From the Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104
Received for publication, October 6, 2000, and in revised form, November 14, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Viral infection is one environmental factor that
may initiate Insulin-dependent diabetes mellitus is an autoimmune
disease characterized by the selective destruction of insulin-secreting Double-stranded RNA (dsRNA), formed during viral replication, is an
active component of a viral infection that triggers antiviral responses
in infected cells (8). The antiviral response includes the expression
of type 1 interferons (9), nitric oxide production (10), macrophage
IL-1 release (11), and a general inhibition of protein translation (12,
13). Similar to a viral infection, the synthetic dsRNA molecule
polyinosinic-polycytidylic acid (poly(I-C)) also activates these
antiviral responses (14-16). In vivo, administration of
poly(I-C) to diabetes-resistant and -prone BioBreeding rats results in
the induction and acceleration of diabetes, respectively (17, 18).
Although viral infection has been implicated in the development of
autoimmune diabetes, the response of islets and specifically Materials--
CMRL-1066 tissue culture medium,
L-glutamine, penicillin, streptomycin, and rat recombinant
IFN- Islet Isolation and Culture--
Islets were isolated from male
Harlan Sprague-Dawley rats by collagenase digestion as described
previously (21). Following isolation, islets were cultured overnight in
complete CMRL-1066 (CMRL-1066 containing 2 mM
L-glutamine, 10% heat-inactivated fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin) under an atmosphere
of 95% air and 5% CO2 at 37 °C. Prior to each experiment, islets were washed 3 times in complete CMRL-1066, counted,
and then cultured for an additional 3 h at 37 °C. Experiments were initiated by the addition of poly(I-C) and IFN-
The Islet Isolation Core Facility at Washington University School of
Medicine and the Diabetes Research Institute at the University of Miami
provided human islets. Isolated human islets were cultured for 3 days
at 37 °C in complete CMRL-1066 prior to experimentation. Where
indicated, human islets were pretreated for 30 min with IRAP prior to
incubation with cytokines and poly(I-C).
Purification of Insulin Secretion--
Islets (220/ml of complete CMRL-1066)
were cultured for 40 h with the indicated concentrations of
poly(I-C), rat IFN- Islet Viability--
Islets (25/500 µl of complete CMRL-1066)
were cultured for 96 h in 24-well microtiter plates with the
indicated concentrations of poly(I-C), IFN- Western Blot Analysis--
Rat or human islets (120/400 µl of
complete CMRL-1066), cultured for the indicated times with poly(I-C),
rat or human IFN- Northern Blot Analysis--
Rat islets (900/3 ml complete
CMRL-1066) were cultured for 18 h at 37 °C with poly(I-C), rat
IFN- Nitrite Determination--
Nitrite production was determined by
mixing 50 µl of culture medium with 50 µl of Griess reagent (29).
The absorbance at 540 nm was measured, and nitrite concentrations were
calculated from a sodium nitrite standard curve.
RT-PCR--
RT-PCR analysis of IL-1 Immunoprecipitation and Immunofluorescence--
For
immunoprecipitations, rat islets (500 islets/ml of methionine-deficient
minimum Eagle's medium) were treated for 18 h with poly(I-C) + IFN-
Immunohistochemistry was performed as described previously (20). In
brief, islets were isolated, dispersed into individual cells by trypsin
treatment as stated above, and centrifuged onto slides. The cells were
fixed in 4% paraformaldehyde containing 0.1% Triton X-100 for 30 min
and then blocked for 1 h with 5% bovine serum albumin (in 0.1 M phosphate-buffered saline). ICE was identified using
rabbit anti-mouse ICE (1:40 dilution) antiserum specific for the p10
active form of ICE, IL-1 IL-1 Mediates dsRNA + IFN- IL-1 Mediates dsRNA + IFN- IRAP Prevents dsRNA + IFN- dsRNA Stimulates IL-1 mRNA Accumulation and Protein Expression
in Primary
To confirm that IL-1 is expressed at the protein level in response to
dsRNA + IFN- Immunohistochemical Identification of Islet Cellular Sources of
IL-1
As expected, poly(I-C) also stimulates IL-1
The RINm5F cell IL-1 bioassay, which is specific for active IL-1 (14,
33), was used to quantitate the levels of IL-1 released by islets in
response to poly(I-C) and IFN- IFN- ICE Activation Is Required for dsRNA + IFN- dsRNA + IFN- dsRNA Stimulates IL-1 A number of mechanisms have been proposed to explain how viral
infections may induce autoimmune diabetes. One hypothesis, termed
"molecular mimicry" suggests that viruses may contain protein structural motifs that mimic host proteins (38, 39). In this model, an
immune response against the invading pathogen will eliminate the
pathogen but may also cross-react with one or more host proteins that
share similar determinants with the infecting agent. Thus, the disease
process would continue even after the invading virus is cleared from
the host. A second hypothesis, termed "bystander activation,"
suggests that viral infection of target tissue may 1) modify surface
antigens into immunogenic forms, 2) stimulate the induction of antigen
expression, or 3) induce the release of sequestered antigens during
target cell lysis (40).
Experimentally, direct evidence supporting molecular mimicry or
bystander activation as mechanisms of virally induced
insulin-dependent diabetes mellitus has been difficult to
obtain, in part, because the host immune response may have already
cleared the viral determinant by the time the disease manifests itself.
However, viruses such as Coxsackie B4 have been isolated from the
pancreata of acutely diabetic individuals (41). Although Coxsackie B4
virus protein P2-C shares significant sequence homology with the islet
antigen glutamic acid decarboxylase (GAD65; see Ref. 42), and infection of mice with Coxsackie B4 induces diabetes (43), a recent report by
Horwitz et al. (44), supports bystander activation as the mechanism for Coxsackie B4-induced diabetes. Diabetes induced by
Coxsackie B4 appears to occur as a result of local inflammation, tissue
damage, and the release of islet antigens followed by re-stimulation of
autoreactive T-cells.
