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INTRODUCTION |
Insulin-dependent diabetes mellitus is an autoimmune
disease characterized by an inflammatory reaction in and around
pancreatic islets followed by selective destruction of insulin
secreting
-cells (1). The mechanisms that lead to the development of autoimmune diabetes are unknown; however, the expression of the inducible form of nitric oxide synthase
(iNOS)1 by
-cells and the
resulting production of nitric oxide may be one factor that mediates
-cell dysfunction and eventual
-cell death. We and others (2-4)
have shown that treatment of isolated rat islets for 18 h with
IL-1 stimulates the time- and concentration-dependent expression of iNOS and production of nitrite that correlates with a
potent inhibition of glucose-stimulated insulin secretion. In the
presence of IFN-
, the concentration of IL-1 required to induce iNOS
expression by
-cells is reduced 10-fold (5). Recently, Okamoto and
co-workers (6) have examined the development of diabetes in transgenic
mice that express iNOS under control of the rat insulin promotor (6).
These mice spontaneously develop diabetes in a nitric
oxide-dependent manner that occurs in the absence of
insulitis. Administration of the iNOS inhibitor, aminoguanidine (AG)
(200 mg/kg/2 times daily) prevents the spontaneous development of
diabetes in these mice (6). These results support an effector role for
nitric oxide in mediating
-cell damage during the development of
autoimmune diabetes.
Viruses have been implicated as one environmental factor that may
initiate or trigger an autoimmune reaction that targets and destroys
-cells in genetically susceptible individuals (7-11). Mouse models
of virus-induced autoimmune diabetes implicate increased cytokine and
iNOS expression and nitric oxide production in the development of the
disease. Encephalomyocarditis virus-induced diabetes in DBA/2 mice
correlates with an increased level of IL-1
and TNF mRNA
expression in islets as determined by in situ hybridization of pancreatic sections (12). Daily administration of antiserum specific
for IL-1
or TNF-
(0.5 mg/mouse) starting on the day of viral
infection attenuates encephalomyocarditis virus-induced diabetes in
DBA/2 mice (12). In addition, encephalomyocarditis virus stimulates
iNOS mRNA expression in islets at early stages of infection, and
iNOS expression persists until the onset of diabetes in DBA/2 mice.
Daily administration of the iNOS inhibitor, AG, (at 2 mg/mouse/day)
significantly attenuates the development of encephalomyocarditis
virus-induced diabetes (12). These findings implicate cytokines and
nitric oxide in the development of viral-induced diabetes.
dsRNA, formed during viral replication, is an active component of a
viral infection that stimulates antiviral activities in infected cells
(13). In vivo, dsRNA (in the form of poly(IC)) stimulates
the development of diabetes in diabetes-resistant BioBreeding (BB)
rats, and accelerates the development of diabetes in diabetes prone BB
rats (14-16). These findings indicate that viral infection and dsRNA
modulate islet function; however, the mechanisms associated with
viral-induced
-cell dysfunction are unknown.
The goal of these studies was to determine whether dsRNA directly
modulates islet function and viability. We show that in combination
with IFN-
, dsRNA stimulates the time- and concentration dependent expression of iNOS and production of nitric oxide by rat islets. We provide evidence that the islet cellular source of iNOS
in response to poly(IC) + IFN-
is the
-cell. In addition, we show
that poly(IC) + IFN-
induces islet degeneration and inhibits insulin
secretion by rat islets and
-cells purified by
fluorescence-activated cell sorting (FACS), and that these effects are
mediated by increased nitric oxide production. These results indicate,
for the first time, that the viral replicative intermediate, dsRNA (in
combination with IFN-
) directly modulates islet viability and
-cell function by a mechanism that involves
-cell production of
nitric oxide.
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EXPERIMENTAL PROCEDURES |
Materials and Animals--
CMRL-1066 tissue culture medium,
L-glutamine, penicillin, streptomycin, and rat recombinant
IFN-
were from Life Technologies, Inc. Fetal calf serum was obtained
from Hyclone (Logan, UT). Male Sprague-Dawley rats (250-300 g) were
purchased from Harlan (Indianapolis, IN). Polyinosinic-polycytidylic
acid (poly(IC)), aminoguanidine hemisulfate (AG), and collagenase type
XI were from Sigma Chemical Co. [
-32P]dCTP and
enhanced chemiluminescence 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 was 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 (Dept. of Pathology,
University of Alabama, Birmingham, AL), respectively. All other
reagents were from commercially available sources.
