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-Activated Cell Death Pathways in NIT-1 Insulinoma Cells and Primary Pancreatic ß Cells1
The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, Parkville, 3050, Victoria, Australia
Address all correspondence and requests for reprints to: Dr. T. W. H. Kay, Walter and Eliza Hall Institute of Medical Research, P.O. Royal Melbourne Hospital, Victoria, 3050, Australia. E-mail: kay{at}wehi.edu.au
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Abstract |
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(TNF) is a potential mediator of ß cell
destruction in insulin-dependent diabetes mellitus. We have studied
TNF-responsive pathways leading to apoptosis in ß cells. Primary ß
cells express low levels of the type I TNF receptor (TNFR1) but do not
express the type 2 receptor (TNFR2). Evidence for TNFR1 expression on
ß cells came from flow cytometry using monoclonal antibodies specific
for TNFR1 and TNFR2 and from RT-PCR of ß cell RNA. NIT-1 insulinoma
cells similarly expressed TNFR1 (at higher levels than primary ß
cells) as detected by flow cytometry and radio-binding studies. TNF
induced NF-
B activation in both primary islet cells and NIT-1 cells.
Apoptosis in response to TNF was observed in NIT-1 cells whereas
apoptosis of primary ß cells required both TNF and interferon-
(IFN). Apoptosis could be prevented in NIT-1 cells by expression of
dominant negative Fas-associating protein with death domain (dnFADD).
Apoptosis in NIT-1 cells was increased by coincubation with IFN,
which also increased caspase 1 expression. These data show that
TNF-activated pathways capable of inducing apoptotic cell death are
present in ß cells. Caspase activation is the dominant pathway of
TNF-induced cell death in NIT-1 cells and may be an important mechanism
of ß cell damage in insulin-dependent diabetes mellitus. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and interferon (IFN), released by T
cells and activated macrophages, are present in the inflammatory
infiltrate of pancreatic islets and have a potential role in ß cell
destruction. These cytokines have damaging effects on ß cells
in vitro and are particularly potent when present in
combination. IL-1ß appears to result in ß cell cytotoxicity mainly
via the induction of nitric oxide (NO) synthase and NO production
(reviewed in Refs. 3, 4), an effect that is potentiated by TNF
and IFN (5, 6, 7). There is also evidence for DNA-strand breaks, a
characteristic feature of apoptotic cell death, in ß cells treated
with IL-1 and other cytokines (8, 9, 10), and features of apoptosis have
been identified in ß cells of nonobese diabetic (NOD) mice (11, 12).
TNF also has cytotoxic effects on ß cells, although generally only
in combination with other cytokines such as IFN. Whether there are
direct effects of TNF on ß cells remains unclear. TNF, with
other stimuli, can activate macrophages present in intact pancreatic
islets leading to their secretion of IL-1ß, which may account for
TNF-induced ß cell cytotoxicity (13). TNF receptor expression on
ß cells has not previously been directly studied.
In non-ß cells, TNF has been shown to bind to two receptors, p55
(TNFR1) and p75 (TNFR2), which have unrelated intracellular domains
(14). While apoptosis can be induced by TNFR1, the capacity of TNFR2 to
signal cell death is less well defined (14, 15), although this is
likely to occur particularly in response to membrane-bound TNF (16).
Apoptosis induced by TNFR1 and Fas (CD95), both members of the TNFR
superfamily, involves the activation of members of the
IL-1ß-converting enzyme (ICE) family of cysteine proteases (mammalian
homologues of the CED-3 death protein in Caenorhabditis
elegans), now known as "caspases" (17, 18, 19). The role of
caspases in TNF and Fas mediated apoptosis was established following
observations that caspase inhibitors, such as the cowpox-virus encoded
crmA (cytokine response modifier A) protein (20) and baculovirus p35
(21), prevent cell death induced by both these molecules (22, 23, 24).
Caspase activation and apoptosis induction by both TNFR1 and Fas involves the ligand-dependent recruitment of various intracellular signalling molecules to the receptors. Both TNFR1 and Fas contain a cytoplasmic motif termed the "death domain," which is essential for Fas and TNF-induced cytotoxicity and receptor oligomerization (25). The death domain is shared by a number of Fas- and TNFR1-associated proteins, including TRADD (26), FADD/MORT-1 (27, 28), and RIP (29), and is involved in their recruitment to the TNFR. FADD, which interacts directly with the Fas receptor and with the TNFR1 via TRADD, is essential in signaling apoptosis via its interaction with caspase 8 (FLICE) (30, 31)
In this study, we have investigated TNF receptor expression and
TNF-activated cell death pathways in ß cells. Where possible, primary
ß cells have been used. Cytotoxic effects of TNF were studied in
the NIT-1 ß cell line to directly look at TNF signaling in these
cells, and avoid the possible influence of nonendocrine cells present
in primary islet cultures. The use of insulinoma cell lines, which are
relatively easy to transfect, also enabled us to explore the role of
caspases in the TNF-induced apoptosis of NIT-1 cells by transfection
with a dominant negative mutant of FADD. The NIT-1 ß cell line (32)
is derived from the spontaneously diabetic nonobese diabetic (NOD)
mouse and therefore carries the NOD MHC and other NOD genes potentially
relevant to ß cell recognition and killing by autoreactive T
cells.