A common determinant of RNA and DNA genome virus families is the
formation of dsRNA during viral replication (45). Virus-derived single-stranded RNAs may also contain extensive dsRNA secondary structure (45). dsRNA is an active component of a viral infection that
stimulates antiviral responses in infected cells (8). The synthetic
dsRNA molecule poly(I-C) has been shown to induce an antiviral response
that is similar to the antiviral response activated following viral
infection (8, 16). Since viruses from both RNA and DNA genome families
have been associated with the development of autoimmune diabetes (3,
4), the synthetic dsRNA molecule, poly(I-C), was used to examine
mechanisms of virally induced islet dysfunction and degeneration. In
this study, we show that poly(I-C), in the presence of IFN- As evidenced by immunohistochemical analysis, poly(I-C) stimulates
IL-1 Results presented in this study also indicate that the Similar to rat islets, we also show that a combination of dsRNA and
IFN- In summary, this study provides direct evidence for endocrine -cell damage during the development of autoimmune
diabetes. Formed during viral replication, double-stranded RNA (dsRNA)
activates the antiviral response in infected cells. In combination,
synthetic dsRNA (polyinosinic-polycytidylic acid, poly(I-C)) and
interferon (IFN)-
stimulate inducible nitric-oxide synthase (iNOS)
expression, inhibit insulin secretion, and induce islet degeneration.
Interleukin-1 (IL-1) appears to mediate dsRNA + IFN-
-induced islet
damage in a nitric oxide-dependent manner, as the
interleukin-1 receptor antagonist protein prevents dsRNA + IFN-
-induced iNOS expression, inhibition of insulin secretion, and
islet degeneration. IL-1
is synthesized as an inactive precursor
protein that requires cleavage by the IL-1
-converting enzyme (ICE)
for activation. dsRNA and IFN-
stimulate IL-1
expression and ICE
activation in primary
-cells, respectively. Selective ICE inhibition
attenuates dsRNA + IFN-
-induced iNOS expression by primary
-cells. In addition, poly(I-C) + IFN-
-induced iNOS expression and
nitric oxide production by human islets are prevented by interleukin-1
receptor antagonist protein, indicating that human islets respond to
dsRNA and IFN-
in a manner similar to rat islets. These studies
provide biochemical evidence for a novel mechanism by which viral
infection may initiate
-cell damage during the development of
autoimmune diabetes. The viral replicative intermediate dsRNA
stimulates
-cell production of pro-IL-1
, and following cleavage
to its mature form by IFN-
-activated ICE, IL-1 then initiates
-cell damage in a nitric oxide-dependent fashion.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cells found in pancreatic islets of Langerhans (1). Triggering events that precipitate
-cell damage have remained elusive; however, evidence supports a role for viral infection in the initiation of
autoimmune diabetes. Viruses have been isolated from pancreata, and
virus-specific IgM antibodies have been identified in newly diagnosed
diabetic patients (2, 3). Diabetes can be induced in genetically
susceptible strains of mice, rats, and primates by infection with
encephalomyocarditis virus, Coxsackie B4 virus, Kilham's rat virus,
rubella virus, and retrovirus (3, 4). Encephalomyocarditis-induced
diabetes in mice is associated with increased expression of
iNOS1 and macrophage-derived
cytokines IL-1
, IL-12, and TNF-
. Administration of neutralizing
antisera for IL-1
or TNF or of the iNOS-selective inhibitor
aminoguanidine attenuates diabetes development in this mouse model (5).
In vitro studies have identified cytokine-stimulated iNOS
expression and nitric oxide production by
-cells as one mechanism by
which IL-1 inhibits insulin secretion and induces islet damage (6,
7).
-cells
to a viral insult has been poorly defined. We have shown that dsRNA, in
combination with IFN-
, inhibits glucose-stimulated insulin secretion
and induces islet degeneration in a nitric oxide-dependent manner (19). Alone, neither poly(I-C) nor IFN-
stimulates iNOS expression or inhibits insulin secretion by rat islets (19). In
combination with IFN-
, dsRNA also activates macrophages, stimulating iNOS expression, nitric oxide formation, and IL-1 release (11). Islets
contain 5-10 resident macrophages that express and release IL-1 when
activated in response to TNF + LPS. Local accumulation of IL-1 in
islets stimulates the expression of iNOS and production of nitric oxide
by
-cells resulting in a potent inhibition of insulin secretion and
islet degeneration (20). In this report, we show that the inhibitory
and destructive effects of dsRNA + IFN-
on insulin secretion and
islet viability are mediated by the intra-islet production of IL-1.
Furthermore, we show that
-cells themselves are a source of IL-1 in
response to dsRNA and that
-cell production of IL-1 leads to IL-1-
and nitric oxide-dependent inhibition of
-cell function.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
were from Life Technologies, Inc. Fetal calf serum was obtained
from HyClone (Logan, UT). Male Harlan Sprague-Dawley rats (250-300 g)
were purchased from Harlan Breeders (Indianapolis, IN). Poly(I-C) and
collagenase type XI were from Sigma. [
-32P]dCTP and
enhanced chemiluminescence (ECL) reagents were purchased from Amersham
Pharmacia Biotech. Human recombinant IL-1
was from Cistron
Biotechnology (Pine Brook, NJ). Horseradish peroxidase-conjugated donkey anti-rabbit IgG, FITC-conjugated donkey anti-guinea pig, and
CY3-conjugated donkey anti-rabbit secondary antibodies were obtained
from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Rabbit
antiserum specific for the C-terminal 27 amino acids of mouse
macrophage iNOS was a gift from Dr. Thomas Misko (G. D. Searle,
St. Louis, MO). iNOS and cyclophilin cDNAs were gifts from Dr.
Charles Rodi (Monsanto Corporate Research, St. Louis, MO) and Dr. Steve
Carroll (Department of Pathology, University of Alabama, Birmingham,
AL), respectively. Guinea pig anti-human insulin antibody was from
Linco Research, Inc. (St. Louis, MO), and goat anti-rat IL-1
antibody was from R & D Systems (Minneapolis, MN). All other reagents
were from commercially available sources.
followed by
culture for the indicated times. Where indicated, islets were pretreated for 30 min with IRAP prior to the addition of poly(I-C) and
IFN-
.
-Cells by Fluorescence-activated Cell Sorting
(FACS)--
Islets isolated from 12 rats were cultured overnight
(~1200 islets/3 ml) in complete CMRL-1066 media under an atmosphere
of 95% air and 5% CO2 at 37 °C. Islets were then
dispersed into individual cells by treatment with trypsin (1.0 mg/ml)
in Ca2+- and Mg2+-free Hanks solution at
37 °C for 3 min as stated previously (21). Dispersed islet cells
were incubated for 60 min at 37 °C in complete CMRL-1066 prior to
cell sorting. Islet cells were purified as described previously (22)
using a FACSTAR + flow cytometer (Becton Dickinson, San Jose, CA). The
cells were illuminated at 488 nm, and emission was monitored at
515-535 nm. This procedure results in
- and
-cell purity of
90-95 and 80-85%, respectively. For RT-PCR analysis of IL-1
and
IL-1
mRNA expression, narrow gated windows were used to enhance
-cell purity, which was greater than 98% based on post-sort FACS
analysis and immunohistochemical analysis of insulin-containing cells
(data not shown).
