| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Cell Death in the Nonobese Diabetic Mouse Is Fas Independent1
*
Autoimmunity and Transplantation Division, The Walter and Eliza Hall Institute of Medical Research, P.O. Royal Melbourne Hospital, Victoria, Australia; and
Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO 63104
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
cells
may be important in the development of autoimmune diabetes in the
nonobese diabetic (NOD) mouse. To address this, pancreatic islets from
NOD mice were analyzed by flow cytometry to directly identify which
cells express Fas and Fas ligand (FasL) ex vivo and after in vitro
culture with cytokines. Fas expression was not detected on cells
isolated from young (35 days) NOD mice. In vitro, incubation of NOD
mouse islets with both IL-1 and IFN-
was required to achieve
sufficient Fas expression and sensitivity for islets to be susceptible
to lysis by soluble FasL. In islets isolated from older (
125 days)
NOD mice, Fas expression was detected on a limited number of cells
(1–5%). FasL was not detected on cells from either NOD or
Fas-deficient MRLlpr/lpr islets. Also, both NOD
and MRLlpr/lpr islets were equally susceptible
to cytokine-induced cell death. This eliminates the possibility that
cytokine-treated murine islet cells commit "suicide" due to
simultaneous expression of Fas and FasL. Last, we show that NO is not
required for cytokine-induced Fas expression and Fas-mediated apoptosis
of islet cells. These findings indicate that cells can be killed by
Fas-dependent cytotoxicity; however, our results raise further doubts
about the clinical significance of Fas-mediated cell destruction
because few Fas-positive cells were isolated immediately before the
development of diabetes. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
or
combinations of IFN- and TNF-
or IL-1 in various cells lines. The
ligand for Fas (FasL)3
is sufficient for induction of apoptosis of Fas-expressing cells and is
more effective as a membrane-bound protein although it is functional in
soluble form. The Fas-FasL interaction leads to activation of an
intracellular signaling pathway that results in cell death by
caspase-dependent mechanisms (reviewed in Refs. 1 and
2).
Type 1 insulin-dependent diabetes mellitus (IDDM) is a T cell-mediated
disease in which recent evidence, mostly from the nonobese diabetic
(NOD) mouse model, suggests that pancreatic cells are destroyed by
apoptotic mechanisms (3, 4, 5). It has been suggested that
Fas may play a role in cell apoptosis in IDDM both in humans and in
the NOD mouse. Much of the evidence for this comes from in vitro
studies with isolated human or mouse islets of Langerhans. IL-1 has
been shown to up-regulate Fas expression on cells, resulting in
apoptosis after the addition of agonist anti-Fas Ab
(6, 7, 8). IL-1 in combination with IFN- has also been
shown to up-regulate inducible NO synthase (iNOS) expression in rodent
and human cells, leading to NO-dependent islet dysfunction
(9, 10). One study suggested that NO is able to regulate
Fas expression on human cells, and not on other cell types within
the islet (11), providing a potential mechanism for
selective cell destruction in IDDM.
The spontaneous mouse mutant lpr (lymphoproliferation)
lacks functional Fas expression due to a mutation in the gene
(12). When this mutation is crossed onto a NOD genetic
background (NODlpr/lpr), the mice do not develop
diabetes or insulitis, either spontaneously or in T cell adoptive
transfer experiments, suggesting that Fas expression is required for
disease (13, 14). However, recent reports suggest that Fas
plays only a minor role in cell destruction, as Fas-deficient islet
grafts are not protected from diabetogenic T cells (15, 16). Immunohistochemical staining of human pancreas sections
revealed Fas expression in islets of diabetic but not normal subjects,
with FasL expression on infiltrating T cells, suggesting a mechanism
for cell destruction (11). In mice, Chervonsky et al.
found that Fas expression on islet cells of old NOD mice (12 wk) was
up-regulated after transfer of a diabetogenic T cell clone; however,
Fas expression in the spontaneous NOD mouse model was not examined
(13). Also, Fas expression was found on cells from
syngeneic islets grafted into diabetic NOD mouse recipients, and this
correlated with expression of inflammatory cytokines (17).
However, the data in all of these studies using two-color
immunohistochemical staining are difficult to interpret, especially
when looking for Fas, FasL, and apoptosis in infiltrated islets where
staining on the infiltrating cells may interfere with the cell
staining. Flow cytometry is a more powerful technique for these
studies, where specific cell types can be easily identified.