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Materials and Methods |
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promoter. The Flag sequence is MDYKDDDDK. NIT-1 cells were transfected
by electroporation using a Gene Pulser apparatus (Bio-Rad Laboratories, Inc., Hercules, CA). A single pulse at 250 mV was
delivered from a 960-µF capacitor to the cells in serum-free medium.
Two days later, transfected cells were selected in 2 µg/ml puromycin
(Sigma Chemical Co.). The optimal transfection conditions
and concentration of selective agent were determined for NIT-1 cells
before transfection.
Islet isolation
Islets of Langerhans were isolated from 5- to 7-week-old
nonobese diabetic (NOD) mice of either sex, as previously described
(33) by the method of Lake et al. (34), which involves
cannulation of the common bile duct and distension of the pancreas with
3 ml of 1.3 U/ml collagenase (collagenase P, Roche Molecular Biomedicals, Mannheim, Germany) followed by purification of
islets on a BSA gradient. Approximately 200 islets per pancreas were
obtained using this method, and usually 4–8 mice were used per
experiment. For flow cytometric analysis, islet cells were identified
by the method of Pipeleers (35). Briefly, islets were dispersed into
single cells by brief incubation with 0.2% trypsin (Calbiochem, San Diego, CA), 10 mM EDTA in HBSS. Dispersed
islets were then washed free of trypsin and allowed to recover in DMEM
plus 10% FCS for 0.5–1 h before staining. Islet cells were usually
analyzed on the day of isolation or occasionally at later times. If
analyzed after the day of isolation, they were incubated in low glucose
(2.5 mM) DMEM for 16–48 h before being dispersed. ß
cells under both these conditions have high autofluorescence due to
increased intracellular FAD levels allowing them to be distinguished
from other intraislet cells for analysis or sorting (35). No
differences were observed between islets dispersed on the day of
isolation or subsequently. All high autofluorescence islet cells
stained with the monoclonal antibody A2B5 (36) and >85% were positive
for insulin by indirect immunofluorescence (not shown).
Pseudo-islets were made by dispersing isolated islets with
trypsin, as above, and then incubating the islets undisturbed for 7
days (37).
Apoptosis assay
Cells were seeded in 24-well plates at a density of
105 cells per well (for NIT-1 cells in DME/NEAA with 10%
FCS; for primary islet cells in CMRL 1066 plus 10% FCS) and incubated
for 24 h with the following reagents: recombinant murine TNF (1
mg = 2.6 x 107 U), human TNF, murine IFN
(1 mg = 8.0 x 106 U) (all obtained from
Genentech, Inc., South San Francisco, CA), recombinant
human IL-1ß (Genzyme Corp., Cambridge, MA). Routinely,
100 ng/ml of TNF and 100 U/ml of IFN was used for apoptosis
experiments. Quantitation of apoptosis was performed according to the
method described by Nicoletti et al. (38). In brief, cells
were harvested and resuspended in hypotonic fluorochrome solution (50
µg/ml propidium iodide in 0.1% sodium citrate with 0.1% Triton
X-100) and incubated at 4 C in the dark in polypropylene tubes for 1–6
days, before being analyzed on the flow cytometer (FACScan,
Becton Dickinson and Co.). The assay measures fragmented
nuclei and therefore greater than one fragmented nucleus can be derived
from one apoptotic cell. The level of TNF-mediated apoptosis was
determined by the formula 100x (% number of apoptotic cells with
cytokine - % number of background apoptotic cells)/(100 -
% number of background apoptotic cells). This formula is
analogous with that used for measuring cytotoxicity in chromium release
assays and allows results from experiments with different levels of
basal apoptosis to be combined.
Additionally, staining of cells with FITC-Annexin V (Trevigen, Inc., Gaithersburg, MD) was also used as a measure of apoptosis. Cells were treated with cytokines for 6–48 h and then stained with FITC-Annexin-V and propidium iodide (PI) according to the manufacturer’s instructions.