, and IRAP. The islets were isolated and washed
three times in Krebs-Ringer bicarbonate buffer (KRB: 25 mM
Hepes, 115 mM NaCl, 24 mM NaHCO3, 5 mM KCl, 1 mM MgCl2, 2.5 mM CaCl2, and 0.1% bovine serum albumin, pH
7.4) containing 3 mM D-glucose, and insulin
secretion was performed as described (23). Medium insulin content was
determined by radioimmunoassay (24).
, and IRAP. Islet
degeneration was determined in a double-blind manner by phase-contrast
microscopic analysis. Islet degeneration is characterized by the loss
of islet integrity, disintegration, and partial dispersion of islets as
described previously (23, 25, 26).
, and IRAP were isolated, lysed, and protein
separated by SDS-gel electrophoresis as described (23). Detection of
iNOS was by ECL according to the manufacturer's specifications
(Amersham Pharmacia Biotech) and as described previously (23).
, IRAP, and cycloheximide (CHX) as indicated. After culture, the
islets were washed 3 times with 0.1 M phosphate-buffered
saline, pH 7.4, and total RNA was isolated using the RNeasy kit
(Qiagen, Inc., Chatsworth, CA). RNA (5-10 µg) was denatured,
fractionated, and transferred to Duralon UV nylon membranes
(Stratagene, La Jolla, CA) as described (23). Membranes were hybridized
to a 32P-labeled probe specific for rat iNOS or cyclophilin
(27). The cDNA probe was radiolabeled with
[
-32P]dCTP by random priming using the Prime-a-Gene
nick translation system from Promega (Madison, WI). iNOS cDNA probe
corresponds to bases 509-1415 of the rat iNOS coding region.
Cyclophilin was used as an internal control for RNA loading.
Hybridization and autoradiography were performed as described
previously (28).
and IL-1
mRNA
accumulation by rat and human islets (100/condition) and FACS-purified
- and
-cells (100,000 cells/condition) was performed as described
previously (20, 30). GAPDH mRNA accumulation was used as a control
for PCRs, and total RNA isolated from rat islets treated for 4 h
with TNF + LPS was used as a positive control for islet expression of
IL-1
and IL-1
. We have previously shown that resident macrophages are the source of IL-1 in response to TNF + LPS (20).
. [35S]Methionine (500 µCi) was added, and the
islets were cultured for 6 additional h. The islets were isolated, and
lysed, and IL-1 was immunoprecipitated using hamster anti-IL-1
- and
hamster anti-IL-1
-specific antisera as described (20).
was identified using goat anti-rat IL-1
antiserum (1:20 dilution), and insulin was identified using guinea pig
anti-human insulin (1:200 dilution). Secondary antibodies included
FITC- or CY3-conjugated donkey anti-rat, donkey anti-guinea pig, and
donkey anti-mouse antisera (1:200 dilution). All figures for
immunohistochemistry were at a × 40 magnification.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-induced iNOS Expression and Nitric
Oxide Formation by Rat Islets--
To determine whether the endogenous
production of IL-1 is required for dsRNA + IFN-
-induced damage,
islets were incubated for 40 h with dsRNA and IFN-
in the
presence or absence of the interleukin-1 receptor antagonist protein
(IRAP). IRAP competes with IL-1 for receptor binding and thereby
prevents IL-1-induced signaling events (31). Treatment of rat islets
with dsRNA + IFN-
results in a 2.5-fold increase in nitrite
production (poly(I-C) + IFN-
treated, 30 pmol/islet; untreated, 12 pmol/islet). In a concentration-dependent manner, IRAP
prevents poly(I-C) + IFN-
-induced nitrite formation with maximal
~70% inhibition at 1-10 µg/ml (Fig. 1a). We have recently shown
that dsRNA + IFN-
-induced iNOS mRNA accumulation and protein
expression are maximal following 18- and 40-h incubations,
respectively, and that concentrations of 50 µg/ml poly(I-C) + 150 units/ml IFN-
stimulate maximal iNOS expression and nitrite
formation by rat islets (19). As shown in Fig. 1b, IRAP
prevents dsRNA + IFN-
-induced iNOS mRNA accumulation following
an 18-h incubation. IRAP also prevents dsRNA + IFN-
-induced iNOS
protein expression following a 40-h incubation (Fig. 1c). Alone, neither IFN-
nor dsRNA stimulates iNOS expression or nitrite formation by rat islets (Fig. 1b and Ref. 19). Consistent
with a requirement for IL-1 production, dsRNA + IFN-
-induced iNOS mRNA accumulation requires de novo protein synthesis.
CHX, at a concentration (10 µM) that inhibits islet total
protein synthesis by greater than 95% (32), prevents dsRNA + IFN-
-induced iNOS mRNA accumulation by rat islets (Fig.
1b). Importantly, CHX (at 10 µM) does not
alter the levels of iNOS mRNA that accumulate in response to 1 unit/ml IL-1, nor does CHX inhibit glucose-stimulated insulin secretion
by rat islets (6, 32). These results show that de novo
protein synthesis is required for dsRNA + IFN-
-induced iNOS
expression by rat islets and suggest that dsRNA + IFN-
-induced iNOS
expression and nitrite formation is mediated by the intra-islet release
of IL-1.
View larger version (22K):
[in a new window]
Fig. 1.
IRAP inhibits dsRNA + IFN- -induced iNOS expression and nitrite
formation by isolated rat islets. Islets were pretreated for 30 min with IRAP, and then 50 µg/ml poly(I-C) and 150 units/ml IFN-
were added. Following a 40-h incubation at 37 °C, the media were
removed, and nitrite production (a) and iNOS protein
expression (c) were determined as described under
"Experimental Procedures." b, rat islets were pretreated
for 30 min with IRAP, followed by the addition of poly(I-C), IFN-
,
and cycloheximide (CHX), and the islets were cultured for
18 h. iNOS mRNA accumulation was examined by Northern analysis
as described under "Experimental Procedures." Results are the
average ± S.E. of three independent experiments for nitrite
production (a), or representative of three independent
experiments for the analysis of iNOS expression (b and
c).
