(Received for publication, September 18, 1996, and in revised form, March 5, 1997)
From The Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, Saint Louis, Missouri 63104
The purpose of this study was to
evaluate the effects of interferon- (IFN-
) alone and in
combination with interleukin 1
(IL-1
) on inducible nitric-oxide
synthase (iNOS) mRNA and protein expression, nitrite production,
and insulin secretion by islets of Langerhans. Treatment of rat islets
with IL-1
results in a concentration-dependent increase in the
production of nitrite that is maximal at 5 units/ml. Individually, 0.1 unit/ml IL-1
or 150 units/ml rat IFN-
do not stimulate iNOS
expression or nitrite production by rat islets; however, in
combination, these cytokines induce the expression of iNOS and the
production of nitrite to levels similar in magnitude to the individual
effects of 5 units/ml IL-1
. The islet
-cell, selectively
destroyed during insulin-dependent diabetes mellitus,
appears to be one islet cellular source of iNOS as 150 units/ml rat
IFN-
and 0.1 unit/ml IL-1
induced similar effects in primary
-cells purified by fluorescence-activated cell sorting and in the
rat insulinoma cell line, RINm5F. iNOS expression and nitrite
production by rat islets in response to 150 units/ml rat IFN-
and
0.1 unit/ml IL-1
are correlated with an inhibition of insulin
secretion and islet degeneration that are prevented by the iNOS
inhibitor aminoguanidine. The mechanism by which IFN-
increases the
sensitivity of
-cells for IL-1-induced iNOS expression appears to be
associated with an increase in the stability of iNOS mRNA. Last,
cellular damage during physical dispersion of islets results in the
release of sufficient amounts of IL-1
to induce iNOS expression and
nitrite production in the presence of exogenously added rat IFN-
.
The cellular source of IL-1
under these conditions is believed to be
resident islet macrophages as depletion of macrophages prior to
dispersion prevents IFN-
-induced iNOS expression and nitrite
formation by dispersed islet cells. These studies show that the
T-lymphocyte cytokine, IFN-
, increases the sensitivity of rat islets
to the effects of IL-1
on iNOS expression and nitrite production by
10-fold, in part, through the stabilization of iNOS mRNA. Our
studies also support an effector role for IFN-
, in concert with
resident islet macrophage release of IL-1
, in mediating
-cell destruction during the development of autoimmune diabetes.
Insulin-dependent diabetes mellitus is an autoimmune
disease characterized by the selective destruction of insulin secreting -cells found in islets of Langerhans. Many lines of evidence support
a role for the involvement of cytokines as effector molecules that
participate in the development of diabetes. Mandrup-Poulsen et
al. (1) first showed that treatment of isolated rat islets with
conditioned media derived from activated mononuclear cells results in a
potent inhibition of insulin secretion followed by islet destruction.
The active component of this conditioned media was determined to be the
cytokine IL-11 (2). IL-1-induced inhibition
of insulin secretion is both time- and
concentration-dependent and requires mRNA transcription and new protein synthesis (3). Recently, IL-1-induced inhibition of
insulin secretion has been attributed to the expression of iNOS and
increased production of nitric oxide by
-cells (4, 5).
Southern et al. (6) first demonstrated that treatment of rat
islets with IL-1 and tumor necrosis factor results in an inhibition
of insulin secretion that is attenuated by the nitric-oxide synthase
inhibitor nitro-L-arginine methyl ester. We and others (7-9) have shown that IL-1
-induced inhibition of insulin secretion and IL-1
-induced nitrite production by rat islets are completely prevented by
NG-monomethyl-L-arginine
(NMMA) and aminoguanidine (AG). The expression of iNOS by rat islets
has been demonstrated at the level of mRNA and protein (10-12).
Immunohistochemical colocalization of iNOS and insulin demonstrates
that IL-1
selectively induces the expression of iNOS by
-cells
(12). The inhibitory and destructive effects of IL-1
on islet
function and viability are mediated, in part, by the ability of nitric
oxide to target and inhibit the enzymatic activity of mitochondrial
enzymes, including aconitase and the electron transport chain complexes
I and II (7, 13, 14). Treatment of rat islets for 18 h with
IL-1
results in an 80% inhibition of aconitase activity that is
prevented by NMMA (7, 13). IL-1
has also been shown to reduce islet
cellular levels of ATP and to inhibit glucose oxidation in a nitric
oxide-dependent manner (15).