TNF binding assay
Cells (3–5 x 106/well) were incubated with 40
ng/ml 125I-TNF- (70,000 cpm/ng) with or without 200-fold
excess of unlabeled human or mouse TNF for 2 h on ice, as
previously described (39). At 40 ng/ml greater than 90% of receptors
will be saturated (40). Ten replicates of each reaction were counted
and the results expressed as mean ± SD.
Electrophoretic mobility (gel) shift assay
Cells grown in 10 cm3 plates were stimulated with
100 U/ml mTNF for 30 min. Nuclear extracts were prepared by lysis of
cells in a buffer containing 10 mM HEPES, pH 7.9, 10
mM KCl, 1.5 mM MgCl2, with protease
inhibitors (0.5 mM phenylmethylsulfonyl fluoride (PMSF), 10
µg/ml leupeptin) and 0.5 mM dithiothreitol (DTT). The
lysate was then vortexed with 25 µl 10% NP40 and centrifuged. The
pellet was resuspended in 420 mM NaCl, 20 mM
HEPES, pH 7.9, 1.5 mM MgCl2, 0.2 mM
EDTA, glycerol, protease inhibitors and DTT and centrifuged at 12,000
rpm for 5 min at 4 C. One pmol of annealed double stranded
oligonucleotide probe (representing the NF-B consensus sequence:
5'-GTACGAGGGGACTTTCCGA-3') was labeled by filling in the ends with
Klenow polymerase and [-32P]dATP, and purified on a
spin column. Electrophoretic mobility shift assays were performed by
incubating 2 µl of extract with 0.5 µg of poly(dI-dC) in a binding
buffer (13.3 mM HEPES (pH 7.6), 0.07 mM EDTA,
3.3 mM MgCl2, 34 mM KCl, 1
mM DTT, 10% glycerol) for 15 min on ice. Labeled NF-B
oligonucleotide probe was added (15,000 cpm), and the mixture was
incubated for a further 15 min. The samples were separated on a 5%
nondenaturing polyacrylamide gel at 200V for 30 min, dried (80 C), and
exposed to x-ray film (Hyperfilm-MP, Amersham Pharmacia Biotech, Buckinghamshire, UK).
Flow cytometry
Cells were analyzed for TNF receptor expression by standard flow
cytometry techniques. The following primary monoclonal antibodies were
used: anti-TNFR1 and anti-TNFR2 (both rat IgG1) obtained from Dr. W.
Buurman (University of Limburg, Maastricht, The Netherlands).
Anti-TNFR1 and anti-TNFR2 staining was followed by biotinylated goat
antirat Ig (PharMingen) followed by
streptavidin-phycoerythrin (Caltag).
Immunoprecipitation and Western Blotting
We harvested 6–8 x 107 cells by scraping,
washing with PBS and lysing in a buffer containing 50 mM
Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA, 1%
Triton X-100, 1 mM PMSF and 10 µg/ml leupeptin. Lysates
(
250 µl) were incubated with 2 µl of 3 mg/ml anti-Flag Ab (M2,
Eastman Kodak Co., New Haven, CT) overnight at 4 C and
then mixed with 20 µl of protein A-Sepharose CL-4B (Amersham Pharmacia Biotech) for a further 2 h. The beads were then
washed twice with a buffer containing 50 mM Tris-HCl (pH
8.0), 150 mM NaCl, 1% NP-40, 1 mM PMSF, then
heated to 95 C for 5 min in SDS-loading buffer. Following 12.5%
SDS-page, immunoprecipitated Flag-FADD was detected by Western
blotting. The gel was transferred at 100 V for at least 1 h to
PVDF membrane, blocked in 5% milk in PBS-0.05% Tween-20, and then
incubated with anti-Flag antibody. After washing the membrane was
incubated with HRP-conjugated sheep antimouse immunoglobulin (Silenus,
Hawthorn, Australia) and developed using ECL (Amersham Pharmacia Biotech).
RT-PCR
RT was carried out on total RNA isolated from NIT cells and
primary islets using RNAzol B. Twenty microliters of reaction mixture
contained 1 µg RNA, 200 U M-MLV reverse transcriptase (Promega Corp., Madison, WI), 320 µM dNTPs (Pharmacia Biotech, Uppsala, Sweden), 25 U RNasin (Promega Corp.), 0.5 µg random hexamers, 10 mM Tris-HCl (pH
8.4), 30 mM KCl, and 2 mM MgCl2.