-induced Inhibition of
Glucose-stimulated Insulin Secretion and Islet Degeneration--
We
have previously shown that dsRNA + IFN-
inhibits glucose-stimulated
insulin secretion and induces islet degeneration in a nitric
oxide-dependent manner (19). To determine whether the inhibitory and destructive effects of dsRNA + IFN-
are mediated by
the intra-islet release of IL-1, rat islets were incubated for 40 (insulin secretion) or 96 h (islet viability) with 50 µg/ml poly(I-C) + 150 units/ml IFN-
in the presence or absence of IRAP. As
shown in Fig. 2, dsRNA + IFN-
inhibits
glucose-stimulated insulin secretion and induces the degeneration of
89% of islets. IRAP prevents the inhibitory effects of dsRNA + IFN-
on glucose-stimulated insulin secretion and attenuates islet
degeneration by ~75%. These results indicate that dsRNA + IFN-
-induced inhibition of insulin secretion, and induction of islet
degeneration is mediated by IL-1.
View larger version (23K):
[in a new window]
Fig. 2.
IRAP prevents dsRNA + IFN- -induced inhibition of glucose-stimulated
insulin secretion and islet degeneration. a, isolated
rat islets were pretreated for 30 min with or without IRAP, poly(I-C)
and IFN-
were then added and the islets cultured for 40 h at
37 °C. The islets were isolated, and glucose-stimulated insulin
secretion was examined as described under "Experimental
Procedures." b, isolated rat islets were pretreated for 30 min with IRAP, poly(I-C) and IFN-
were then added, and islet
morphological damage was examined following a 96-h culture at 37 °C
as described under "Experimental Procedures." Results for
a and b are the average ± S.E. of three
independent experiments.
-induced iNOS Expression by
FACS-purified
-Cells--
To examine whether
-cells are a source
of IL-1 in response to dsRNA + IFN-
, primary
-cells purified by
fluorescence-activated cell sorting (FACS) were incubated with
poly(I-C) and IFN-
in the presence or absence of IRAP. Following a
40-h incubation,
-cells express high levels of iNOS in response to
dsRNA + IFN-
(Fig. 3). IRAP prevents
dsRNA + IFN-
-induced iNOS expression by FACS-purified
-cells,
indicating that
-cells may be one islet cellular source of IL-1.
Alone, neither dsRNA nor IFN-
induces iNOS expression by
FACS-purified
-cells, and dsRNA and IFN-
, alone or in
combination, fail to induce iNOS expression by FACS-purified
-cells (data not shown).
View larger version (18K):
[in a new window]
Fig. 3.
dsRNA + IFN- -induced
iNOS expression by FACS-purified
-cells is
prevented by IRAP. Primary
-cells purified by FACS were
pretreated for 30 min with or without IRAP. Poly(I-C) and IFN-
were
then added, and the cells were cultured for 40 additional h at
37 °C. The cells were isolated by centrifugation, and iNOS
expression was examined by Western blot analysis as described under
"Experimental Procedures." Results are representative of three
independent experiments.
-Cells and Rat Islets--
Two isoforms of IL-1 have
been identified, IL-1
and IL-1
. To determine the isoform(s) of
IL-1 expressed by
-cells, the effects of dsRNA and IFN-
on
IL-1
and IL-1
mRNA accumulation were examined by reverse
transcriptase-polymerase chain reaction (RT-PCR) and
immunoprecipitation. Treatment of FACS-purified
-cells with
poly(I-C) or poly(I-C) + IFN-
results in IL-1
mRNA
accumulation following an 18-h incubation (Fig.
4a). FACS-purified
-cells fail to express either IL-1
or IL-1
(data not shown), and
FACS-purified
-cells fail to express IL-1
in response to either
poly(I-C), IFN-
, or poly(I-C) + IFN-
(Fig. 4a). In rat
islets, poly(I-C) and poly(I-C) + IFN-
stimulate both IL-1
and
IL-1
mRNA accumulation that is first apparent following a 12-h
incubation and that persists for up to 24 h (Fig. 4b).
Resident macrophages appear to be the islet cellular source of IL-1
,
as dsRNA + IFN-
fails to stimulate IL-1
mRNA accumulation in
islets depleted of resident macrophages (data not shown) and primary
-cells purified by FACS (Fig. 4a). These results indicate
that dsRNA alone stimulates IL-1
expression by primary
-cells.
View larger version (38K):
[in a new window]
Fig. 4.
dsRNA stimulates IL-1 mRNA accumulation
in FACS-purified -cells and rat islets.
a, FACS-purified
-cells were cultured for 18 h at
37 °C with poly(I-C) and IFN-
, alone or in combination. Total RNA
was isolated, and RT-PCR was used to evaluate IL-1
and IL-1
mRNA accumulation as described under "Experimental Procedures."
b, IL-1
and IL-1
mRNA accumulation in rat islets
treated with 50 µg/ml poly(I-C), 1 unit/ml IL-1, and 150 units/ml
IFN-
at 37 °C for 12, 18, 24, and 40 h was examined by
RT-PCR. GAPDH mRNA accumulation is shown as an internal control for
the RT-PCRs. c, isolated rat islets were treated for 18 h with 50 µg/ml dsRNA + 150 units/ml IFN-
.
[35S]Methionine (500 µCi) was added, and the islets
were cultured for an additional 6 h. The islets were isolated;
IL-1
and IL-1
were immunoprecipitated; and immunoprecipitates
were resolved by SDS-15% polyacrylamide gel electrophoresis and
visualized by fluorography as described under "Experimental
Procedures." Results for a-c are representative of three
independent experiments.
, IL-1
and IL-1
were sequentially
immunoprecipitated from [35S]methionine-labeled rat
islets using hamster monoclonal antisera previously used to
immunoprecipitate both the pro-forms and mature forms of IL-1
and IL-1
from activated macrophages (33). Poly(I-C) + IFN-
stimulates the expression of immunoprecipitable pro-IL-1
from
isolated rat islets following a 24-h incubation (Fig. 4c). We were unable to immunoprecipitate IL-1
from rat islets, a finding that is consistent with IL-1
expression by the limited number of
macrophages found in rat islets (~10/islet). Also, we were unable to
immunoprecipitate the mature form of IL-1
from islets, consistent
with its release following proteolytic processing (Fig. 4c
(34)).