Islet Isolation and Culture--
Islets were isolated from male
Sprague-Dawley rats by collagenase digestion as described previously
(17). 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. Before each
experiment, islets were washed three 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(IC), cytokines, and
iNOS inhibitors, followed by culture for the indicated times.
Islet Dispersion and Macrophage Depletion--
Isolated rat
islets were dispersed into individual cells by treatment with trypsin
(1 mg/ml) in Ca2+- and Mg2+-free Hanks'
solution at 37 °C for 3 min as stated previously (5, 17). For
pseudoislet formation, dispersed rat islets were cultured for 7 days at
37 °C to allow for endocrine cell reaggregation (18). Alternatively,
intact rat islets were cultured for 7 days in complete CMRL-1066 at
24 °C in an atmosphere of 95% air and 5% CO2 (19, 20).
Islets were removed from the 24 °C culture, washed three times with
fresh complete CMRL-1066, and then cultured for 2 days at 37 °C in
complete CMRL-1066 (19, 20). Experiments were conducted as described
above for freshly isolated islets.
Purification of
-Cells by FACS--
Islets isolated from 12 rats were cultured overnight (~1,200/3 ml) in complete CMRL-1066
under an atmosphere of 95% air and 5% CO2 at 37 °C.
Islets were then dispersed into individual cells as stated above.
Dispersed islet cells were incubated for 60 min at 37 °C in complete
CMRL-1066 before cell sorting. Islet cells were purified as described
previously (21-23) 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. The sorting process yielded a 95% pure
population of
-cells and an 80-85% pure population of
-cells.
Insulin Secretion--
Islets (220/ml of complete CMRL-1066)
were cultured for 40 h with the indicated concentrations of
poly(IC), IL-1, IFN-
, and AG. 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 (5). FACS-purified
-cells were
treated for 24 h with the indicated concentrations of IL-1,
poly(IC), IFN-
, and AG. The cells were washed three times with KRB
containing 3 mM D-glucose and 0.1% bovine
serum albumin.
-cells were aliquoted into 96-well microtiter plates
(15,000 cells/250 µl of KRB containing either 3 mM or 20 mM D-glucose and 0.1% bovine serum albumin), and insulin secretion was performed as described (24). The
phosphodiesterase inhibitor, theophylline, was also included to elevate
cAMP levels, which are required for glucose-stimulated insulin
secretion from purified
-cells (23, 25). Medium insulin content was
determined by radioimmunoassay (26).
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(IC), IL-1, IFN-
, and AG. 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 (4, 5, 19).
Western Blot Analysis--
Rat islets (120/400 µl of complete
CMRL-1066), cultured for the indicated times with poly(IC), IL-1, and
rat IFN-
were isolated, lysed, and proteins were separated by
SDS-gel electrophoresis as described (5). Detection of rat iNOS was by
enhanced chemiluminescence according to manufacturer's specifications
and as described previously (5).
Northern Blot Analysis--
Rat islets (900/3 ml of complete
CMRL-1066) were cultured for the indicated times at 37 °C with
poly(IC) (50 µg/ml), IL-1
(1 unit/ml), and IFN-
(150 units/ml).
After culture, the cells were washed three times with 0.1 M
phosphate-buffered saline (pH 7.4), and total RNA was isolated using
the RNeasy kit (QIAGEN, Inc., Chatsworth, CA). Total cellular RNA
(5-10 µg) was denatured, fractionated, and transferred to Duralon UV
nylon membranes (Stratagene, La Jolla, CA) as described (5). 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).
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.
Densitometry and Image Analysis--
Autoradiograms were scanned
into NIH Image version 1.59 using a COHU high performance CCD camera
(Brookfield, WI), and densities were determined using NIH Image version
1.59 software. Phosphorimaging analysis of rat islet mRNA
accumulation levels was performed using a Molecular Dynamics
PhosphorImager and Molecular Dynamics ImageQuant Software version 3.3 (Molecular Dynamics, Inc.).
Statistics--
Statistical comparisons were made between groups
using a one-way analysis of variance. Significant differences between
treatment groups compared with untreated controls (indicated by *;
p < 0.05) were evaluated using a Scheffe's F-test
post hoc analysis.