PCR was carried out in a 20 µl reaction solution containing 10
mM Tris-HCl (pH 8.0), 30 mM KCl, 2
mM MgCl2, 8 pmol each of upstream and
downstream primers, 320 µM dNTPs and 1 U Taq
polymerase (Perkin Elmer). Amplification for 30 cycles
(ICE) and 23 cycles (actin) was done for 1 min each at 95 C, 58 C, and
70 C. The primers 5'-GATTCTAAAGGAGGACATCC-3' (upstream primer) and
5'-GTACATAAGAATGAACTGGA-3' (downstream primer) amplify a 930-bp segment
of the murine caspase 1 gene. The primers
5'-GTGGGCCGCCCTAGGCACCA-3' and
5'-CTCTTTGATGTCACGCACGATTTC-3' amplify a 530-bp segment of
the murine actin gene. The primers 5'-CGGACATGGGTCTCCCCACCG-3' and
5'-AACCCTGCATGGCAG-3' amplify a 550-bp segment of the murine TNFR1
cDNA. The TNFR1 primers span an intron within the gene making the cDNA
amplification product easily distinguishable from the genomic product.
The PCR products were electrophoresed on a 1% agarose gel and blotted
to nylon membrane (Magna, MSI, Westborough, MA) overnight. Southern
blots were then probed with [-32P]dATP-labeled ICE or
actin cDNA.
Statistics
Unless otherwise stated, statistical comparisons were carried
out by one-way ANOVA with Bonferroni post tests using the GraphPad
Prism program.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, as a
positive control (Fig. 1A
). These cells
stained positively for TNFR1 and TNFR2 (Fig. 1A, iv). Primary mouse ß
cells, identified by high autofluorescence and side-scatter on flow
cytometry, expressed low levels of TNFR1 but no detectable TNFR2 (Fig. 1A, i, and Table 1). The fluorescence
profile indicating TNFR1 expression represented a small but
statistically different shift when compared with isotype control
staining (P < 0.01 for the ratio of the medians, one
sample t test). As a negative control for this, islets were
isolated from TNFR1 gene-targeted (TNFR1-/-) mice (41). No shift in
the fluorescence profile was seen when ß cells from TNFR1-/- mice
were stained with anti-TNFR1 compared with an isotype control antibody
(Fig. 1A, ii, and Table 1), consistent with the idea that the staining
of the wild-type ß cells does indeed represent low levels of TNFR1
expression. A further possibility was that the staining of
wild-type islets might have been due to contamination of the
electronic gate set for ß cells with other cells. This is unlikely
because >95% of the high autofluorescence cells in this gate can be
stained with the monoclonal antibody A2B5 and are therefore endocrine
cells and >85% of these stain for intracellular insulin by
fluorescence microscopy (not shown). To further address the possibility
of contaminating nonß cells we analyzed TNFR expression on NIT-1
insulinoma cells which are purely of ß cell lineage. NIT-1 cells
showed cell surface staining for TNFR1 at a lower level than positive
control L929 cells but higher than primary ß cells (Fig. 1A, iii).
Like primary ß cells they showed no detectable expression of
TNFR2.
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) that was nevertheless reproducible and
statistically significant (P < 0.05, by Student’s
t test). Competition studies using human TNF, which binds
mouse TNFR1 but not mouse TNFR2 (42), confirmed this to be due to TNFR1
rather than TNFR2. Consistent with the flow cytometric analysis, the
number of receptors on NIT-1 cells was lower than on L929 cells that
have 1000 TNF receptors per cell (Grell, M., unpublished data). This
assay was not carried out for primary ß cells because of the number
of cells required. In related experiments, a NIT-1 cell clone which was
found not to respond to TNF in apoptosis and EMSA assays, was
isolated. This clone showed no specific binding of TNF in the
radio-binding assay and had no evidence for TNFR1 expression by flow
cytometry (not shown). This unusual clone confirms the specificity of
the assays for TNFR1 expression as does the negative binding of
anti-TNFR2.
We also analyzed NIT-1 cells and primary ß cells for TNFR1 mRNA by
RT-PCR. Primary ß cells were isolated by FACS-sorting isolated islet
cells into high- and low- autofluorescence populations (see
Materials and Methods). After the cells were collected, RNA
was isolated from the two populations; because of the very low numbers
of sorted cells, accurate RNA quantitation was not possible. Despite
this, there was evidence for TNFR1 mRNA expression in NIT-1 cells,
primary ß cells and primary nonß islet cells. (Fig. 1C)
TNF-dependent NF-B activation in NIT-1 and primary islet
cells
To demonstrate functional activity of TNFRs on ß cells, we
assessed the ability of TNF to induce activation of NF-B by EMSA
(Fig. 2). Both TNF and IL-1 induced
NF-B in NIT-1 cells as demonstrated by nuclear translocation and
binding to a radiolabeled NF-B-binding oligonucleotide (Fig. 2). To
reduce (but not eliminate) the possibility of the EMSA result being
influenced by islet nonendocrine cells such as macrophages and
endothelial cells, primary islets were disaggregated and reaggregated
to form pseudoislets. NF-B activation by TNF was also observed in
these cells indicating that cells within pseudoislets express
functional TNFRs consistent with the flow cytometry data (Fig. 2).