Although a large body of in vitro evidence suggests that Fas may
participate in cell destruction, it remains to be determined
whether Fas is involved in the natural progression of diabetes in the
NOD mouse, which has different mechanisms of disease from adoptive
transfer models. Furthermore, the lpr mutation has multiple
effects such as lymphadenopathy, constitutive up-regulation of FasL,
and dysregulation of T cell populations (18, 19), which
makes it difficult to conclude that Fas is required for the development
of diabetes in NOD mice. Additionally, some reagents that have been
used to examine FasL expression have been shown to be nonspecific
(20), making it difficult to confirm that Fas-FasL
participates in cell destruction during the development of
diabetes.
In this study, we have used techniques to directly address some of
these problems with the literature. We have defined Fas expression on
cells from NOD mice of different ages by flow cytometry, where
cells can easily be distinguished from other islet cells and
infiltrating cells. We present evidence indicating that up-regulation
of Fas expression on the majority of NOD mouse cells does not
correlate with the onset of autoimmune diabetes, although expression on
a few cells suggests that some Fas-mediated lysis may occur. We have
also used MRLlpr/lpr islets to demonstrate that
killing of cells by Fas and FasL coexpression is not important in
cytokine-induced damage.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
NOD/Lt and MRLlpr/lpr mice were housed in the animal facility at the Walter and Eliza Hall Institute of Medical Research. The prevalence of diabetes of NOD/Lt mice at our institution is 70–75% of females and 15% of males by 300 days.
Recombinant murine IFN- (used at 100 U/ml) was obtained from
Genentech (South San Francisco, CA), and recombinant human IL-1
(10–100 U/ml) from Genzyme (Cambridge, MA). Soluble human FasL (100
ng/ml) was kindly provided by Dr. J. Tschopp (University of Lausanne,
Lausanne, Switzerland).
NG-monomethyl-L-arginine
(NMMA) (3 mM) and sodium nitroprusside (SNP) (100 µM) were purchased
from Sigma (St. Louis, MO). Z-VAD-fmk (1 µg/ml) was provided by Dr.
G. Vairo (Walter and Eliza Hall Institute)
Islet isolation
Islets of Langerhans were isolated from mice by pancreas digestion with collagenase P (Boehringer Mannheim, Mannheim, Germany) followed by a BSA density gradient (First Link, U.K.), as previously described (21, 22). Islets were hand picked and cultured at 37°C in 5% CO2 in CMRL Medium-1066 (Life Technologies, Gaithersburg, MD) supplemented with 10% FCS and antibiotics.
Flow cytometry
For flow cytometric analysis, islets were either stained on the
day of isolation or cultured with cytokines in DMEM containing 2.5 mM
glucose, 10% FCS, and antibiotics. This glucose concentration was used
to allow for detection of cells on the basis of high
autofluorescence due to intracellular flavin adenine dinucleotide
(23). There is no evidence that expression of cell-surface
molecules is altered by incubation of islets in low glucose, and we
have used the islet endocrine cell marker A2B5 (24) on
islets incubated in normal (10 mM) glucose to show this (not shown). We
have examined the cell types present in the autofluorescent populations
of islets analyzed by flow cytometry. Autofluorescent cells were sorted
(FACStar+; Becton Dickinson, Mountain View, CA),
fixed onto slides by cytospin, and stained by direct
immunofluorescence. Eighty to 90% of the autofluorescent cells stained
positive for insulin, and all were positive for A2B5, suggesting that
the cells are all of islet endocrine nature (not shown).
Islets for staining were dispersed into single cells with 0.2% trypsin (Calbiochem, La Jolla, CA), 10 mM EDTA in HBSS, and allowed to recover in culture medium for 1 h before staining with mAb. Islet cell suspensions were stained using standard procedures. Cells were finally washed and resuspended in balanced salt solution with 2% FCS and 1 µg/ml propidium iodide to stain dead cells. Analysis was performed on a FACScan (Becton Dickinson). Hamster anti-mouse Fas (Jo2; PharMingen, San Diego, CA) was used for analysis of surface Fas expression, with hamster anti-human B anti-human 2 as an isotype control Ab for background staining. This was followed by biotinylated anti-hamster Ig (PharMingen) and PE-conjugated streptavidin (Caltag Laboratories, Burlingame, CA).
Apoptosis assay
Quantitation of apoptosis was determined according to the method
of Nicoletti et al. (25). After 3–5 days in culture with
cytokines, islets were dispersed into a single-cell suspension with
trypsin, followed by recovery in complete medium at 37°C for 30 min.
The cells were then resuspended in a hypotonic fluorochrome solution
(50 µg/ml propidium iodide, 0.1% sodium citrate, 0.1% Triton X-100)
and kept overnight in the dark at 4°C. Cells were then analyzed on
the flow cytometer. This assay measures fragmented nuclei, and
therefore greater than one fragment can be derived from one apoptotic
cell. The level of induced apoptosis over spontaneous was determined by
the formula 100 x (% number of apoptotic cells with
cytokine - % number of background apoptotic cells)/(100 -
% number of background apoptotic cells). This formula allows results
from experiments with different levels of basal apoptosis to be
combined. Data from at least three individual experiments were pooled.