Autoimmune diabetes is associated with a local inflammatory reaction
(insulitis) in and around pancreatic islets. In an activated state,
T-lymphocytes and macrophages, primary cellular components of islet
insulitis, release high levels of IL-1 and IFN-, respectively. Although the effects of IL-1 on islet function have been examined in
detail, few studies have investigated the effects of IFN-
on
-cell function and viability. Many lines of evidence support a role
for IFN-
in the development of autoimmune diabetes. This evidence
includes 1) IFN-
mRNA expression in islets correlates with the
development of insulitis and diabetes in the nonobese diabetic mouse
(16); 2) transgenic mice expressing IFN-
under control of the
insulin promoter develop insulitis and diabetes (17); and 3) monoclonal
antiserum specific for IFN-
attenuates the development of diabetes
in the nonobese diabetic mouse (18, 19). In this study we have examined
the effects of IFN-
alone and in combination with IL-1
on iNOS
expression, nitrite formation, and islet function and viability. Alone,
IFN-
does not modulate islet function or viability; however, IFN-
increases the sensitivity of rat islets to IL-1
by stabilizing
IL-1-induced iNOS mRNA expression resulting in the increased
production of nitric oxide. The increased sensitivity of rat islets for
IL-1 results in the inhibition of
-cell function and islet
destruction at concentrations of IL-1
that alone have no effect on
islet viability or function.
RINm5F cells were obtained from
Washington University Tissue Culture Support Center (St. Louis, MO).
RPMI Medium 1640 containing 1 × L-glutamine,
CMRL-1066 tissue culture medium, L-glutamine, penicillin,
streptomycin, and rat and human 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). Human islets were obtained from the Islet Isolation
Core Facility at Washington University School of Medicine (St. Louis,
MO) and the Diabetes Research Institute at the University of Miami
(Miami, FL). Aminoguanidine hemisulfate (AG) and collagenase type XI
were from Sigma. [
-32P]dCTP and enhanced
chemiluminescence reagents were purchased from Amersham Corp. 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
(Department of Pathology, Washington University, St. Louis, MO),
respectively. Mouse recombinant interleukin 1 receptor antagonist
protein (IRAP) was a gift from Dr. Charles Hall (Upjohn, Kalamazoo,
MI). All other reagents were from commercially available sources.
Islets were isolated from male Sprague Dawley rats by collagenase digestion as described previously (20). 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. Human islets were incubated for 48 h at 37 °C in complete CMRL-1066 before the initiation of experiments. 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 then initiated by the addition of cytokines and iNOS inhibitors, followed by culture for the indicated times.
For experiments using RINm5F cells, cells were removed from growth flasks by treatment with 0.05% trypsin, 0.02% EDTA at 37 °C. Cells were washed 2 times with complete CMRL-1066, plated at a concentration of 200,000 cells/200 µl of complete CMRL-1066 in 96-well microtiter plates, and then cultured at 37 °C for 2-3 h prior to cytokine treatment. Experiments were initiated by the addition of cytokines, and the RINm5F cells were then incubated for the indicated times at 37 °C.
Islet Dispersion and Macrophage DepletionIsolated rat islets were 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). The dispersed islet cells were counted and then immediately aliquoted into 24-well microtiter plates (100,000 cells/well in 400 µl of complete CMRL-1066) and cultured for 1 h at 37 °C. For macrophage depletion studies, islets were cultured for 7 days at 24 °C followed by 2 days at 37 °C prior to cell dispersion (24). Where indicated, islet cells were pretreated for 30 min with IRAP; cytokines were then added, and the islet cells were incubated for 24 h.
Purification ofIslets isolated from 10 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 as stated above. 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 (11,
13, 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. The sorting process yielded a 95% population of -cells and an 80-85% population of
-cells.
Islets (220/ml of complete CMRL-1066)
were cultured for 40 h with the indicated concentrations of
IL-1, IFN-
, and aminoguanidine (AG). The islets were then
isolated and washed 3 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. Groups of 20 islets were counted into 10- × 75-mm borosilicate test tubes and preincubated for 30 min at 37 °C
with shaking in 200 µl of the same buffer. The preincubation buffer
was removed, and glucose-stimulated insulin secretion was initiated by
the addition of 200 µl of KRB containing either 3 or 20 mM D-glucose. Islets were then incubated at
37 °C for 30 min, the incubation buffer was removed, and insulin
content was determined by radioimmunoassay (23).