Pseudoislets are not pure ß cells and it is possible that islet cells
other than ß cells might contribute to TNF-dependent NF-B
activation.
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induces apoptosis in NIT-1 cells was observed to induce cell death in NIT-1 cells.
TNF was cytotoxic to NIT-1 cells at concentrations as low as 0.01
ng/ml (data not shown), but was routinely used at a concentration of
100 ng/ml, which was at least 10-fold higher than the concentration
needed for maximal cytotoxicity. Cell death in NIT-1 cells following
TNF treatment was evident by their rounded and shrunken appearance
(Fig. 3A). Quantitation of the level of
cell death in cytokine-treated NIT-1 cells was determined using flow
cytometric assessment of DNA fragmentation, a feature of apoptotic cell
death, as illustrated in Fig. 3B. This assay confirmed that TNF leads
to significant DNA fragmentation in NIT-1 cells (P <
0.001 compared with untreated cells) (Fig. 3C). Furthermore, Annexin V
binding to NIT-1 cells increased after TNF treatment (Fig. 3B, ii),
again consistent with cell death due to apoptosis. Plasma membrane
alterations leading to the exposure of phosphatidylserine (to which
Annexin V binds) also occur in necrotic cell death; however, the
combination of Annexin V binding and PI exclusion is typical of early
apoptosis (43). IFN increased the levels of TNF-induced apoptosis
(P < 0.001, compared with TNF alone), although it did
not on its own induce cell death in NIT-1 cells (P >
0.05). Human TNF also induced apoptosis in NIT-1 cells (not shown),
confirming a role for the TNFR1 in apoptosis induction. IL-1 did not
induce apoptosis in NIT-1 cells.
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in primary mouse
ß cells. Addition of IFN as well as TNF was required to induce
convincing apoptosis in primary islet. It was observed microscopically
that FACS-purified islet endocrine cells (sorted on the basis of A2B5
expression) underwent cell death following incubation with TNF and
IFN (Fig. 3D). Similarly, increased numbers of fragmented nuclei
were observed in unfractionated islet cells with TNF and IFN (Fig. 3E).
Caspase inhibitors protect NIT-1 cells from TNF-induced
apoptosis
To address the role of caspases in TNF-mediated apoptosis of NIT-1
cells, they were transfected with a dominant negative mutant of FADD
(dnFADD), which lacks the death-effector domain. This FADD mutant has
been shown to be an effective inhibitor of apoptosis in other cell
types. Following selection in puromycin, cells expressing dnFADD were
identified by immunoprecipitation and Western blotting with anti-Flag
mAb (Fig. 4A). Several independent clones
expressing dnFADD (clones F1, F15, F17) were protected from TNF-induced
apoptosis (Fig. 4B) (P < 0.001). Clone F19, though
transfected with the Flag-FADD vector did not express detectable
transfected protein and was not protected from apoptosis. NIT-1
cells transfected with the control vector without a cDNA insert (clone
C1) were similarly unprotected from cytokine-induced apoptosis. TNFR1
expression was confirmed on these selected clones by flow cytometry and
TNF-inducible NF-B activity (not shown). The involvement of the
caspase pathway in TNF-induced death of NIT-1 cells further points to
an apoptotic mechanism of cell death, rather than necrosis in which
caspases play no role.
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increased sensitivity to TNF-induced apoptosis but
did not induce apoptosis on its own, we speculated that IFN may
increase expression of factors in the caspase pathway. NIT-1 cells were
analyzed for the expression of ICE at the RNA level using RT-PCR, both
constitutively and following treatment with IFN and TNF. Low
levels of ICE transcript were detectable in NIT-1 cells in the absence
of cytokines, and its expression was increased by IFN (Fig. 5). Similarly, ICE expression in primary
ß cells was also induced by treatment with IFN (Fig. 5). Levels of
mRNA of another ICE family member caspase 3 (CPP32) was also analyzed
in NIT-1 cells by RT-PCR and was found to be constitutively present in
substantial levels in NIT-1 cells (results not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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can interact directly with mouse primary
ß cells and insulinoma cell lines and can signal both NF-B
activation and apoptotic cell death. Primary ß cells expressed TNFR1
at low levels, which were at the limit of detection by flow cytometry.