--
To confirm directly that
-cells are a source of
IL-1
, islets were treated for 18 h with poly(I-C) or poly(I-C) + IRAP, isolated, and dispersed into individual cells, and IL-1
expressing cells were identified by immunocytochemistry. As shown in
Fig. 5a, treatment of
rat islets with poly(I-C) results in IL-1
expression (red fluorescence), and IL-1
immunoreactivity localizes with
insulin-containing cells (green fluorescence), as indicated
by the yellow fluorescence following double exposure. To
control for IL-1
binding to cell membranes, rat islets were treated
for 18 h with poly(I-C) + IRAP. Under these conditions, IRAP
should antagonize the interactions of IL-1
with its surface
receptors on
-cells. As shown in Fig. 5b, IL-1
expression (green fluorescence) localizes to
insulin-containing cells (red fluorescence), as indicated by
the yellow following double exposure. Similar results were
obtained from islets treated for 18 h with poly(I-C) + IFN-
and
poly(I-C) + IFN-
+ IRAP (data not shown). In addition, IL-1
was
not expressed in untreated islets nor was it expressed in islets
treated for 18 h with IFN-
(data not shown). Whereas
-cells
express high levels of IL-1
, less than 1% of islet cells express
this cytokine in response to poly(I-C).
View larger version (46K):
[in a new window]
Fig. 5.
Immunohistochemical identification
of -cells as an islet cellular source of
IL-1
in response to dsRNA. Rat islets
were treated for 18 h with 50 µg/ml poly(I-C) (a),
poly(I-C) and 10 µg/ml IRAP (b), or for 5 h with 10 µg/ml LPS + 10 ng/ml TNF (c). The islets were isolated and
dispersed into individual cells, and IL-1
was identified by
immunofluorescence using goat anti-rat IL-1
and CY-3-conjugated
donkey anti-goat secondary antisera (red fluorescence,
a and c), or FITC-conjugated donkey anti-goat
secondary antisera (green fluorescence, b).
Insulin was identified using guinea pig anti-human insulin and
FITC-conjugated donkey anti-guinea pig secondary antisera
(green fluorescence, a and c), or
CY-3-conjugated donkey anti-guinea secondary antisera (red
fluorescence, b). Results are representative of three
independent experiments.
expression by a second
population of islet cells that appear to be resident macrophages. As
shown in Fig. 5b, IL-1
immunoreactivity localizes with a
large highly vacuolized cell containing a high degree of macrophage morphology. To confirm that these cells are macrophages, islets were
treated for 5 h with TNF + LPS. We have previously shown that
macrophages are the sole islet cellular source of IL-1
under these
conditions (20). As shown in Fig. 5c, a 5-h incubation of
islets with TNF + LPS results in the expression of IL-1
(red fluorescence) by resident macrophages. The morphology
of the IL-1
expressing macrophages in Fig. 5, b and
c, are nearly identical. Similar numbers of macrophages
express IL-1
in response poly(I-C) as compared with TNF + LPS. These
findings provide direct support for
-cell expression of IL-1
and
that resident islet macrophages also produce this cytokine in response
to poly(I-C).
. In this experiment, islets were
treated for 40 h with 50 µg/ml poly(I-C), 150 units/ml IFN-
,
or both poly(I-C) and IFN-
; the culture supernatant was isolated and
IL-1 levels quantitated. Alone, neither IFN-
nor poly(I-C) stimulate
IL-1 release by islets; however, poly(I-C) + IFN-
stimulates the
accumulation of 32.6 ± 1.2 pg/ml of IL-1. We have previously
shown that this level of IL-1 is sufficient to stimulate iNOS
expression by
-cells in the presence of IFN-
(23).
Induces ICE Activation in
-Cells--
The
interleukin-1
-converting enzyme (ICE or caspase-1) is required for
cleavage of inactive pro-IL-1
to the active mature cytokine. ICE is
present in the cytoplasm of cells as an inactive 45-kDa enzyme that
self-cleaves into p10 and p20 subunits to form an active tetramer (34).
IFN-
has been shown to induce ICE expression by NIT cells (Ref. 35
and data not shown) and activates ICE in
-cells (Fig.
6a). By using antiserum
specific for active ICE (p10 subunit; red fluorescence), we
show that a 24-h incubation of rat islets with IFN-
stimulates ICE
activation. Active ICE (red fluorescence) localizes to
insulin-containing cells (green fluorescence) as evidenced
by the intense yellow fluorescence upon double exposure (Fig.
6a). IFN-
activates ICE in greater than 90% of
insulin-containing cells. Active ICE was not detected in untreated or
poly(I-C)-treated islet cells, and poly(I-C) did not further enhance or
inhibit IFN-
-induced ICE expression or activation in
-cells (data
not shown). In addition, we did not detect cross-reactivity or
nonspecific binding of the secondary antibodies.
View larger version (38K):
[in a new window]
Fig. 6.
ICE activation is required for dsRNA + IFN- -induced iNOS expression by
FACS-purified
-cells. a, rat
islets were treated with or without 150 units/ml IFN-
for 24 h.
The islets were dispersed into individual cells, and active ICE was
identified by immunofluorescence using rabbit anti-mouse ICE antiserum
specific for the p10 active form of ICE, and CY-3-conjugated donkey
anti-rabbit secondary antisera (red fluorescence).
Insulin-containing cells were identified using guinea pig anti-human
insulin and FITC-conjugated donkey anti-guinea pig secondary antiserum
(green fluorescence). b, FACS-purified
-cells
were preincubated for 30 min with the indicated concentrations of the
ICE-selective inhibitor YVAD-FMK (Enzyme System Products, Livermore,
CA). Poly(I-C) (50 µg/ml) and IFN-
(150 units/ml) were then added,
and the cells were cultured for 40 h at 37 °C.
-Cells were
isolated, and iNOS expression was evaluated by Western blot analysis as
described under "Experimental Procedures." Results for both
a and b are representative of three independent
experiments.
-induced iNOS
Expression by
-Cells--
Since dsRNA stimulates IL-1
expression
and IFN-
activates ICE in
-cells, we examined whether IL-1
processing by ICE is required for
-cell expression of iNOS in
response to dsRNA + IFN-
. As shown in Fig. 6b, the
ICE-selective peptide inhibitor benzyloxycarbonyl-Tyr-Val-ala-Asp(OMe)-fluoromethyl ketone (YVAD-FMK) attenuates poly(I-C) + IFN-
-induced iNOS expression by FACS-purified
-cells in a concentration-dependent manner with a
maximal ~80% inhibition observed at 100-200 µM
(determined by densitometry). Me2SO, the vehicle
control for YVAD-FMK, does not inhibit dsRNA + IFN-
-induced iNOS
expression (Fig. 6b), and 200 µM YVAD-FMK does
not inhibit IL-1-induced iNOS expression by primary
-cells (data not
shown). The latter experiment was used to control for nonspecific
inhibitory actions of YVAD-FMK on proteolytic cleavage events that are
required for iNOS expression (such as I
B degradation; see Ref. 6).