Islets (25/500 µl of complete CMRL-1066)
were cultured for 96 h in 24-well microtiter plates with the
indicated concentrations of IL-1, IFN-
, and aminoguanidine. 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 (14, 24, 25).
RINm5F cells (400,000/400 µl of
complete CMRL-1066), cultured in 24-well microtiter plates with the
indicated concentrations of IL-1 and IFN-
for 24 h at
37 °C, were washed 3 times with 0.1 M phosphate-buffered
saline (PBS), pH 7.4, followed by the addition of 25 µl of sodium
dodecyl sulfate (SDS) sample mix (0.25 M Tris-HCl, 20%
-mercaptoethanol, and 4% SDS). The lysed cells were then
transferred to 1.5-ml microcentrifuge tubes, and the individual wells
of the microtiter plate were rinsed with 15 µl of distilled
H2O which was then added to the corresponding lysed samples. The samples were boiled for 4 min followed by the addition of
4 µl of loading dye (0.05% bromphenol blue in 80% glycerol). Rat
islets (150/400 µl of complete CMRL-1066) were cultured for 40 h
with the indicated concentrations of IL-1
and IFN-
at 37 °C under an atmosphere of 95% air and 5% CO2. The islets
were isolated by centrifugation (6,000 × g, 3 min) and
washed 3 times with 0.1 M PBS. Islets were lysed by the
addition of 25 µl of SDS sample mix and 15 µl of distilled
H2O, boiled for 4 min, followed by the addition of 4 µl
loading dye. Proteins were separated by SDS-gel electrophoresis using
standard conditions (26) and transferred to Nitrocell nitrocellulose
membranes (Pharmacia Biotech Inc.) under semi-dry transfer conditions.
Blots were blocked overnight in TBST (20 mM Tris, 500 mM NaCl, and 0.1% Tween 20, pH 7.5) containing 5% nonfat
dry milk. Blots were washed one time with TBST and then incubated for
1.5 h at room temperature with rabbit anti-mouse iNOS (1:2000
dilution) in TBST containing 1% nonfat dry milk. Following incubations
with the primary antisera, blots were washed 4 times with TBST (5 min/wash) and then incubated for 1 h at room temperature with
horseradish peroxidase-conjugated donkey anti-rabbit secondary antibody
at a dilution of 1:7000. The blots were washed 3 times in TBST and once
in 0.1 M PBS at room temperature. Detection of rat iNOS was
by enhanced chemiluminescence according to manufacturer's specifications (Amersham Corp.).
RINm5F cells (10 × 106 cells/3 ml complete CMRL-1066) were cultured for 6 and
12 h at 37 °C with the indicated concentrations of IL-1 and
IFN-
. For the mRNA stability experiments, RINm5F cells (10 × 106 cells/3 ml of complete CMRL-1066) were cultured for
6 h in the presence of the indicated concentrations of IL-1 and
IFN-
. Actinomycin D (1 µM) was then added, and the
cells were cultured for an additional 6 h. After culture, the
cells were washed 3 times with 0.1 M PBS, pH 7.4, and total
RNA was isolated using the RNeasy kit (Qiagen, Inc., Chatsworth, CA).
Total cellular RNA (10-20 µg) was denatured and fractionated by gel
electrophoresis using a 1.0% agarose gel containing 2.2 M
formaldehyde. RNA was transferred by capillary action in 20 × SSC
(3 M NaCl, 0.3 M sodium citrate, pH 7.0) to Duralon UV nylon membranes (Stratagene, La Jolla, CA), and the 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. 28 S RNA band or cyclophilin was used as an internal control
for RNA loading. Hybridization and autoradiography were performed as
described previously (28).
Autoradiograms were scanned into NIH Image version 1.59 using a COHU high performance CCD camera (Brookfield, WI). Densities were determined using NIH Image version 1.59 software. PhosphorImaging analysis of RINm5F cell mRNA stability experiments was performed using a Molecular Dynamics PhosphorImager and Molecular Dynamics ImageQuant Software Version 3.3 (Molecular Dynamics, Inc.). For Western blot data, autoradiograms were scanned into NIH Image and then imported into Canvas 3.5 (Deneba Software, Miami, Fl) for the preparation of figures.
Nitrite DeterminationNitrite 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.