All experiments were conducted on islets isolated from 
40-day-old
NOD mice.
Nitrite determination
Nitrite was detected in the cultures by mixing 50 µl supernatant with 50 µl Griess reagent (26). Absorbances were read at 540 nm, and nitrite concentration was calculated off a standard curve with sodium nitrite.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
cells following exposure
to cytokines
To confirm that Fas expression can be induced by cytokines on
murine cells, islets isolated from young (40 days) NOD mice were
cultured in vitro with combinations of the cytokines IFN- and
IL-1. Fas expression was then examined on cells by flow
cytometry. Alone, IL-1 (10 U/ml) stimulates low levels of Fas
expression on NOD cells (Fig. 1
A), and this is enhanced by
the addition of 100 U/ml IFN- (Fig. 1B). IFN- alone
did not significantly up-regulate cell expression of Fas (Fig. 1A) when compared with either untreated islets or islets
isolated from MRLlpr/lpr mice, which are
deficient in Fas (Fig. 1). Fas expression was induced by IL-1 and IL-1
plus IFN- after a 24-h incubation with cytokines and was not further
increased with longer incubations (not shown). These data are
consistent with the findings of Yamada et al., which showed Fas
up-regulation on murine islets by IL-1 (6). The finding
that cytokines up-regulate Fas is not unique to NOD cells. We have
also analyzed Fas expression after cytokine treatment of islets
isolated from C57BL/6 mice with the same results (not shown).
|
To demonstrate that Fas expression on cells is functional,
islets were incubated with cytokines plus soluble FasL (sFasL)
(27), and apoptosis was measured by propidium iodide
staining of fragmented nuclei. Concentrations of sFasL between 1 and
1000 ng/ml were examined, and sFasL-induced killing was maximal at 100
ng/ml (not shown). This concentration was used for all subsequent
experiments. IFN- (100 U/ml) and IL-1 (10 U/ml) induced a 2- to
4-fold increase in DNA fragmentation of islet cells over background
after a 4-day culture (Fig. 2,
A and B). This cell death was attenuated by the
iNOS inhibitor NMMA (3 mM), indicating that cell death induced by
cytokines (in the absence of sFasL) is due to the increased production
of NO (Fig. 2). In the presence of IFN- and IL-1 (to up-regulate
Fas) and sFasL, the level of cell death increased 8- to 10-fold over
background, demonstrating that Fas expression induced by cytokines is
functional. When NMMA was added with cytokines and sFasL,
cytokine-induced NO-mediated cell death was inhibited, but
FasL-mediated death was still observed (Fig. 2A). Alone,
sFasL did not induce islet cell death compared with untreated islets,
indicating the absence of functional Fas expressed constitutively on
the islet cells of young NOD mice. FasL-mediated death was inhibited by
the caspase inhibitor Z-VAD-fmk, demonstrating that the islet cells are
killed by a caspase-dependent mechanism. Fas-deficient
MRLlpr/lpr islets were used as a control for
Fas-independent cytokine-induced cell death (Fig. 2C). These
islets were found to be as susceptible to NO-dependent cell death as
NOD islets, but this apoptosis did not increase upon addition of sFasL
to the lpr islets, as expected.
|
Cytokines induce the up-regulation of iNOS and the production of
NO by cells in vitro, and it has been demonstrated that NO, either
induced by cytokines or exogenously supplied by donor compounds, is
able to prime human cells for Fas-mediated killing by up-regulation
of Fas (11). The mechanism by which NO modulates Fas
expression by human islets is unknown. We have examined whether NO
could regulate murine cell expression of Fas and directly modulate
Fas-sFasL-mediated islet cell killing by analyzing the effects of
endogenously produced or exogenously added NO on both Fas expression
and FasL-mediated islet cell death. To confirm that cytokines stimulate
NO production by NOD mouse islets, we show in Fig. 3 that incubation with IL-1 plus IFN-
for 24 h results in the increased production of nitrite and
nitrite production is attenuated by NMMA. To determine whether NO
participates in cytokine regulation of Fas expression, the effect of
NMMA on IL-1 plus IFN--induced Fas expression was examined by flow
cytometry.
|
A). Also, the exogenous
addition of NO using the NO donor compound SNP does not induce Fas
expression on cells (Fig. 4B), despite detection of
222 ± 98.7 pmol nitrite in the culture supernatant after 24-h
incubation with SNP. These results indicate that NO does not
participate in up-regulation of Fas. Thus, while NO is able to cause
damage to the islets, it does not appear to be involved in Fas-mediated
destruction of NOD islets.
|
(after 4-day culture), again showing that
NO is unable to affect Fas expression (Fig. 4
C). The fact
that a high level of exogenous NO is unable to induce cell death
suggests that NO production by islet cells themselves is required for
damage to occur.