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RESULTS |
dsRNA + IFN-
Stimulates the Time- and
Concentration-dependent Expression of iNOS and Production
of Nitrite by Rat Islets--
To determine whether poly(IC) stimulates
nitric oxide formation, rat islets were incubated for 40 h with
1-100 µg/ml poly(IC) and 150 units/ml IFN-
. Alone, neither
IFN-
(5) nor poly(IC) stimulates nitrite production by rat islets;
however, in combination with IFN-
, poly(IC) stimulates the
concentration-dependent increase in nitrite formation that
is maximal at 50 µg/ml poly(IC) (Fig. 1a). The level of nitrite
produced in response to 50-100 µg/ml poly(IC) + IFN-
is similar
in magnitude to the levels produced in response to 1 unit/ml IL-1.

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Fig. 1.
Effects of poly(IC) and
IFN- on nitrite production, iNOS mRNA, and
protein expression by rat islets. Panel a, rat islets
(120/400 µl of complete CMRL) were incubated for 40 h with the
indicated concentrations of poly(IC), 150 units/ml IFN- , and 1 unit/ml IL-1. Nitrite production was determined on the culture medium
as described under "Experimental Procedures." Panel b,
rat islets (900/3 ml of complete CMRL-1066) were cultured for 6, 12, and 18 h with 50 µg/ml poly(IC) and 150 unit/ml IFN- . Total
RNA was isolated and probed for iNOS and cyclophilin by Northern
analysis as stated under "Experimental Procedures." iNOS mRNA
levels were quantitated by phosphorimaging analysis (arbitrary
phosphorimaging units) using cyclophilin as an internal control for RNA
loading. Panel c, rat islets (120/400 µl of complete CMRL)
were incubated for 40 h with the indicated concentrations of IL-1,
poly(IC), and IFN- . The islets were isolated, and the expression of
iNOS was determined by Western blot analysis as described under
"Experimental Procedures." Results for nitrite are the average ± S.E. of six independent experiments, and iNOS mRNA and protein
expression are representative of three independent experiments,
respectively. Statistical significance, p < 0.05 versus control (*) as indicated.
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The effects of poly(IC) + IFN-
on iNOS mRNA accumulation and
protein expression are shown in Fig. 1, b and c.
Alone, poly(IC) and IFN-
do not induce iNOS mRNA accumulation by
rat islets (data not shown); however, in combination, poly(IC) + IFN-
induce the time-dependent accumulation of iNOS
mRNA that is first apparent at 6 h and maximal following an
18-h incubation period (Fig. 1b). Similarly, poly(IC) and
IFN-
alone do not induce iNOS protein expression by rat islets (data
not shown, and Ref. 5); however, poly(IC) + IFN-
induce the
time-dependent expression of iNOS protein that is first
apparent following a 24-h incubation period and maximal following a
48-h incubation period (Fig. 1c). The stimulatory effects of
poly(IC) + IFN-
on iNOS expression by rat islets are delayed in
comparison to IL-1-induced iNOS expression, which is first apparent
following a 6-h incubation period and maximal following a 24-h
incubation period (Ref. 5 and Fig. 1c). In addition,
poly(IC) + IFN-
stimulate iNOS expression to levels ~2-fold higher
than the maximal level of iNOS expressed in response to IL-1 (compare
iNOS expression following a 48-h incubation period with poly(IC) + IFN-
to a 24-h incubation period with IL-1). These findings provide
the first evidence that dsRNA, in combination with IFN-
, stimulates
the time- and concentration-dependent expression of iNOS
expression and nitrite production by rat islets.
dsRNA + IFN-
Inhibit Glucose-stimulated Insulin Secretion and
Induce Islet Degeneration in a Nitric Oxide-dependent
Manner--
Previous studies have shown that nitric oxide mediates the
inhibitory and destructive actions of cytokines (such as IL-1 and IL-1 + IFN-
) on islet function and viability (4, 30-34). We therefore
examined the effects of dsRNA, alone and in combination with IFN-
,
on glucose-stimulated insulin secretion. Treatment of rat islets for
40 h with 50 µg/ml poly(IC) + 150 units/ml IFN-
results in
the inhibition of glucose-stimulated insulin secretion to levels
comparable with the inhibitory effects induced by 1 unit/ml IL-1 (Fig.