StatisticsStatistical comparisons were made between groups using a one-way analysis of variance. Significant differences between treatment groups compared with untreated controls (p < 0.05) were evaluated using a Scheffe's F test posthoc analysis.
To determine if
IFN- modulates iNOS expression, the effects of rat IFN-
, alone
and in combination with IL-1
, on nitrite production and iNOS
expression by isolated rat islets were examined. Incubation of rat
islets for 40 h with IL-1
results in a
concentration-dependent increase in the production of
nitrite (Fig. 1A). IL-1
induces the first
detectable increase in nitrite production by rat islets at 0.5 units/ml
IL-1
with maximal nitrite production observed at 1 and 5 units/ml
IL-1
(data not shown for 1 unit/ml IL-1
). Alone, rat IFN-
(concentrations from 1.5 to 150 units/ml) does not stimulate nitrite
formation by rat islets; however, in the presence of 0.1 unit/ml
IL-1
(which alone does not stimulate nitrite formation), rat IFN-
induces the concentration-dependent production of nitrite by rat
islets. The levels of nitrite produced in response to 0.1 unit/ml
IL-1
and 150 units/ml rat IFN-
are similar in magnitude to the
levels produced by rat islets treated with 5 units/ml IL-1
. Rat
IFN-
also slightly increases (~8%) the production of nitrite by
rat islets stimulated with 5 units/ml IL-1
as compared with 5 units/ml IL-1
alone.
The effects of rat IFN- and IL-1
on iNOS expression from the same
islets used in Fig. 1A are shown in Fig. 1B.
Alone, 0.1 unit/ml IL-1
or 150 units/ml rat IFN-
do not induce
the expression of iNOS by rat islets; however, a combination of 0.1 unit/ml IL-1
and 150 units/ml rat IFN-
stimulates the expression
of iNOS to levels similar in magnitude to the expression of iNOS
induced by 5 units/ml IL-1
. Also, the combination of 5 units/ml
IL-1
and 150 units/ml rat IFN-
induce iNOS expression to levels
that exceed those induced by the treatment of rat islets with 5 units/ml IL-1
. This effect is consistent with the ability of rat
IFN-
to increase the level of nitrite produced by rat islets in
response to maximal concentrations of IL-1
.
These results indicate that rat IFN-, in combination with IL-1
at
concentrations that alone do not induce iNOS expression, stimulate the
expression of iNOS by rat islets to levels that are similar to the
individual effects of maximal concentrations of IL-1
. For
convenience, we have defined 0.1 unit/ml IL-1
as submaximal and 1 and 5 units/ml IL-1
as maximal concentrations of IL-1
.
Our
previous studies have shown that nitric oxide mediates the inhibitory
effects of IL-1 on glucose-stimulated insulin secretion (8). The
effects of rat IFN-
and IL-1
on insulin secretion by rat islets
were examined to determine if nitric oxide production, stimulated by
submaximal concentrations of IL-1
in the presence of rat IFN-
, is
associated with an inhibition of insulin secretion. Treatment of rat
islets with a maximal concentration of IL-1
results in a complete
inhibition of glucose-stimulated insulin secretion (Table
I). We have previously shown that IL-1
-induced inhibition of insulin secretion is prevented by the NOS inhibitors NMMA
and AG and that iNOS inhibitors do not modulate glucose-stimulated insulin secretion in the absence of cytokines (8, 9). Incubation of
islets for 40 h with a combination of rat IFN-
and a submaximal concentration of IL-1
also results in a complete inhibition of insulin secretion that is prevented by AG (Table I). Individually, submaximal IL-1
or rat IFN-
do not inhibit glucose-stimulated insulin secretion. The lack of an inhibitory effect of submaximal IL-1
or rat IFN-
on insulin secretion is consistent with the inability of these cytokines to stimulate nitrite production by rat
islets. These findings indicate that treatment of rat islets with a
submaximal concentration of IL-1
, in the presence of rat IFN-
,
results in an inhibition of insulin secretion that is mediated by the
production of nitric oxide.
|
The effects of rat IFN-, alone and in combination with IL-1
, on
islet viability are also shown in Table I. Incubation of islets for
96 h with a maximal concentration of IL-1
results in the
complete degeneration of islets. Islet degeneration is characterized by
loss of islet integrity and islet dispersion (14, 24, 25). The
destructive effects of IL-1
on islet viability are completely
prevented by the iNOS inhibitor aminoguanidine (AG), indicating that
nitric oxide participates in IL-1
-induced islet degeneration. Alone,
rat IFN-
(150 units/ml) or a submaximal concentration of IL-1
do
not induce islet degeneration; however, in combination these cytokines
stimulate islet degeneration to levels similar to the individual
effects of maximal concentrations of IL-1
alone. The destructive
effects of submaximal concentrations of IL-1
, in combination with
rat IFN-
, are completely prevented by AG. These findings indicate
that islet degeneration stimulated by rat IFN-
and submaximal
concentrations of IL-1
is mediated by the production of nitric
oxide.