FasL is not detectable on cells of NOD mice
Several groups have reported expression of FasL on human and mouse
islets, proposing direct self-killing as a mechanism for cell
destruction (8, 28). These studies have been conducted
using immunohistochemical techniques with antisera that may not be
specific for FasL (20). We have analyzed FasL expression
on cells by flow cytometry, which is a more sensitive method, using
MFL-3 (hamster anti-FasL Ab), which has been shown to be specific
for FasL (29). Islets were isolated from NOD mice of
between 40 and 120 days of age and stained for FasL expression. As a
positive control, islets from rat insulin promoter (RIP)-FasL
transgenic animals were stained at the same time (30).
While a high level of FasL staining was consistently detected on
RIP-FasL transgenic cells, indicating that the process of islet
isolation does not prevent FasL detection, we did not observe FasL
staining on cells isolated from nontransgenic littermates or from
either young or old NOD mice (Fig. 5).
FasL was also not detected by immunohistochemical analysis (L. Stephens
and P. Augstein, unpublished observations). In addition, we were unable
to detect FasL staining after incubation of islets with the cytokines
IFN- and IL-1 (not shown).
|
cells, we cannot
rule out the possibility that low levels, undetectable by flow
cytometry, are expressed, particularly as detection of FasL on
activated T cells is difficult (not shown). To further explore this
possibility, we analyzed cytokine-induced cell death in islets
deficient in Fas expression. MRLlpr/lpr and NOD
islets were equally susceptible to cytokine-mediated cell death (Fig. 2C). If FasL were constitutively expressed on cells and
Fas were induced by cytokines, the level of cell death in the normal
NOD islets with cytokines alone would be expected to be higher than
that seen in the MRLlpr/lpr islets. This is not
the case. Thus direct autolysis of cells by Fas/FasL cannot occur
in NOD mouse islets. We have also repeated this experiment with
NODlpr/lpr islets with the same result (not
shown). As expected, cell death of lpr islets was not
enhanced by the addition of sFasL (Fig. 2C).
Relevance of Fas-mediated apoptosis of cells in vivo
These results and those of others have clearly demonstrated that
Fas expression is induced on murine cells by cytokines in vitro.
However, the relevance of Fas expression to the induction of autoimmune
diabetes in the NOD mouse is unclear. Evidence for the role of Fas in
IDDM comes primarily from NODlpr/lpr mice, which
do not develop insulitis or diabetes (13, 14, 15). In these
NODlpr/lpr experiments, the importance of Fas
expression on cells and Fas-mediated cell death is unclear. In
NOD mice, IFN- and IL-1 are expressed within the islet during the
development of autoimmune diabetes. To examine the role of cytokines
and Fas during the progression of islet insulitis and diabetes
development, we assessed Fas expression on cells of NOD mice at
various stages of disease.
A group of 60 female NOD mice was set aside to monitor diabetes
incidence and Fas expression on islets. Five mice per age group were
killed for islet isolation at 120, 125, 130, and 135 days of age. Blood
glucose measurements (range 5.2–9.3 mmol/L) were determined before
islet isolation from individual mice. Although these mice were
nondiabetic, some had elevated blood glucose measurements that suggests
they were close to diabetes, and many other mice in the cohort were
also progressing to diabetes. Additionally, the number of islets
isolated from each mouse was determined (between 17 and 370 per mouse),
and the mice with low islet numbers may have been prediabetic,
undergoing cell destruction and islet loss. Islets were stained for
Fas expression, and levels were compared with an isotype control Ab,
Fas staining on Fas-deficient MRLlpr/lpr islets,
and on islets from 35-, 70-, and 90-day-old NOD mice. A high level of
Fas expression was detected on infiltrating T cells. Even though there
were relatively few infiltrating cells in the young islets, the level
of Fas staining on T cells did not change as the mice became older and
developed more severe insulitis (Fig. 6).
T cell Fas staining was used as an internal positive control and shows
that Fas expression is maintained during the islet isolation
procedure.
|
B. Fas expression was only
observed on 1–5% of the cells from mice of 125 days of age, and
the level of staining was not as high as that seen after in vitro
culture with cytokines. This is more apparent when the histograms of
cell Fas staining are overlaid with that of isotype control
staining (Fig. 6B). Diabetes incidence in the mice not
killed for islet isolation (n = 47) during the
experiment was 75% at 135 days of age (Fig. 6C). This
indicates that we analyzed Fas expression on islets during a period
when many of the mice are developing diabetes. Islets from mice 70–90
days of age were also studied with similar results (not shown).