2a). AG completely prevents
poly(IC) + IFN-
-induced inhibition of insulin secretion suggesting
that the inhibitory effects are mediated by nitric oxide. Individually,
neither poly(IC) nor IFN-
inhibits glucose-stimulated insulin
secretion by rat islets (data not shown). In addition, we have
previously shown that AG alone does not inhibit glucose-stimulated
insulin secretion by rat islets (34). These results indicate that
nitric oxide mediates the inhibitory actions of dsRNA + IFN-
on
glucose-stimulated insulin secretion by rat islets.

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Fig. 2.
Effects of poly(IC) and
IFN- on glucose-stimulated insulin secretion
and islet degeneration by rat islets. Panel a, rat
islets (220/ml of complete CMRL) were treated for 40 h with the
indicated concentrations of IL-1, poly(IC), IFN- , and AG. The islets
were isolated and glucose-stimulated insulin secretion was examined.
Panel b, rat islets (25/500 µl of CMRL) were incubated for
96 h with the indicated concentrations of IL-1, poly(IC), IFN- ,
and AG and then islet degeneration was examined by phase-contrast
microscopy in a double-blind manner as described under "Experimental
Procedures." Results for insulin secretion are the average ± S.E. of four independent experiments and islet degeneration are the
average ± S.E. of three independent experiments. Statistical
significance, p < 0.05 versus control (*)
as indicated.
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Treatment of rat islets with IL-1, or the combination of IL-1 + IFN-
, for 96 h induces islet degeneration that is characterized by the loss of islet integrity, disintegration, and partial dispersion of islets into individual cells (4, 5, 19). The effects of dsRNA on
islet viability are shown in Fig. 2b. Rat islets were incubated for 96 h with IL-1, poly(IC), IFN-
, and poly(IC) + IFN-
in the presence or absence of AG. Individually, poly(IC) and
IFN-
do not induce islet degeneration; however, the combination of
poly(IC) + IFN-
stimulates islet degeneration to levels comparable with the destructive effects of IL-1 (Fig. 2b). Islet
destruction appears to be mediated by nitric oxide as AG completely
prevents islet degeneration in response to IL-1 (Ref. 5 and Fig.
2b) and poly(IC) + IFN-
. These findings provide evidence
that dsRNA, in combination with IFN-
, induces islet degeneration in
a nitric oxide-dependent manner.
Resident Macrophages Are not Required for the Inhibitory Actions of
dsRNA + IFN-
on Islet Function--
We have recently shown that
poly(IC) + IFN-
activate resident mouse macrophages, stimulating
iNOS expression, nitric oxide formation, and IL-1 release (35). In
addition, activation of resident macrophages by treatment of islets
with TNF + LPS results in the expression of iNOS, production of nitric
oxide, and inhibition of insulin secretion by a mechanism associated
with the intraislet release of IL-1 (20, 24). Because poly(IC) + IFN-
activates macrophages, stimulating nitric oxide production and
IL-1 release, and resident islet macrophage IL-1 release (in response
to TNF + LPS) results in the inhibition of insulin secretion, we
examined the role of islet macrophages in poly(IC) + IFN-
-induced
iNOS expression, nitric oxide formation, and inhibition of insulin secretion by depleting rat islets of this cell population. Rat islets
were dispersed into individual cells and then allowed to reaggregate
during a 7 day culture at 37 °C. These reaggregated islet cells,
termed pseudoislets, are composed of only endocrine cells (18). As
shown in Fig. 3, the combination of
poly(IC) + IFN-
stimulates the production of nitrite and expression
of iNOS to levels comparable with IL-1-induced nitrite formation and
iNOS expression by rat pseudoislets (Fig. 3, a and
b, respectively). As a control for macrophage depletion, we
show that TNF + LPS fail to stimulate iNOS expression or nitrite
production by rat pseudoislets. We have confirmed these findings using
rat islets depleted of resident macrophages by culturing for 7 days at
24 °C. Previous studies have shown that these culture conditions deplete greater than 95% of islet macrophages (19, 20). As shown in
Fig. 3c, a 40 h incubation period of macrophage-depleted rat
islets (by culturing for 7 days at 24 °C) with poly(IC) + IFN-
results in an ~3-fold increase in nitrite production and a potent
inhibition of insulin secretion. The stimulatory effects of poly(IC) + IFN-
on nitrite formation and inhibitory effects on insulin
secretion are similar in magnitude to the actions of IL-1. Importantly,
TNF + LPS fails to inhibit insulin secretion or induce nitrite
formation, indicating that resident macrophages have been depleted by
the 7 day culture at 24 °C. These results suggest that resident
islet macrophages are not required for poly(IC) + IFN-
-induced iNOS
expression, nitrite formation, or the inhibition of insulin secretion
by rat islets.