In addition, YVAD-FMK did not alter
-cell viability as determined by
trypan blue exclusion (data not shown). These results indicate that
proteolytic processing of IL-1
by IFN-
-activated ICE is required
for dsRNA + IFN-
-induced iNOS expression by primary
-cells.
-induced iNOS Expression and Nitric Oxide Formation
by Human Islets Is Dependent on the Intra-islet Production of
IL-1--
To determine whether dsRNA + IFN-
stimulates iNOS
expression and nitric oxide formation, human islets were incubated with poly(I-C), in the presence or absence of IFN-
, for 48 h. Alone, neither poly(I-C) (Fig. 7, a
and b) nor IFN-
(Refs. 30, 36, and 37 and data not shown)
stimulate nitric oxide production or iNOS expression by human islets.
However, in the presence of IFN-
, poly(I-C) stimulates the
concentration-dependent production of nitric oxide and
expression of iNOS that is maximal at 400 µg/ml (Fig. 7, a
and b). The levels of iNOS expressed and nitrite produced in
response to 400 µg/ml poly(I-C) + IFN-
are similar in magnitude to
the levels induced in response to a 48-h incubation with IL-1 + IFN-
(minimal combination of cytokines required to stimulate iNOS expression
by human
-cells; see Refs. 30, 36, and 37). Poly(I-C) + IFN-
-induced iNOS expression and nitric oxide production appears to
require the intra-islet production of IL-1. As shown in Fig. 7,
a and b, IRAP prevents dsRNA + IFN-
-induced nitric oxide production and iNOS expression by human islets. These findings indicate that poly(I-C) + IFN-
stimulates iNOS expression and nitric oxide production by human islets and that nitric oxide production under these conditions is dependent on the local release of
IL-1 in human islets.
View larger version (26K):
[in a new window]
Fig. 7.
IRAP prevents poly(I-C) + IFN- -induced nitrite production and iNOS
expression by human islets. Human islets were treated for 48 h with IL-1 (75 units/ml), IFN-
(750 units/ml), IRAP (25 µg/ml),
and poly(I-C) as indicated. The islets were then isolated, and nitrite
production was determined on the culture medium (a), and
Western blot analysis was used to examine iNOS expression by the
isolated islet (b). Results are the average ± S.E. of
three experiments from three independent human islet isolations for
nitrite production (a), and representative of three
independent experiments from three independent human islet isolations
for iNOS expression (b).
and IL-1
mRNA Accumulation in Human
Islets--
To determine the isoform(s) of IL-1 expressed in response
to dsRNA and IFN-
, human islets were incubated for 6, 12, and
24 h with 400 µg/ml poly(I-C) and 750 units/ml IFN-
, alone
and in combination. The islets were isolated, and IL-1
and IL-1
mRNA accumulation was examined by RT-PCR. As shown in Fig.
8, poly(I-C) and poly(I-C) + IFN-
stimulate the mRNA accumulation of both IL-1
and IL-1
in
human islets. IL-1
and IL-1
mRNA accumulation is first
apparent following a 6-h incubation and persists for up to 24 h.
Alone, IFN-
fails to stimulate IL-1 expression by human islets (data
not shown), a finding that is consistent with the inability of this
cytokine to induce iNOS and IL-1 expression by rat islets.
View larger version (46K):
[in a new window]
Fig. 8.
Poly(I-C) stimulates IL-1 expression by human
islets. Human islets were treated for the indicated times with
poly(I-C) (400 µg/ml) or poly(I-C) + IFN- (750 units/ml). Total
RNA was isolated, and RT-PCR was used to evaluate IL-1
and IL-1
mRNA accumulation as described under "Experimental Procedures."
GAPDH mRNA accumulation is shown as an internal control for the
RT-PCRs. Results are representative of three independent experiments
from three independent human islet isolations.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
stimulates iNOS expression and nitrite formation, inhibits insulin
secretion, and induces rat islet degeneration by an
IL-1-dependent mechanism.
-Cells, selectively destroyed
during the development of autoimmune diabetes, are one islet cellular
source of IL-1 in response to dsRNA. IFN-
appears to participate in
dsRNA + IFN-
-induced iNOS expression by activating ICE, the enzyme
responsible for processing IL-1
from the inactive pro-form to
the active mature cytokine. We show that IFN-
stimulates ICE
activation (processing from inactive pro-ICE to the active mature
enzyme) and that selective inhibition of ICE using YVAD-FMK prevents
poly(I-C) + IFN-
-induced iNOS expression by FACS-purified
-cells.
expression in both insulin-containing cells as well as resident
macrophages. It is not surprising that resident macrophages are a
source of IL-1 in islets, as we have previously shown that poly(I-C)
stimulates IL-1 expression and release from mouse peritoneal macrophages and RAW 264.7 cells (11, 46). Unexpectedly, poly(I-C) also
stimulates
-cells to express and produce this proinflammatory cytokine that has been implicated in
-cell destruction (6, 7). Less
than 1% of islet macrophages and
-cells appear to express IL-1 in
response to poly(I-C). Although this is a small proportion of islet
cells, previous studies have shown that activation of resident
macrophages (which compose ~0.1% of islet cells) in both rat and
human islets results in the local production of IL-1 to levels
sufficient to stimulate iNOS expression, nitric oxide production, and
inhibit insulin secretion (20, 30, 47). In addition, the amount of IL-1
released by islets treated with poly(I-C) + IFN-
(~30 pg/ml) is an
amount that we have shown to be sufficient to stimulate
-cell
expression of iNOS in the presence of IFN-
(23).
-cells
themselves are capable of producing sufficient levels of IL-1 to
inhibit
-cell function and to stimulate iNOS expression. Treatment of FACS-purified
-cells with poly(I-C) + IFN-
results in iNOS expression in an IL-1-dependent manner. In addition,
poly(I-C) + IFN-
-induced inhibition of glucose-stimulated insulin
secretion by FACS-purified
-cells is dependent on nitric oxide
production (19). Although
-cells are capable of producing sufficient
levels of IL-1 to impair
-cell function (based on the FACS-purified
-cell studies), both
-cells and macrophages could contribute to
poly(I-C) + IFN-
islet damage by releasing IL-1 locally in islets.