Therefore, unless Fas-expressing cells are very short-lived and not
detected in this experiment, it is unlikely that a large number of
cells express Fas before the onset of autoimmune diabetes, raising
doubt whether Fas-mediated lysis of cells plays an important role
in cell destruction in NOD mice. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
cells have
generally been restricted to immunohistochemical analysis, which can be
difficult to interpret due to the presence of many cell types within
NOD islets on histological sections. We have avoided this problem by
using alternative techniques that enable us to identify specific cell
types within the islet expressing Fas and FasL. We have found that
while Fas is expressed at high levels by infiltrating lymphocytes, only
a limited number cells from prediabetic NOD mice express Fas at low
levels. It has been suggested that nondestructive insulitis progresses
throughout the life of the NOD mouse, but that cell destruction is
rapid and occurs immediately before the onset of diabetes
(31). If this were the case and Fas-mediated lysis were a
mechanism for cell death, we would expect to see large numbers of
Fas-positive cells in mice close to the time that diabetes occurs,
which we did not observe.
Alternative mechanisms of cell destruction include soluble factors
such as cytokines and NO, as well as perforin released by CTL. Perforin
and FasL are believed to be the major mechanisms of killing by CTL. It
is known that perforin plays a role in the effector phase of cell
destruction as diabetes is reduced and delayed in perforin knockout
mice in the presence of infiltrate similar to that of wild-type mice
(32). However, it is likely that a combination of factors
is important for diabetes to occur in the NOD mouse. We have shown that
class I MHC expression on cells is required for insulitis, also
implicating CD8+ T cells in initiation of disease
(33, 34).
Our data suggest that the cells we isolated from NOD mice may not
have been exposed to high levels of IL-1 in vivo because levels of Fas
expression achieved in vitro by addition of IL-1 and IFN- were not
observed on cells from infiltrated islets. IFN- is present in the
infiltrated islet (35) at sufficient levels to uniformly
up-regulate class I MHC, for example. We have shown that NOD mice of
increasing age and degree of insulitis have gradually increasing levels
of class I MHC expression on cells and that mice with cells
unresponsive to IFN- have no such class I MHC up-regulation, despite
the presence of insulitis (22). This shows that the rise
in class I MHC seen on cells is due to local IFN-. Both resident
and infiltrating macrophages are thought to produce IL-1
(36, 37, 38). As both IL-1 and IFN- are required for in
vitro up-regulation of functional Fas, either IL-1 concentrations may
be limiting or factors which inhibit IL-1-iduced Fas expression may be
present within the islet. It is also possible that cells in close
proximity to macrophages have up-regulated Fas expression, and that
these cells may be rapidly destroyed by FasL-bearing T cells. This
could explain the low numbers of Fas-positive cells observed in NOD
islets.
We have demonstrated that cell "suicide" due to cell
expression of both Fas and FasL does not occur. cells did not
appear to express FasL while mice expressing FasL transgenically
displayed high levels of FasL. These results suggested that FasL
expression was maintained throughout the islet isolation procedure and
we were not missing FasL expression on normal NOD cells due to
technical problems. We cannot rule out the possibility that FasL is
expressed at levels undetectable by flow cytometry, although our data
show that such levels are not functional. In the absence of soluble
FasL, IL-1 and IFN- kill MRLlpr/lpr islets to
the same extent as normal NOD islets in a NO-dependent fashion. Thus,
it is unlikely that FasL is constitutively expressed on cells and
the cells are lysed due to up-regulation of Fas by cytokines, as has
been suggested by others (8). FasL is most likely
expressed on activated T cells within the infiltrate.
IFN- is required for Fas-mediated lysis of islets in vitro. IL-1 and
sFasL do not cause islet cell death in vitro, even though IL-1 is able
to up-regulate low levels of Fas expression on cells. IFN- on
its own has little effect on Fas expression, but in combination with
IL-1 leads to enhanced Fas up-regulation in vitro. It is possible that
the high levels of Fas induced by IL-1 plus IFN- are required for
activated cell death. Humans with a mutation in the Fas gene have the
rare disease autoimmune lymphoproliferative syndrome, despite having
one normal copy of Fas. It has been suggested that the mutant gene acts
as a dominant negative receptor (39, 40); however, it is
also possible that insufficient levels of Fas are expressed with only
one functional copy of the gene. Although no phenotype is observed in
mice heterozygous for the lpr mutation, decreased killing of
cells from these mice has been observed in vitro (41). Our
data support the possibility that high levels of Fas are required for
killing. In addition to its effect on Fas expression, IFN- is known
to regulate caspase-1 expression (42, 43), and may also
activate other intracellular cell death signaling molecules. Our data
show that caspases are important mediators of cell destruction, as
inhibition of caspases with Z-VAD-fmk prevented cytokine-induced
apoptosis due to Fas-FasL interaction.