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Fig. 3.
Effects of poly(IC) and
IFN- on nitrite formation, iNOS expression,
and insulin secretion by macrophage-depleted rat islets.
Panels a and b, rat pseudoislets (120/400 µl
complete CMRL) were incubated for 40 h with 1 unit/ml IL-1, 10 ng/ml TNF, 10 µg/ml LPS, 50 µg/ml poly(IC), and 150 units/ml
IFN- as indicated. Nitrite production was determined on the culture
supernatant (panel a) and iNOS protein expression was
determined by Western blot analysis (panel b) as described
under "Experimental Procedures." Panel c, rat islets
(220/ml of CMRL), cultured for 7 days at 24 °C to deplete
macrophages, were incubated for 40 h with 1 unit/ml IL-1, 10 ng/ml
TNF, 10 µg/ml LPS, 50 µg/ml poly(IC), and 150 units/ml IFN- as
indicated. The islets were isolated for glucose-stimulated insulin
secretion, and nitrite production was determined on the culture medium.
Results for nitrite (panels a and c) are the
average ± S.E. of three and four independent experiments,
respectively. iNOS expression (panel b) is representative of
three independent experiments. Glucose-stimulated insulin secretion
(panel c) are the average ± S.E. of three independent
experiments containing three replicates/condition. Statistical
significance, p < 0.05 versus control (*)
as indicated.
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dsRNA + IFN-
Stimulate iNOS Expression by FACS-purified
-Cells--
Because resident macrophages are not required for dsRNA + IFN-
-induced iNOS expression by rat islets, the effects of dsRNA + IFN-
on
- and
-cell expression of iNOS were examined.
- and
-cells purified by FACS were treated for 40 h with IL-1, poly(IC), IFN-
, and poly(IC) + IFN-
. As shown in Fig.
4, poly(IC) + IFN-
induce
-cell
expression of iNOS to levels that are ~2-fold higher than the levels
of iNOS expressed in response to 1 unit/ml IL-1. Alone, neither
poly(IC) nor IFN-
induces iNOS expression by primary
-cells, and
poly(IC) + IFN-
(alone or in combination) do not induce iNOS
expression by primary
-cells. These results indicate that the
-cell is one islet cellular source of iNOS in response to poly(IC) + IFN-
.

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Fig. 4.
Effects of poly(IC) and
IFN- on iNOS expression by FACS-purified
- and -cells. -
and -cells (200,000 cells/200 µl of complete CMRL-1066) purified
by FACS were incubated with the indicated concentrations of IL-1,
poly(IC), and IFN- for 40 h. - and -cell iNOS expression
was determined by Western blot analysis as described under
"Experimental Procedures." Results are representative of three
independent experiments.
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Our previous studies have shown that treatment of FACS-purified
-cells for 18 h with IL-1 results in an inhibition of
glucose-stimulated insulin secretion that is attenuated by coincubation
with AG (22, 24). Treatment of primary
-cells with poly(IC) + IFN-
for 24 h results in a complete inhibition of
glucose-stimulated insulin secretion (Fig.
5). AG prevents poly(IC) + IFN-
-induced inhibition of insulin secretion suggesting that the
inhibitory effects are mediated by nitric oxide. In addition, we have
previously shown that AG does not inhibit insulin secretion by purified
-cells in the absence of cytokine treatment (22, 24). These results show that dsRNA, in combination with IFN-
, is able to act directly on the
-cell, stimulating iNOS expression and inhibiting insulin secretion in a nitric oxide-dependent manner.

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Fig. 5.
Effects of poly(IC) and
IFN- on glucose-stimulated insulin secretion
by FACS-purified -cells. -cells
purified by FACS were incubated for 24 h with the indicated
concentrations of IL-1, poly(IC), IFN- , and AG. The cells were then
isolated, and glucose-stimulated insulin secretion was performed as
described under "Experimental Procedures." Results are the
average ± S.E. of four independent experiments containing three
replicates/condition. Statistical significance, p < 0.05 versus control (*) as indicated.