Other primary cells, such as lymphocytes, were found to express
similarly low levels of TNFR1 by flow cytometry (our
unpublished observations). TNFR1 but not TNFR2 receptors are expressed
on mouse ß cells and ß cell lines, as determined by flow cytometric
analysis, radio-binding studies and the effects of human TNF. This
pattern of TNFR expression is similar to that seen in many
nonhematopoietic cells and is consistent with the observation that the
TNFR1 promoter has sequences suggesting ubiquitous expression, although
the level of expression varies in different tissues (44). TNFR1 is able
to transduce signals that regulate gene expression and cytotoxicity and
its expression makes ß cells potentially capable of undergoing a full
range of TNF-induced responses. TNF-mediated apoptosis of NIT-1 cells was inhibited by dnFADD and also by crmA (not shown), an observation consistent with work done in other cell lines but not previously in ß cells, which indicates an essential role for caspases in this process (23, 24, 45). The ability of caspase inhibitors to protect NIT-1 cells from the cytotoxic effects of TNF clearly shows that these cells are undergoing apoptosis rather than necrosis, another mode of cell death in which caspases do not play a role.
Although we have shown that nuclear fragmentation can also be induced
in primary ß cells by cytokines, the role of caspase pathways in this
is as yet unproven. This will require expression of caspase inhibitors
in primary ß cells. Our study, however, indicates that in principle,
TNF can damage ß cells by this mechanism because they express both
TNFR1 and caspases. Consistent with this, primary ß cells have been
shown to be susceptible to apoptosis mediated by Fas, which uses a very
similar intracellular pathway (46, 47). In our hands, however, TNF
cannot on its own cause apoptosis in primary ß cells. We speculate
that whether cell death occurs or not in a given cell population
depends on the balance between synthesis of pro- and antiapoptotic
factors and that this balance is tipped further toward apoptosis in
NIT-1 cells than in primary ß cells in which IFN is also required.
It is conceivable that in disease states this balance between
expression of pro- and antiapoptotic genes may be altered (for example
by coexpression of IFN or other regulators of the apoptosis pathway)
leading to TNF-induced ß cell apoptosis. The role of receptor number
in this balance is uncertain; however it is possible that the very low
expression of TNFR1 on primary ß cells may be a factor in their
resistance to apoptosis mediated by TNF alone. For the IL-1 receptor, a
relationship between receptor number and effects of IL-1 has been
described (48).
Coincubation with IFN substantially increased the level of
TNF-induced apoptosis of both NIT-1 cells and primary ß cells, but
IFN did not on its own affect cell viability. IFN is known to
cooperate and at times synergize with TNF in many biological responses
and combinations of cytokines have previously been reported to enhance
DNA fragmentation in ß cell lines compared with single cytokines (8).
We did not observe any increase in TNFR1 expression with IFN
treatment (not shown). One hypothesis based on our results is that
IFN may enhance TNF-mediated apoptosis of NIT-1 cells at the level
of transcriptional regulation of caspases. The promoter region of
murine ICE has been shown to have a putative IRF-1 binding site (49),
and overexpression of IRF-1 led both to increased ICE expression and
increased sensitivity to apoptosis in T cells (50). STAT1, another
IFN-regulated transcription factor has also recently been shown to
regulate caspases and sensitivity to TNF-induced apoptosis (51).
Up-regulation of ICE transcription by cytokines has previously also
been reported in a macrophage cell line (52). Therefore up-regulation
of ICE may provide one possible explanation for the synergistic effect
of IFN in TNF-mediated cell death. IFN may also be needed for the
transcription of other proteins involved in the signaling pathway
leading to TNF-induced apoptosis in NIT-1 cells and primary ß cells,
and much remains to be understood about the precise signaling molecules
involved. Other important caspases including caspase 8 may also be
regulated by cytokines.
The role of TNF in ß cell destruction in vivo remains
uncertain. It is known to be produced in infiltrated islets of NOD mice
(53) and local neutralization by expression of soluble TNFR1 reduces
insulitis and prevents diabetes (54). Transgenic mice in which TNF
is produced by ß cells develop severe insulitis but do not develop
diabetes (55) but this may be due to down-regulation of cytotoxic
effects of TNF with chronic exposure (56). Experiments in which NOD
mice are injected with TNF or neutralizing antibodies to TNF fail
to differentiate between the effects of TNF in mediating ß cell
destruction and its immunoregulatory role (57).
In summary, we have shown evidence that pathways leading to TNF-induced
apoptosis are present in primary ß cells and NIT-1 cells and that
TNF kills NIT-1 cells via the caspase pathway. Killing via this
pathway is potentially similar but not identical in NIT-1 cells and
primary ß cells, which also require IFN possibly to increase the
expression of key members of the caspase pathway. The identification of
the caspase pathway and its likely role in ß cell destruction paves
the way for therapeutic strategies designed to inhibit this pathway,
testable in transgenic mice. Such strategies may be applicable to ß
cell replacement therapies, such as engineered ß cell lines or islet
transplants, and are also likely to contribute further to understanding
the role and action of mediators such as TNF, IL-1ß, and Fas/Fas
Ligand in insulin-dependent diabetes mellitus pathogenesis.