Islets contain a
heterogeneous population of both endocrine and nonendocrine cells, of
which the insulin-secreting -cell is selectively destroyed during
the development of autoimmune diabetes. To determine if rat IFN-
increases the sensitivity of
-cells for IL-1
-induced iNOS
expression, we have examined the effects of rat IFN-
alone, and in
combination with IL-1
, on nitrite production and iNOS expression by
primary rat
- and
-cells purified by FACS. As shown in Fig.
2, treatment of primary
-cells with submaximal
IL-1
in the presence of rat IFN-
stimulates the production of
nitrite to levels similar in magnitude to the effects of maximal
concentrations of IL-1
alone. Also, nitrite production by primary
-cells incubated with maximal concentrations of IL-1
, or the
combination of maximal IL-1
and rat IFN-
, are virtually
identical.
The effects of rat IFN- and IL-1
on iNOS expression correlate
with the effects of these cytokines on nitrite production by primary
-cells. As shown in Fig. 2B, submaximal concentrations of
IL-1
or rat IFN-
do not stimulate the expression of iNOS by
primary
-cells. However, in combination, these cytokines stimulate the expression of iNOS to levels that are slightly higher than the
effects of maximal concentrations of IL-1
on iNOS expression by
primary
-cells. Also shown in Fig. 2 are the effects of IL-1
and
rat IFN-
on nitrite formation and iNOS expression by primary rat
-cells. Individually or in combination, IL-1
and rat IFN-
do
not stimulate the production of nitrite or the expression of iNOS by
primary
-cells. These experiments demonstrate that the combination
of submaximal concentrations of IL-1
in the presence of rat IFN-
stimulates the expression of iNOS by primary
-cells, suggesting that
the
-cell is one islet cellular source of iNOS under these
conditions.
We
have examined the time-dependent production of nitrite and
iNOS mRNA accumulation using RINm5F cells. RINm5F cells represent a
homogeneous population of -cells that respond to IL-1 and IFN-
in
a manner similar to the effects of these cytokines on iNOS expression
by intact islets. As shown in Fig. 3A, a
maximal concentration of IL-1
stimulates the
time-dependent production of nitrite that is first apparent
at 6 h, then progresses linearly from 6 to 24 h, with little
increase in the level of nitrite from 24 to 48 h. Individually,
rat IFN-
or submaximal concentrations of IL-1
do not stimulate
the production of nitrite by RINm5F cells at any time point examined;
however, the combination of these cytokines induces the
time-dependent production of nitrite by RINm5F cells that
is similar to the effects of maximal concentrations of IL-1
alone.
Nitrite production induced by the combination of submaximal concentrations of IL-1
in the presence of rat IFN-
is first detected 6 h after the addition of cytokines (control, 2.9 ± 0.6 pmol/2000 cells versus IL-1
+ IFN-
, 4.3 ± 0.6 pmol/2000 cells) and increases linearly from 6 to 24 h. The
rate by which IL-1
alone or the combination of IL-1
and rat
IFN-
stimulate nitrite formation by RINm5F cells was determined by
linear regression of nitrite data from 6 to 24-h time points shown in
Fig. 3A. The rate of nitrite formation induced by the
combination of submaximal IL-1
and rat IFN-
is reduced compared
with the individual effects of maximal IL-1
(2.5 pmol of nitrite/h
versus 3.6 pmol nitrite/h, respectively). Also, the maximal
level of nitrite produced in response to submaximal IL-1
and rat
IFN-
is ~20-30% less than that induced by maximal IL-1
alone.