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 |
DISCUSSION |
The biochemical events that precipitate the initial destruction of
pancreatic
-cells during the development of autoimmune diabetes have
remained elusive. It has been proposed that viral infection of islets
may "trigger" or initiate an autoimmune process in genetically
predisposed individuals by stimulating the initial destruction of
-cells (7-11). However, few studies have examined the effects of
viral infection on the function and viability of isolated islets. In
this study, we have examined the effects of the viral replicative
intermediate, dsRNA, on islet function and viability. dsRNA is the
active component of a viral infection that stimulates antiviral
responses in infected cells (13). We show that treatment of rat islets
with dsRNA + IFN-
results in the time- and concentration- dependent
expression of iNOS and production of nitric oxide. dsRNA + IFN-
-induced nitric oxide production results in a potent inhibition
of insulin secretion and islet degeneration. One islet cellular source
of iNOS appears to be the
-cell, because poly(IC) + IFN-
induces
iNOS expression and inhibits insulin secretion by primary
-cells
purified by FACS. In addition, treatment of primary
-cells with
dsRNA + IFN-
results in the inhibition of insulin secretion. AG
prevents the inhibitory effects of dsRNA + IFN-
on
-cell function
indicating that the destructive effects are mediated by
-cell
production of nitric oxide. These results suggest that one potential
mechanism by which a viral infection may mediate the initial damage to
-cells during the development of autoimmune diabetes is by the
induction of iNOS expression by
-cells followed by nitric
oxide-mediated inhibition of
-cell function and eventual islet
damage. In support of this hypothesis, we have recently shown that
incubation of rat islet cells with dsRNA + IFN-
stimulates an
~3-4-fold increase in both
-cell DNA damage (colocalization of
insulin with DNA damage assessed by immunohistochemistry and
TdT-mediated dUTP nick-end labeling staining) and islet cell necrosis
(determined by acridine orange/ethidium bromide staining), and that
both effects are attenuated by
NMMA.2
Viral infection and poly(IC) are classic activators of macrophages,
stimulating antiviral responses such as type I interferon production
and nitric oxide production (36-44). We have recently shown that
activation of resident mouse macrophages, by treatment with poly(IC) + IFN-
, results in the induction of iNOS, the production of nitric
oxide, and the release of IL-1 (35). In addition, treatment of rat
islets with TNF + LPS (conditions known to activate macrophages)
results in a potent inhibition of insulin secretion (20, 24). The
inhibitory effects of TNF + LPS on insulin secretion are mediated by
intraislet IL-1 release followed by IL-1-induced iNOS expression by
-cells. Resident macrophages appear to be the source of IL-1, as a
7-day culture of rat islets at 24 °C (conditions known to deplete
islets of class II+ lymphoid cells, see Refs. 19 and 20)
prevents TNF + LPS-induced iNOS expression, nitric oxide formation and
the inhibitory effects on insulin secretion. Immunocytochemical
colocalization of IL-1
with the macrophage surface marker, ED1,
directly supports the resident islet macrophage as the islets cellular
source of IL-1 (20). Similar to the effects of TNF + LPS, poly(IC) + IFN-
may activate resident islet macrophages, stimulating nitric
oxide production and IL-1 release; however, our studies indicate that resident macrophages are not required for the inhibitory effects of
poly(IC) + IFN-
on islet function and viability. Removal of resident
macrophages by culture for 7 days at 24 °C or pseudoislet formation
does not inhibit poly(IC) + IFN-
-induced iNOS expression, nitric
oxide production or the inhibition of insulin secretion. In addition,
poly(IC) + IFN-
stimulates iNOS expression and inhibit insulin
secretion by
-cells purified by FACS. These results indicate that
the inhibitory and destructive effects of dsRNA on rat islet function
are not dependent upon resident macrophage production of nitric oxide
or IL-1 but appear to be associated with a direct interaction of dsRNA
with the
-cell. These findings provide evidence for a novel
biochemical mechanism by which a viral infection may trigger the
initial damage to
-cells leading to the development of autoimmune
diabetes. Viral infection of
-cells and the accumulation of the
viral replicative intermediate, dsRNA, in combination with IFN-
supplied by peri-insulitic T-cells, would result in the induction of
iNOS and the production of nitric oxide by
-cells, followed by
nitric oxide-mediated inhibition of
-cell function and eventual
-cell damage.