However, we have previously shown that poly(I-C) + IFN-
stimulates
iNOS expression by macrophage-depleted rat islets and by FACS-purified
-cells (19). These findings provide the first evidence that primary
endocrine cells are capable of producing the proinflammatory cytokine
IL-1 and that the levels of IL-1 produced are sufficient to inhibit
-cell function (independent of macrophage IL-1 production). In
addition, these findings also suggest that IL-1 may participate in the
antiviral response activated by dsRNA in
-cells.
stimulate IL-1
, IL-1
, and iNOS expression and nitric
oxide production by human islets. iNOS expression and nitric oxide
production in response to dsRNA + IFN-
appears to be dependent on
the local production of IL-1 in human islets. Antagonism of the IL-1
receptor using IRAP prevents dsRNA + IFN-
-induced iNOS expression
and nitric oxide production by human islets. Presently, it is not clear
if
-cells, resident macrophages, or both are cellular sources of
IL-1 in human islets. However, based on the effects of poly(I-C) + IFN-
on IL-1 expression by rat islets, it is likely that both cell
types contribute to poly(I-C)-induced IL-1 expression in human islets.
Also, since rat and human islets express iNOS in an
IL-1-dependent manner, it is likely that the mechanisms by
which poly(I-C) + IFN-
stimulate iNOS expression by human islets may
be similar to those activated in rat islets.
-cell
expression of IL-1 as a novel mechanism by which viral infection may
mediate
-cell damage in autoimmune diabetes. Our findings suggest
that dsRNA, the active component of a viral infection that activates
the antiviral response, stimulates IL-1
expression by
-cells and
IL-1
and IL-1
expression by macrophages. IL-1
requires
proteolytic processing for activation (34), an event that appears to be
mediated by IFN-
-induced ICE activation in
-cells. Active ICE
cleaves inactive pro-IL-1
to the active mature cytokine resulting in
the release of IL-1
by
-cells followed by autocrine or paracrine
stimulation of adjacent
-cells to express iNOS and produce nitric
oxide (Fig. 9). The local release of IL-1 by resident macrophages may also contribute to poly(I-C) + IFN-
-induced iNOS expression by islets. In support of this model, we
have recently shown that Coxsackie B4 infection of isolated rat islets
results in iNOS expression.2
Although our findings do not distinguish between bystander activation or molecular mimicry as a mechanism of virally induced autoimmune diabetes, these findings support a novel mechanism by which a viral
infection may stimulate
-cell damage that could result in
autoimmunity directed against
-cells.
View larger version (36K):
[in a new window]
Fig. 9.
Schematic model of dsRNA + IFN- -induced
-cell
damage.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Jessica Gorman for expert technical assistance and Drs. Paul Lacy, Mark Buller, Michael Moxley, Claudette Klein, and Abdul Waheed for advice and critical review of this manuscript. We also thank Dr. Byeong Chang (Amgen Inc.) for the gift of IRAP; the Islet Isolation Core Facility and the Diabetes Research and Training Center at Washington University School of Medicine for supplying human islets and performing insulin RIAs; and the Diabetes Research Institute at the University of Miami for supplying human islets.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants DK-52194 and AI44458 and a Career Development award from the Juvenile Diabetes Foundation International (to J. A. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, St. Louis University, 1402 South Grand Blvd.,
St. Louis, MO 63104. Tel.: 314-577-8165; Fax: 314-577-8156; E-mail:
corbettj@slu.edu.
Published, JBC Papers in Press, December 6, 2000, DOI 10.1074/jbc.M009159200
2 J. A. Corbett, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
iNOS, inducible
nitric-oxide synthase;
IL, interleukin;
IFN-, interferon-
;
dsRNA, double-stranded RNA;
TNF, tumor necrosis factor;
LPS, lipopolysaccharide;
FITC, fluorescein isothiocyanate;
RT-PCR, reverse
transcriptase-polymerase chain reaction;
IRAP, interleukin-1 receptor
antagonist protein;
ICE, IL-1
-converting enzyme;
FACS, fluorescence-activated cell sorting;
CHX, cycloheximide;
FMK, fluoromethyl ketone;
YVAD-FMK, benzyloxycarbonyl-Tyr-Val-Ala-Asp(OMe)-fluoromethyl ketone;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Gepts, W. (1965) Diabetes 14, 619-633[Medline] [Order article via Infotrieve] |
2. | Bach, J.-F. (1994) Endocr. Rev. 15, 516-541[Abstract] |
3. | Yoon, J.-W. (1995) Diabetes Metab. Rev. 11, 83-107[Medline] [Order article via Infotrieve] |
4. | von Herrath, M. G., and Oldstone, M. B. A. (1996) Curr. Opin. Immunol. 8, 878-885[CrossRef][Medline] [Order article via Infotrieve] |
5. | Hirasawa, K., Jun, H. S., Maeda, K., Kawaguchi, Y., Itagaki, S., Mikami, T., Baek, H. S., Doi, K., and Yoon, J.-W. (1997) J. Virol. 71, 4024-4031[Abstract] |
6. | Corbett, J. A., and McDaniel, M. L. (1996) in Nitric Oxide: Principles and Action (Lancaster, J. R., Jr., ed) , pp. 177-217, Academic Press, Inc., San Diego, CA |
7. | Mandrup-Poulsen, T. (1996) Diabetologia 39, 1005-1029[CrossRef][Medline] [Order article via Infotrieve] |
8. | Jacobs, B. L., and Langland, J. O. (1996) Virology 219, 339-349[CrossRef][Medline] [Order article via Infotrieve] |
9. | Kerr, I. M., and Stark, G. R. (1992) J. Interferon Res. 12, 237-240[Medline] [Order article via Infotrieve] |
10. | Karupiah, G., Xie, Q., Buller, M. L., Nathan, C., Duarte, C., and MacMicking, J. D. (1993) Science 261, 1445-1448[Medline] [Order article via Infotrieve] |
11. |
Heitmeier, M. R.,
Scarim, A. L.,
and Corbett, J. A.
(1998)
J. Biol. Chem.
273,
15301-15307 |
12. |
Wu, S.,
and Kaufman, R. J.
(1997)
J. Biol. Chem.
272,
1291-1296 |
13. |
de Haro, C.,
Mendez, R.,
and Santoyo, J.