Although nitrite production by RINm5F cells in response to submaximal
concentrations of IL-1 in combination with rat IFN-
is similar to
the effects of maximal IL-1
(in terms of the time dependence), the
effects of these two conditions on iNOS mRNA accumulation are
different. As shown in Fig. 3B, iNOS mRNA accumulation in response to a maximal concentration of IL-1
is 2-fold higher than
the effects of submaximal concentrations of IL-1
and rat IFN-
following a 6-h incubation. However, following a 12-h incubation, the
levels of iNOS mRNA that accumulate in response to submaximal concentrations of IL-1
and rat IFN-
are nearly identical to the
levels observed following a 6-h exposure, whereas maximal IL-1
-induced iNOS mRNA accumulation is reduced to near
background levels.
The persistence of iNOS mRNA accumulation following a 12-h exposure
of RINm5F cells with submaximal concentrations of IL-1 in
combination with IFN-
compared with maximal concentrations of
IL-1
alone (Fig. 3B) suggests that IFN-
may stabilize
IL-1-induced iNOS mRNA. To examine this question, an analysis of
iNOS mRNA stability using the transcriptional inhibitor actinomycin
D was performed. In Fig. 3C, RINm5F cells were incubated for
6 h in the presence of IFN-
and maximal or submaximal
concentrations of IL-1
or with maximal concentrations of IL-1
alone. Actinomycin D was then added, and the RINm5F cells were cultured
for 6 additional h. As shown in Fig. 3C, ~70%
IL-1
-induced iNOS mRNA is degraded in the 6-h incubation
following the addition of actinomycin D; however, only ~30% iNOS
mRNA is degraded in the presence of maximal concentrations of IL-1
in combination with IFN-
, and only ~40% iNOS mRNA is degraded
in the presence of submaximal concentrations of IL-1 in combination
with IFN-
. These data suggest a role for IFN-
in the
stabilization of IL-1
-induced iNOS mRNA that results in the
persistence of iNOS mRNA accumulation after a 12-h exposure to
these cytokines (Fig. 3B). Whereas maximal IL-1
-induced
iNOS mRNA accumulation is ~2-fold greater than for submaximal
IL-1
in the presence of INF-
, the increase in iNOS mRNA
stability afforded by IFN-
ultimately results in similar levels of
iNOS protein expression (data not shown) and nitrite production under both conditions.
Islets contain resident macrophages
that are known to express and release IL-1. We have previously shown
that endogenous release of IL-1 within islets results in an inhibition
of insulin secretion that is mediated by -cell expression of iNOS
(12). To further examine the extent to which the presence of IFN-
is
able to increase the sensitivity of islets to IL-1, islets were
dispersed into individual cells and treated with varying concentrations
of IFN-
or with a maximal concentration of IL-1
alone (Fig.
4). Islet dispersion involves the treatment of intact
islets with trypsin, an experimental manipulation that results in the
destruction of 2-3% islet cells (based on trypan blue exclusion, data
not shown). As shown in Fig. 4A, treatment of dispersed
islet cells with rat IFN-
stimulates nitrite formation and iNOS
expression (inset) in a concentration-dependent
manner. The interleukin-1 receptor antagonist protein (IRAP), which
competes with IL-1 for receptor binding (33), completely prevents
IFN-
-induced nitrite production and iNOS expression by dispersed
islet cells. These findings indicate that sufficient levels of IL-1
are released during dispersion to stimulate iNOS expression in the
presence of IFN-
.
The cellular source of the IL-1 released during islet cell dispersion
is believed to be the resident islet macrophage. To provide evidence
for the intra-islet macrophage as a source of IL-1, the effects of
macrophage depletion on IFN--induced iNOS expression and nitrite
formation by dispersed islet cells were examined. Macrophage depletion
was accomplished by culturing intact islets for 7 days at 24 °C.
This culture condition has previously been shown to deplete over 95%
of the islet lymphoid population (24). As shown in Fig. 4B,
IFN-
no longer stimulates iNOS expression (inset) or
nitrite production by islet cells dispersed from macrophage-depleted islets. The culture conditions used for macrophage depletion do not
damage islet endocrine cells as maximal concentrations of IL-1 induce
the expression of iNOS and the production of nitrite. These findings
provide evidence for the resident islet macrophage as the cellular
source of IL-1 and indicate that IFN-
increases the sensitivity of
islet cells for iNOS expression stimulated by the endogenous release of
IL-1.
We have also evaluated the effects of human
IFN- and IL-1
on nitrite production by human islets of
Langerhans. We and others (30, 31) have shown previously that the
minimal combination of cytokines required to stimulate iNOS expression
and nitrite formation by human islets is IFN-
and IL-1
.
