessment of
ß-cell necrosis/apoptosis.
It is noteworthy that NO seems to play a necessary role for ß-cell
apoptosis induced by PIC and IL-1ß (present data), as suggested by
the complete protection induced by
NG-monomethyl-L-arginine (MA) against
these agents. These observations indicate that PIC synergizes with
IFN- and IL-1ß for the induction of ß-cell apoptosis by
different mechanisms, i.e. either by NO-independent (PIC
plus IFN-) or NO-dependent (PIC plus IL-1ß) processes. The nature
of the signal transduction used by PIC and IFN- to induce ß-cell
apoptosis remains to be clarified.
We presently observed another PIC effect that may contribute to ß-cell apoptosis, the induction of Fas mRNA. The initiation of autoimmune diabetes in mice probably involves Fas ligand (FasL)-Fas mediated apoptosis, i.e. FasL-expressing T lymphocytes may induce cell death in Fas-expressing ß-cells (50, 51, 52, 53). Fas expression was also detected in ß-cells from patients with recent-onset T1DM, whereas FasL was observed in the islet-infiltrating lymphocytes (54).
In conclusion, the present observations suggest that PIC interacts with
the cytokines IL-1ß and IFN- to induce the expression of ß-cell
genes potentially involved in ß-cell dysfunction/death, namely Fas
and iNOS. Moreover, the viral product in combination with
proinflammatory cytokines leads to ß-cell death by apoptosis.
Previous studies have indicated that cytokines are expressed in the
vicinity of islet ß-cells during virus-induced diabetes in mice
(14). Several viruses can infect human ß-cells
(5), and epidemiological data support a role for viral
infections in the pathogenesis of human T1DM (3, 4, 55).
Taking this and the present data into account, it is conceivable that
prolonged infection of ß-cells by viruses leads to local
inflammation, with both production of cytokines (such as IL-1ß and
IFN-) and eventual islet invasion by mononuclear cells expressing
FasL. In this context, viral products (e.g. dsRNA) acting in
synergism with the locally produced cytokines could contribute to
ß-cell death by Fas up-regulation, intracellular NO production, and
generation of additional proapoptotic signals whose nature remains to
be clarified.
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Acknowledgments |
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Footnotes |
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Received December 19, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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by tetratricopeptide repeat protein and co-chaperone P58(IPK). Mol Cell
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required for cytokine-induced manganese superoxide dismutase expression
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expression in rodent and human islet cells precedes nitric oxide
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and NF-B. Mol Cell Biol 19:4653–4663This article has been cited by other articles:
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D. Liu, A. K. Cardozo, M. I. Darville, and D. L. Eizirik Double-Stranded RNA Cooperates with Interferon-{gamma} and IL-1{beta} to Induce Both Chemokine Expression and Nuclear Factor-{kappa}B-Dependent Apoptosis in Pancreatic {beta}-Cells: Potential Mechanisms for Viral-Induced Insulitis and {beta}-Cell Death in Type 1 Diabetes Mellitus Endocrinology, April 1, 2002; 143(4): 1225 - 1234. [Abstract] [Full Text] [PDF]
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A. K. Cardozo, H. Heimberg, Y. Heremans, R. Leeman, B. Kutlu, M. Kruhoffer, T. Orntoft, and D. L. Eizirik A Comprehensive Analysis of Cytokine-induced and Nuclear Factor-kappa B-dependent Genes in Primary Rat Pancreatic beta -Cells J. Biol. Chem., December 21, 2001; 276(52): 48879 - 48886. [Abstract] [Full Text] [PDF]
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) or to
IL-1ß (50 U/ml), IFN-