(1996)
FASEB. J.
10,
1378-1387 |
14. | Kreil, T. R., and Eibl, M. M. (1996) Virology 219, 304-306[CrossRef][Medline] [Order article via Infotrieve] |
15. | Melkova, Z., and Esteban, M. (1995) J. Immunol. 155, 5711-5718[Abstract] |
16. | Clemens, M. J., and Elia, A. (1997) J. Interferon Cytokine Res. 17, 503-524[Medline] [Order article via Infotrieve] |
17. | Ewel, C. H., Sobel, D. O., Zeligs, B. J., and Bellanti, J. A. (1992) Diabetes 41, 1016-1021[Abstract] |
18. | Sobel, D. O., Newsome, J., Ewel, C. H., Bellanti, J. A., Abbassi, V., Creswell, K., and Blair, O. (1992) Diabetes 41, 515-520[Abstract] |
19. |
Heitmeier, M. R.,
Scarim, A. L.,
and Corbett, J. A.
(1999)
J. Biol. Chem.
274,
12531-12536 |
20. |
Arnush, M. A.,
Scarim, A. L.,
Heitmeier, M. R.,
Kelly, C. B.,
and Corbett, J. A.
(1998)
J. Immunol.
160,
2684-2691 |
21. | McDaniel, M. L., Colca, J. R., Kotagal, N., and Lacy, P. E. (1983) Methods Enzymol. 98, 182-200[Medline] [Order article via Infotrieve] |
22. | Pipeleers, D. G., Int Veld, P. A., Van De Winkel, M., Maes, E., Schuit, F. C., and Gepts, W. (1985) Endocrinology 117, 806-816[Abstract] |
23. |
Heitmeier, M. R.,
Scarim, A. L.,
and Corbett, J. A.
(1997)
J. Biol. Chem.
272,
13697-13704 |
24. | Wright, P. H., Makulu, D. R., Vichick, D., and Sussman, K. E. (1971) Diabetes 20, 33-45[Medline] [Order article via Infotrieve] |
25. | Lacy, P. E., and Finke, E. H. (1991) Am. J. Pathol. 138, 1183-1190[Abstract] |
26. | Corbett, J. A., and McDaniel, M. L. (1994) Biochem. J. 299, 719-724[Medline] [Order article via Infotrieve] |
27. | Brown, R., and Mackey, K. (1997) in Current Protocols in Molecular Biology (Ausubel, F. M. , Brent, R. , Kingston, R. E. , Moore, D. D. , Seidman, J. G. , Smith, J. A. , and Struh, L. K., eds), Vol. 2 , pp. 4.9.1-4.9.13, Green Publishing Associates and Wiley-Interscience, New York |
28. | Burd, P. R., Rogers, H. W., Gordon, J. R., Martin, C. A., Jayaraman, S., Wilson, S. D., Dvorak, A. M., Galli, S. J., and Dorf, M. E. (1989) J. Exp. Med. 170, 245-257[Abstract] |
29. | Green, L. C., Wagner, D. A., Glogowski, J., Skipper, P. L., Wishnok, J. S., and Tannenbaum, S. R. (1982) Anal. Biochem. 126, 131-138[Medline] [Order article via Infotrieve] |
30. |
Arnush, M.,
Heitmeier, M. R.,
Scarim, A. L.,
Marino, M. A.,
Manning, P. A.,
and Corbett, J. A.
(1998)
J. Clin. Invest.
102,
516-526 |
31. | Arend, W. P. (1991) J. Clin. Invest. 88, 1445-1451[Medline] [Order article via Infotrieve] |
32. | Hughes, J. H., Colca, J. R., Easom, R. A., Turk, J., and McDaniel, M. L. (1990) J. Clin. Invest. 86, 856-863[Medline] [Order article via Infotrieve] |
33. |
Hill, J. R.,
Corbett, J. A.,
Kwon, G.,
Marshall, C. A.,
and McDaniel, M. L.
(1996)
J. Biol. Chem.
271,
22672-22678 |
34. | Wilson, K. P., Black, J. A., Thomson, J. A., Kim, E. E., Griffith, J. P., Navia, M. A., Murcko, M. A., Chambers, S. P., Aldape, R. A., Raybuck, S. A., and Livingston, D. J. (1994) Nature 370, 270-274[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Stephens, L. A.,
Thomas, H. E.,
Ming, L.,
Grell, M.,
Darwiche, R.,
Volodin, L.,
and Kay, T. W.
(1999)
Endocrinology
140,
3219-3227 |
36. | Corbett, J. A., Sweetland, M. A., Wang, J. L., Lancaster, J. R., Jr., and McDaniel, M. L. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1731-1735[Abstract] |
37. | Corbett, J. A., Kwon, G., Marino, M., Rodi, C. P., Sullivan, P. M., and McDaniel, M. L. (1996) Am. J. Physiol. 39, C1581-C1587 |
38. | von Herrath, M. G., Evans, C. F., Horwitz, M. S., and Oldstone, M. B. (1996) Immunol. Rev. 152, 111-143[Medline] [Order article via Infotrieve] |
39. | Maclaren, N. K., and Atkinson, M. A. (1997) Mol. Med. Today 3, 76-83[CrossRef][Medline] [Order article via Infotrieve] |
40. | von Herrath, M. G., Homann, D., Gairin, J. E., and Oldstone, M. B. (1997) Biochem. Soc. Trans. 25, 630-635[Medline] [Order article via Infotrieve] |
41. | Notkins, A. L., and Yoon, J.-W. (1984) in Concepts in Viral Pathogenesis (Notkins, A. L. , and Oldstone, M. B. A., eds) , Springer-Verlag Inc., New York |
42. | Atkinson, M. A., Bowman, M. A., Campbell, L., Darrow, B. L., Kaufman, D. L., and Maclaren, N. K. (1994) J. Clin. Invest. 94, 2125-2129[Medline] [Order article via Infotrieve] |
43. | Yoon, J., Onodera, T., and Notkins, A. (1978) J. Exp. Med. 148, 1068-1080[Abstract] |
44. | Horwitz, M. S., Bradley, L. M., Harbertson, J., Krahl, T., Lee, J., and Sarvetnick, N. (1998) Nat. Med. 4, 781-785[Medline] [Order article via Infotrieve] |
45. |
Lengyel, P.
(1987)
EMBO J.
19,
3630-3638 |
46. | Corbett, J. A., and McDaniel, M. L. (1995) J. Exp. Med. 181, 559-568[Abstract] |