Concentrations of IL-1
and human IFN-
that have been used to
stimulate iNOS expression and nitrite production by human islets are
50-75 and 750-1000 units/ml, respectively (30-32). Because rat
IFN-
is effective at reducing the amount of IL-1
required to
stimulate nitrite formation by rat islets, the effects of human IFN-
on IL-1
-induced nitrite formation by human islets were examined. In
the presence of 750 units/ml human IFN-
, IL-1
induces a
concentration-dependent increase in nitrite formation (Fig.
5) by human islets that is first detected at 1 unit/ml
IL-1
and increases linearly to 50 units/ml IL-1
. Nitrite
production stimulated by 75 units/ml IL-1
and 750 units/ml human
IFN-
is slightly lower than the level of nitrite produced by human
islets treated with 50 units/ml IL-1
and 750 units/ml human IFN-
.
In the presence of 75 units/ml human IFN-
and IL-1
at
concentrations from 1 to 75 units/ml, lower levels of nitrite are
produced as compared with 750 units/ml human IFN-
; however, 10 units/ml IL-1
and 75 units/ml human IFN-
induce a 2-fold increase
in nitrite production as compared with untreated human islets. Although
human IFN-
reduces the concentration of IL-1 required to stimulate
iNOS expression by human islets, the concentrations of human IFN-
used in these studies may be higher than those present in and around
islets during the development of diabetes.
In this report we have examined the effects of IFN-, alone and
in combination with IL-1, on iNOS expression and nitrite production by
both rat and human islets of Langerhans. We demonstrate that rat
IFN-
increases the sensitivity of rat islets for IL-1-induced iNOS
expression by 10-fold. Alone, concentrations of IL-1
as low as 0.1 unit/ml (0.57 pM) or 150 units/ml rat IFN-
do not induce
iNOS expression or nitrite production by rat islets, RINm5F cells, or
primary
-cells purified by FACS; however, in combination, these
cytokines induce the expression of iNOS and the production of nitrite
to similar levels induced by maximal concentrations of IL-1
. Also,
IL-1
, alone or in combination with rat IFN-
, does not stimulate
nitrite formation or iNOS expression by primary
-cells, the other
major endocrine cell type found in islets. These results are consistent
with previous studies that have identified the
-cell as the islet
cellular source of iNOS in response to IL-1
(12, 13) and provide the
first direct evidence that IFN-
modulates the function and viability
of primary
-cells by increasing the sensitivity of
-cells for
IL-1
-induced iNOS expression. The combination of IL-1
and rat
IFN-
also results in inhibition of insulin secretion and islet
destruction that are prevented by the iNOS inhibitor AG. These findings
indicate that nitric oxide participates in the inhibitory and
destructive effects of submaximal concentrations of IL-1
plus rat
IFN-
on insulin secretion and islet destruction.
The mechanism by which IFN- increases the sensitivity of
-cells
for iNOS expression and nitrite production in response to IL-1
appears to be associated with an increase in the stability of iNOS
mRNA. Consistent with previous studies (34), maximal concentrations
of IL-1
induce an 8-fold increase in the accumulation of iNOS
mRNA following a 6-h exposure; however, iNOS mRNA accumulation is reduced to near background levels following a 12-h incubation. In
contrast, nearly equivalent levels of iNOS mRNA accumulate in
RINm5F cells treated for 6 or 12 h with submaximal concentrations of IL-1
in combination with rat IFN-
. Stability studies indicate that iNOS mRNA induced by IL-1
and rat IFN-
is approximately 2-fold more stable than the individual effects of IL-1
. These findings indicate that IFN-
may induce the expression of factors, and/or activate factors that stabilize iNOS mRNA, thus preventing iNOS mRNA degradation or that IFN-
may inhibit the activity of factors that are required for down-regulating iNOS mRNA expression, and/or the degradation of iNOS mRNA. These possibilities are
currently under investigation.
It is clear from studies with rat islets that IFN- reduces the
concentration of IL-1
required to stimulate iNOS expression by
-cells; however, it is important to evaluate the effectiveness of
human IFN-
on iNOS expression by human islets treated with maximal
and submaximal concentrations of IL-1
to determine if human islets
respond in a similar manner. In this study we show that IL-1
, at a
concentration as low as 1 unit/ml (5.7 pM), is able to
stimulate high levels of nitrite production by human islets in the
presence of human IFN-
(750 units/ml). We also show that in the
presence of 75 units/ml human IFN-
, as little as 10 units/ml IL-1
is required to induce a 2-fold increase in the level of nitrite
production. These results indicate that IFN-
reduces the
concentration of IL-1
required to stimulate iNOS expression by human
islets in a manner similar to IFN-
's effects on rat islets.