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Acknowledgments |
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Footnotes |
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2 These authors contributed equally
Received September 22, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and
IFN-gamma potentiate the deleterious effects of IL-1ß on mouse
pancreatic islets mainly via generation of nitric oxide. Cytokine 6:399–406[CrossRef][Medline]
and
interferon- inhibit insulin secretion and cause DNA damage in
unweaned-rat islets. Extent of nitric oxide involvement. Diabetes 45:183–189[Abstract]
-induced apoptosis in STAT1-null cells
due to low constitutive levels of caspases. Science 278:1630–1632 and granzyme A in the intra-islet infiltrates. Diabetes 44:112–117[Abstract]
transgene in murine
pancreatic ß cells results in severe and permanent insulitis without
evolution towards diabetes. J Exp Med 176:1719–1731This article has been cited by other articles:
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  T. Dai, M. Patel-Chamberlin, R. Natarajan, I. Todorov, J. Ma, J. LaPage, L. Phillips, C. C. Nast, D. Becerra, P. Chuang, et al. Heat Shock Protein 27 Overexpression Mitigates Cytokine-Induced Islet Apoptosis and Streptozotocin-Induced Diabetes Endocrinology, July 1, 2009; 150(7): 3031 - 3039. [Abstract] [Full Text] [PDF]
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 F. Ortis, A. K. Cardozo, D. Crispim, J. Storling, T. Mandrup-Poulsen, and D. L. Eizirik Cytokine-Induced Proapoptotic Gene Expression in Insulin-Producing Cells Is Related to Rapid, Sustained, and Nonoscillatory Nuclear Factor-{kappa}B Activation Mol. Endocrinol., August 1, 2006; 20(8): 1867 - 1879. [Abstract] [Full Text] [PDF]
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 N. R. Barshes, S. Wyllie, and J. A. Goss Inflammation-mediated dysfunction and apoptosis in pancreatic islet transplantation: implications for intrahepatic grafts J. Leukoc. Biol., May 1, 2005; 77(5): 587 - 597. [Abstract] [Full Text] [PDF]
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 M. Flodstrom-Tullberg, M. Hultcrantz, A. Stotland, A. Maday, D. Tsai, C. Fine, B. Williams, R. Silverman, and N. Sarvetnick RNase L and Double-Stranded RNA-Dependent Protein Kinase Exert Complementary Roles in Islet Cell Defense during Coxsackievirus Infection J. Immunol., February 1, 2005; 174(3): 1171 - 1177. [Abstract] [Full Text] [PDF]
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 Y. Chen, M. M.W. Chong, R. Darwiche, H. E. Thomas, and T. W.H. Kay Severe Pancreatitis with Exocrine Destruction and Increased Islet Neogenesis in Mice with Suppressor of Cytokine Signaling-1 Deficiency Am. J. Pathol., September 1, 2004; 165(3): 913 - 921. [Abstract] [Full Text] [PDF]
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W. Chen, K. V. Salojin, Q.-S. Mi, M. Grattan, T. C. Meagher, P. Zucker, and T. L. Delovitch Insulin-Like Growth Factor (IGF)-I/IGF-Binding Protein-3 Complex: Therapeutic Efficacy and Mechanism of Protection against Type 1 Diabetes Endocrinology, February 1, 2004; 145(2): 627 - 638. [Abstract] [Full Text] [PDF]
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 H. E. Thomas, W. Irawaty, R. Darwiche, T. C. Brodnicki, P. Santamaria, J. Allison, and T. W.H. Kay IL-1 Receptor Deficiency Slows Progression to Diabetes in the NOD Mouse Diabetes, January 1, 2004; 53(1): 113 - 121. [Abstract] [Full Text] [PDF]
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I. Chang, S. Kim, J. Y. Kim, N. Cho, Y.-H. Kim, H. S. Kim, M.-K. Lee, K.-W. Kim, and M.-S. Lee Nuclear Factor {kappa}B Protects Pancreatic {beta}-Cells From Tumor Necrosis Factor-{alpha}-Mediated Apoptosis Diabetes, May 1, 2003; 52(5): 1169 - 1175. [Abstract] [Full Text] [PDF]
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E. Bernal-Mizrachi, W. Wen, M. Shornick, and M. A. Permutt Activation of Nuclear Factor-{kappa}B by Depolarization and Ca2+ Influx in MIN6 Insulinoma Cells Diabetes, December 1, 2002; 51(90003): S484 - 488. [Abstract] [Full Text] [PDF]