Stassi et al. reported that functional Fas expression on human
cells is induced by NO (11). This has also been shown in
other cell types including vascular smooth muscle cells
(44), and NO has been shown to regulate expression of
several other genes such as IL-8 (45) and superoxide
dismutase (46) by various mechanisms. However NO has also
been shown to inhibit Fas-mediated apoptosis by suppressing caspase-3
activation in a cyclic GMP-independent manner (47, 48, 49),
demonstrating the wide range of effects NO has on cells, possibly
depending on its concentration (50). We found that NO had
no effect on either Fas expression or Fas-mediated apoptosis of islet
cells from NOD mice. The differences between our result in mice and
that of Stassi et al. in human islets suggests a species difference in
the effects of cytokines and cell death-inducing agents on islets. We
also found that while cytokine-induced NO production was toxic to
islets in vitro, exogenously supplied NO was not, even at higher
concentrations than those produced by islet cells. cells produce NO
in response to cytokines (37). Also, transgenic mice
expressing iNOS in their cells develop insulin-dependent diabetes
(51). Therefore, it is likely that NO production by the
islet cells themselves is required for toxicity.
We have also presented evidence that Fas is not constitutively
expressed on cells of young NOD mice before the onset of insulitis
and that as insulitis progresses only a few cells express Fas. The
lack of Fas staining on cells from young mice suggests that
Fas-mediated lysis of cells is probably not involved in initiation
of disease. The patchy, low level expression on cells from mice
with insulitis implies that Fas plays only a minor role in the effector
phase of disease. Fas-deficient NODlpr/lpr fetal
pancreas grafts are unable to reverse diabetes in NOD mice as they
undergo T cell-mediated destruction (15). These grafts
appeared slightly less damaged than wild-type NOD grafts, suggesting
that there may be some Fas-mediated cell destruction in autoimmune
diabetes. Therefore, it is possible that the few Fas positive cells
within the islets of old NOD mice are playing a role in cell
destruction, in addition to perforin-dependent lysis, which has been
shown in knockout animals to be required for the effector phase of
autoimmune diabetes. It is likely that other mechanisms of cell
destruction exist, including Fas, TNFR1 (52), and nitric
oxide-mediated damage. The effects of making any one of these deficient
may be difficult to observe in the presence of perforin.
The role of Fas-mediated destruction of cells is very topical, with
many seemingly inconsistent findings being published. Of these
findings, it is clear that Fas-deficient
NODlpr/lpr mice to not develop pathology
(13, 14, 15). However, using NODlpr/lpr
pancreas grafts, it has been suggested that this absence of disease may
be due to other immune defects in the lpr mouse (15, 16). The published data on Fas expression by cells
(11, 13, 17) are consistent with our flow cytometry data
and reveal at best only a limited number of Fas-positive cells.
Analysis of FasL expression by cells (8) was performed
using antisera of doubtful specificity, and there is little functional
evidence for cell FasL expression. As these studies were performed
with human islets, it is possible that the data differ from ours due to
species differences; however, there is no convincing demonstration in
the literature of FasL expression by cells.
In conclusion, we have shown that there is a limited amount of Fas
expression in cells from NOD mice. FasL expression on cells is
undetectable both by flow cytometry and functional analysis using
islets from MRLlpr/lpr mice. This is contrary to
other studies using two-color immunohistochemistry and co-localization
on serial sections as methods for Fas and FasL detection. While we do
not yet have a complete understanding of the role of Fas and FasL in
the development of IDDM, more recent data, including ours, makes a
major role of Fas-FasL interactions in cell death uncertain. Our
inability to find Fas expression on the majority of cells does not
equivocally rule out a role for Fas in cell death in NOD mice. This
will be directly tested by several groups making transgenic NOD mice
with FasL-insensitive cells.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Thomas W. H. Kay, Autoimmunity and Transplantation Division, Walter and Eliza Hall Institute, P.O. Royal Melbourne Hospital, Victoria 3050, Australia. E-mail address:
3 Abbreviations used in this paper: FasL, Fas ligand; NOD, nonobese diabetic; IDDM, insulin-dependent diabetes mellitus; iNOS, inducible NO synthase; SNP, sodium nitroprusside; NMMA, NG-monomethyl-L-arginine; sFasL, soluble FasL; RIP, rat insulin promoter.