We have also evaluated the effects of endogenous IL-1 release on iNOS
expression and nitrite production by rat islets. Macrophages are
believed to play a primary role in the development of autoimmune diabetes. Islets contain approximately 10-15 resident macrophages. Macrophage depletion, by silica treatment or feeding a diet deficient in essential fatty acids, prevents the natural occurrence of diabetes in the Bio Breeding rat and prevents the development of diabetes induced by multiple injections of streptozotocin in CD-1 mice (35-37).
We have previously shown that treatment of rat islets with tumor
necrosis factor + lipopolysaccharide, conditions known to activate
macrophages, results in the release of IL-1 within islets and that IL-1
subsequently stimulates iNOS expression and nitric oxide production by
-cells resulting in the inhibition of
-cell function (12). These
studies have led to the suggestion that activation of resident islet
macrophages may represent a triggering event associated with the
initiation of autoimmune diabetes (12, 38). We show that IFN-
, in
the absence of exogenously added IL-1, induces the expression of iNOS
and the production of nitrite by islets physically dispersed into
individual cells by trypsin treatment. Under these conditions, iNOS
expression and nitrite production are prevented by IRAP, indicating
that during the dispersion process low levels of IL-1 are released from
cells that may be damaged during this procedure. The cellular source of
IL-1 in the dispersed islet cells appears to be resident macrophages.
Macrophage depletion of islets prior to islet dispersion completely
prevents IFN-
-induced nitrite formation and iNOS expression. Cellular mechanisms associated with macrophage release of IL-1 are
incompletely characterized; however, macrophage death, by either
apoptosis or necrosis, is known to result in the release of IL-1 (39,
40). Macrophage damage and IL-1 release during islet dispersion is
consistent with these previous studies showing IL-1 release following
macrophage death.
The release of IL-1, in islets following cellular damage, represents a
novel mechanism that may be associated with the initiation of
autoimmune diabetes. Environmental toxins or viral infections have been
proposed to target and destroy -cells during the initiation of
autoimmune diabetes (41, 42). In contrast, our data support a potential
role for the resident islet macrophage as a target for such an event.
Viral infection of macrophages, or macrophage death stimulated by
chemical toxins, in islets under immune surveillance or peri-insulitis
(conditions associated with the migration but not infiltration of
T-lymphocytes into islets), could result in the release of IL-1 in the
local environment of the islet. In the presence of IFN-
, the release
of low levels of IL-1 would be sufficient to stimulate iNOS expression
and nitric oxide production by
-cells. Nitric oxide production by
-cells would then result in the inhibition of function and the
destruction of
-cells, resulting in the release of autoantigens and
the initiation of a T-cell-mediated immune response. In addition,
tissue damage associated with acute and chronic inflammation and injury
may be mediated by a similar mechanism. Low levels of IL-1 released in
the presence of IFN-
could result in high levels of iNOS expression and nitric oxide production by target cells, leading to target cell
damage. Under these conditions, macrophage production of nitric oxide
may participate in target tissue damage; however, as our studies
indicate, cytokine release by activated resident macrophages and
cytokine-induced iNOS expression by target cells may be the more
important mechanism associated with tissue damage. If our hypothesis is
correct, human macrophage production of nitric oxide is not required
for target cell damage. This interpretation is consistent with the
difficulties in demonstrating human macrophage production of nitric
oxide. In conclusion, our studies show that the T-cell cytokine,
IFN-
, directly inhibits the function and viability of islets by
reducing the concentration of IL-1 required to stimulate iNOS
expression and nitric oxide production by
-cells. These findings
support an effector role for IFN-
, in concert with IL-1, in
mediating the initial destruction of
-cells during the development
of autoimmune diabetes.
We thank Colleen Kelly for expert technical
assistance, Parveen Chand for assistance with FACS purification of
-cells, and Drs. Claudette Klein, Peggy Weidman, and Polly Hansen
for helpful discussion with the preparation of this manuscript. We also
thank the Diabetes Research and Training Center at Washington
University for performing insulin RIAs.