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 M. M. W. Chong, H. E. Thomas, and T. W. H. Kay Suppressor of Cytokine Signaling-1 Regulates the Sensitivity of Pancreatic beta Cells to Tumor Necrosis Factor J. Biol. Chem., July 26, 2002; 277(31): 27945 - 27952. [Abstract] [Full Text] [PDF]
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S. Cottet, P. Dupraz, F. Hamburger, W. Dolci, M. Jaquet, and B. Thorens cFLIP Protein Prevents Tumor Necrosis Factor-{alpha}-Mediated Induction of Caspase-8-Dependent Apoptosis in Insulin-Secreting {beta}Tc-Tet Cells Diabetes, June 1, 2002; 51(6): 1805 - 1814. [Abstract] [Full Text] [PDF]
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J. Huo, R.-H. Luo, S. A. Metz, and G. Li Activation of Caspase-2 Mediates the Apoptosis Induced by GTP-Depletion in Insulin-Secreting (HIT-T15) Cells Endocrinology, May 1, 2002; 143(5): 1695 - 1704. [Abstract] [Full Text] [PDF]
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L. Gunther, P. O. Berberat, M. Haga, S. Brouard, R. N. Smith, M. P. Soares, F. H. Bach, and E. Tobiasch Carbon Monoxide Protects Pancreatic {beta}-Cells From Apoptosis and Improves Islet Function/Survival After Transplantation Diabetes, April 1, 2002; 51(4): 994 - 999. [Abstract] [Full Text] [PDF]
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H. E. Thomas, R. Darwiche, J. A. Corbett, and T. W.H. Kay Interleukin-1 Plus {gamma}-Interferon-Induced Pancreatic {beta}-Cell Dysfunction Is Mediated by {beta}-Cell Nitric Oxide Production Diabetes, February 1, 2002; 51(2): 311 - 316. [Abstract] [Full Text] [PDF]
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L. Carpenter, D. Cordery, and T. J. Biden Inhibition of Protein Kinase C {delta} Protects Rat INS-1 Cells Against Interleukin-1{beta} and Streptozotocin-Induced Apoptosis Diabetes, February 1, 2002; 51(2): 317 - 324. [Abstract] [Full Text] [PDF]
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M. Van de Casteele, B. A. Kefas, Z. Ling, H. Heimberg, and D. G. Pipeleers Specific Expression of Bax-{omega} in Pancreatic {beta}-Cells Is Down-Regulated by Cytokines before the Onset of Apoptosis Endocrinology, January 1, 2002; 143(1): 320 - 326. [Abstract] [Full Text] [PDF]
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M. M.W. Chong, H. E. Thomas, and T. W.H. Kay {gamma}-Interferon Signaling in Pancreatic {beta}-Cells Is Persistent but Can Be Terminated by Overexpression of Suppressor of Cytokine Signaling-1 Diabetes, December 1, 2001; 50(12): 2744 - 2751. [Abstract] [Full Text] [PDF]
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A. Rabinovitch, W. L. Suarez-Pinzon, K. Sooy, K. Strynadka, and S. Christakos Expression of Calbindin-D28k in a Pancreatic Islet {beta}-Cell Line Protects against Cytokine-Induced Apoptosis and Necrosis Endocrinology, August 1, 2001; 142(8): 3649 - 3655. [Abstract] [Full Text] [PDF]
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K. Suk, S. Kim, Y.-H. Kim, K.-A. Kim, I. Chang, H. Yagita, M. Shong, and M.-S. Lee IFN-{{gamma}}/TNF-{{alpha}} Synergism as the Final Effector in Autoimmune Diabetes: A Key Role for STAT1/IFN Regulatory Factor-1 Pathway in Pancreatic {{beta}} Cell Death J. Immunol., April 1, 2001; 166(7): 4481 - 4489. [Abstract] [Full Text] [PDF]
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C. Bonny, A. Oberson, S. Negri, C. Sauser, and D. F. Schorderet Cell-Permeable Peptide Inhibitors of JNK: Novel Blockers of {beta}-Cell Death Diabetes, January 1, 2001; 50(1): 77 - 82. [Abstract] [Full Text]
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N. Giannoukakis, W. A. Rudert, M. Trucco, and P. D. Robbins Protection of Human Islets from the Effects of Interleukin-1beta by Adenoviral Gene Transfer of an Ikappa B Repressor J. Biol. Chem., November 17, 2000; 275(47): 36509 - 36513. [Abstract] [Full Text] [PDF]
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M. R. Heitmeier, M. Arnush, A. L. Scarim, and J. A. Corbett Pancreatic beta -Cell Damage Mediated by beta -Cell Production of Interleukin-1. A NOVEL MECHANISM FOR VIRUS-INDUCED DIABETES J. Biol. Chem., March 30, 2001; 276(14): 11151 - 11158. [Abstract] [Full Text] [PDF]
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