Received for publication March 4, 1999. Accepted for publication May 18, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-cell death responsible for the development of IDDM in the nonobese diabetic (NOD) mouse. Diabetes 46:750.[Abstract]
cell apoptosis in T cell-mediated autoimmune diabetes. Proc. Natl. Acad. Sci. USA 94:213.-cell destruction in pancreatic islets on NOD mice with spontaneous and cyclophosphamide-accelerated diabetes. Diabetologia 41:1381.[Medline]
-cells sorted by flow-cytometry and cultured in vitro. Transpl. Proc. 27:3271.[Medline]
and Fas ligation. Diabetes 47:727.[Abstract]
cells. Diabetologia 39:875.[Medline]
cell death in diabetes: a minor role for CD95. Proc. Natl. Acad. Sci. USA 95:13818. cells detected in accelerated diabetes of NOD mice: no role of Fas-Fas ligand interaction in autoimmune diabetes. Eur. J. Immunol. 29:455.[Medline]
-cell destruction in NOD mice correlates with Fas (CD95) expression on -cells and proinflammatory cytokine expression in islets. Diabetes 48:21.[Abstract]
action on pancreatic cells causes class I MHC upregulation but not diabetes. J. Clin. Invest. 102:1249.[Medline]
cells induces a granulocytic infiltration but does not confer immune privilege upon islet allografts. Proc. Natl. Acad. Sci. USA 94:3943.2-microglobulin transgene expression restores insulitis, but not diabetes, in 2-microglobulin null nonobese diabetic mice. J. Immunol. 157:3688.[Abstract]
gene expression in pancreatic islet-infiltrating mononuclear cells correlates with autoimmune diabetes in nonobese diabetic mice. J. Immunol. 154:4874.[Abstract]
and nitric oxide synthase in pancreatic islets isolated from female and male nonobese diabetic mice. Diabetologia 38:153.[Medline]
cell function by inducing cell expression of inducible nitric oxide synthase. J. Exp. Med. 181:559.-induced apoptosis in STAT1-null cells due to low constitutive levels of caspases. Science 278:1630. cells develop insulin-dependent diabetes without insulitis. J. Biol. Chem. 273:2493. cell toxicity as an effector pathway in autoimmune diabetes. J. Immunol. 162:4598.This article has been cited by other articles:
|
|
 |
|
  M. D. McKenzie, E. M. Carrington, T. Kaufmann, A. Strasser, D. C.S. Huang, T. W.H. Kay, J. Allison, and H. E. Thomas Proapoptotic BH3-Only Protein Bid Is Essential For Death Receptor-Induced Apoptosis of Pancreatic {beta}-Cells Diabetes, May 1, 2008; 57(5): 1284 - 1292. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. S. Mohamood, M. L. Guler, Z. Xiao, D. Zheng, A. Hess, Y. Wang, H. Yagita, J. P. Schneck, and A. R. A. Hamad Protection from Autoimmune Diabetes and T-Cell Lymphoproliferation Induced by FasL Mutation Are Differentially Regulated and Can Be Uncoupled Pharmacologically Am. J. Pathol., July 1, 2007; 171(1): 97 - 106. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Estella, M. D. McKenzie, T. Catterall, V. R. Sutton, P. I. Bird, J. A. Trapani, T. W. Kay, and H. E. Thomas Granzyme B-Mediated Death of Pancreatic {beta}-Cells Requires the Proapoptotic BH3-Only Molecule Bid. Diabetes, August 1, 2006; 55(8): 2212 - 2219. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. D McKenzie, N. L Dudek, L. Mariana, M. M. Chong, J. A Trapani, T. W. Kay, and H. E Thomas Perforin and Fas induced by IFN{gamma} and TNF{alpha} mediate beta cell death by OT-I CTL Int. Immunol., June 1, 2006; 18(6): 837 - 846. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Allison, H. E. Thomas, T. Catterall, T. W. H. Kay, and A. Strasser Transgenic Expression of Dominant-Negative Fas-Associated Death Domain Protein in {beta} Cells Protects against Fas Ligand-Induced Apoptosis and Reduces Spontaneous Diabetes in Nonobese Diabetic Mice J. Immunol., July 1, 2005; 175(1): 293 - 301. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Askenasy, E. S. Yolcu, I. Yaniv, and H. Shirwan Induction of tolerance using Fas ligand: a double-edged immunomodulator Blood, February 15, 2005; 105(4): 1396 - 1404. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J M Mellado-Gil and M Aguilar-Diosdado High glucose potentiates cytokine- and streptozotocin-induced apoptosis of rat islet cells: effect on apoptosis-related genes J. Endocrinol., October 1, 2004; 183(1): 155 - 162. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Augstein, P. Heinke, E. Salzsieder, S. Berg, R. Rettig, C. Salzsieder, and L. C. Harrison Fas Ligand Down-Regulates Cytokine-Induced Fas Receptor Expression on Insulinoma (NIT-1), But Not Islet Cells, from Autoimmune Nonobese Diabetic Mice Endocrinology, June 1, 2004; 145(6): 2747 - 2752. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. M. W. Chong, Y. Chen, R. Darwiche, N. L. Dudek, W. Irawaty, P. Santamaria, J. Allison, T. W. H. Kay, and H. E. Thomas Suppressor of Cytokine Signaling-1 Overexpression Protects Pancreatic {beta} Cells from CD8+ T Cell-Mediated Autoimmune Destruction J. Immunol., May 1, 2004; 172(9): 5714 - 5721. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. Christen, R. Darwiche, H. E. Thomas, T. Wolfe, E. Rodrigo, A. Chervonsky, R. A. Flavell, and M. G. von Herrath Virally Induced Inflammation Triggers Fratricide of Fas-Ligand-Expressing {beta}-Cells Diabetes, March 1, 2004; 53(3): 591 - 596. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
|
|
R. Darwiche, M. M. W. Chong, P. Santamaria, H. E. Thomas, and T. W. H. Kay Fas Is Detectable on {beta} Cells in Accelerated, But Not Spontaneous, Diabetes in Nonobese Diabetic Mice J. Immunol., June 15, 2003; 170(12): 6292 - 6297. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. G. Silva, N. Petrovsky, L. Socha, R. Slattery, P. Gatenby, and B. Charlton Mechanisms of Accelerated Immune-Mediated Diabetes Resulting from Islet {beta} Cell Expression of a Fas Ligand Transgene J. Immunol., May 15, 2003; 170(10): 4996 - 5002. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Hayashi and D. L. Faustman Role of Defective Apoptosis in Type 1 Diabetes and Other Autoimmune Diseases Recent Prog. Horm. Res., January 1, 2003; 58(1): 131 - 153. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Nakayama, M. Nagata, H. Yasuda, K. Arisawa, R. Kotani, K. Yamada, S. A. Chowdhury, S. Chakrabarty, Z. Z. Jin, H. Yagita, et al. Fas/Fas Ligand Interactions Play an Essential Role in the Initiation of Murine Autoimmune Diabetes Diabetes, May 1, 2002; 51(5): 1391 - 1397. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
|
|
K. Maedler, G. A. Spinas, R. Lehmann, P. Sergeev, M. Weber, A. Fontana, N. Kaiser, and M. Y. Donath Glucose Induces {beta}-Cell Apoptosis Via Upregulation of the Fas Receptor in Human Islets Diabetes, August 1, 2001; 50(8): 1683 - 1690. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. I. Darville and D. L. Eizirik Cytokine Induction of Fas Gene Expression in Insulin-Producing Cells Requires the Transcription Factors NF-{kappa}B and C/EBP Diabetes, August 1, 2001; 50(8): 1741 - 1748. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. Christen, T. Wolfe, U. Mohrle, A. C. Hughes, E. Rodrigo, E. A. Green, R. A. Flavell, and M. G. von Herrath A Dual Role for TNF-{{alpha}} in Type 1 Diabetes: Islet-Specific Expression Abrogates the Ongoing Autoimmune Process When Induced Late but Not Early During Pathogenesis J. Immunol., June 15, 2001; 166(12): 7023 - 7032. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
S. DeFranco, S. Bonissoni, F. Cerutti, G. Bona, F. Bottarel, F. Cadario, A. Brusco, G. Loffredo, I. Rabbone, A. Corrias, et al. Defective Function of Fas in Patients With Type 1 Diabetes Associated With Other Autoimmune Diseases Diabetes, March 1, 2001; 50(3): 483 - 488. [Abstract] [Full Text]
|
|
|
|
|
|
J. Allison, H. Thomas, D. Beck, J. L. Brady, A. M. Lew, A. Elefanty, H. Kosaka, T. W. Kay, D. C. S. Huang, and A. Strasser Transgenic overexpression of human Bcl-2 in islet {beta} cells inhibits apoptosis but does not prevent autoimmune destruction Int. Immunol., January 1, 2000; 12(1): 9 - 17. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Bonny, A. Oberson, M. Steinmann, D. F. Schorderet, P. Nicod, and G. Waeber IB1 Reduces Cytokine-induced Apoptosis of Insulin-secreting Cells J. Biol. Chem., May 26, 2000; 275(22): 16466 - 16472. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
