| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO 63104
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and LPS results in a potent inhibition
of glucose-stimulated insulin secretion. The inhibitory actions of
TNF + LPS are mediated by the intraislet production and release of
IL-1 followed by IL-1-induced inducible nitric oxide synthase (iNOS)
expression by ß cells. The IL-1R antagonist protein completely
prevents TNF + LPS-induced nitrite production, iNOS expression and
the inhibitory effects on glucose-stimulated insulin secretion by rat
islets. 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 lymphoid cells) prevents TNF + LPS-induced iNOS
expression, nitrite production, and the inhibitory effects on insulin
secretion. In addition, macrophage depletion also inhibits TNF +
LPS-induced IL-1 and IL-1ß mRNA expression in rat islets.
Immunocytochemical colocalization of IL-1ß with the
macrophage-specific marker ED1 was used to provide direct support for
resident macrophages as the islet cellular source of IL-1. IL-1ß
appears to mediate the inhibitory actions of TNF + LPS on ß cell
function as TNF + LPS-induced expression of IL-1ß is fourfold
higher than IL-1, and Ab neutralization of IL-1ß prevents TNF
+ LPS-induced nitrite production by rat islets. These findings support
a mechanism by which the activation of resident islet macrophages and
the intraislet release of IL-1 may mediate the initial dysfunction and
destruction of ß cells during the development of autoimmune
diabetes. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
IL-1 is one of the predominant cytokines released by macrophages. Mandrup-Poulsen et al. (5, 6) first showed that treatment of rat islets with IL-1 results in a potent inhibition of insulin secretion followed by islet destruction. We and others have shown that the cellular mechanism by which IL-1 mediates an inhibitory effect on islet function and induces islet destruction involves the expression of iNOS3 and the increased production of nitric oxide (7, 8, 9). Treatment of rat islets with IL-1 results in a time-dependent inhibition of insulin secretion that is associated with a similar time-dependent production of nitric oxide (8, 10). Inhibitors of iNOS, NG-monomethyl-L-arginine (NMMA), aminoguanidine (AG), and nitro-L-arginine methylester prevent the inhibitory effects of IL-1 on insulin secretion and the destructive effects of IL-1 on islet viability (7, 8, 11, 12, 13, 14). The inhibitory effects of IL-1-induced nitric oxide production on islet function and viability are mediated, in part, by the targeting and disruption of mitochondrial electron transport (complexes I and II) and islet aconitase activity (8, 15). IL-1-induced inhibition of mitochondrial glucose oxidation to CO2 and aconitase activity are prevented by NMMA and AG (8, 10, 15, 16). The ß cell, selectively destroyed during the development of autoimmune diabetes, appears to be the sole islet cellular source of iNOS following treatment with IL-1. We have shown by immunohistochemical analysis that iNOS expression colocalizes exclusively with insulin in rat islets treated with IL-1 (17).
In 1991, Lacy and Finke (18) showed that depletion of islet lymphoid
cells prevents IFN-
-mediated islet destruction. We have extended
these original observations to show that treatment of rat islets with
TNF + LPS (classical conditions used to activate macrophages)
results in the impairment of ß cell function that is mediated by the
intraislet release of IL-1 and the subsequent expression of iNOS by ß
cells (17). In the current study, the effects of TNF + LPS on the
time dependence and isoform of IL-1 expressed in islets and the effects
of resident islet lymphoid cell depletion on TNF + LPS-induced
inhibition of ß cell function have been evaluated. Depletion of
resident lymphoid cells prevents TNF + LPS-induced iNOS
expression, nitric oxide production, and the inhibitory effects of this
cytokine and endotoxin on glucose-stimulated insulin secretion by rat
islets. In addition, we have identified IL-1ß as the primary isoform
of IL-1 that is expressed in islets under conditions associated with
resident islet macrophage activation. These findings provide a
mechanism by which the activation of resident islet macrophages may
initiate ß cell damage during the development of autoimmune
diabetes.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
CMRL-1066 tissue culture medium, penicillin, streptomycin,
L-glutamine, and PCR oligonucleotide primers were from Life
Technologies (Gaithersburg, MD). FBS was obtained from HyClone (Logan,
UT). Collagenase type XI, LPS, and aminoguanidine hemisulfate (AG) were
from Sigma Chemical Co. (St. Louis, MO). Human rIL-1ß was purchased
from Cistron Biotechnology (Pine Brook, NJ), and human rTNF- was
from Boehringer Mannheim (Indianapolis, IN).
[-32P]-labeled dCTP and enhanced chemiluminescence
reagents were obtained from Amersham (Arlington Heights, IL).
Horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG was
from Jackson ImmunoResearch Laboratories, (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). Murine rIL-1R antagonist protein (IRAP) was a gift from
Dr. Charles Hall (Upjohn, Kalamazoo, MI), and hamster anti-mouse
IL-1 and anti-mouse IL-1ß were gifts from Dr. David Chaplin
(Washington University School of Medicine, St. Louis, MO). All other
reagents were from commercially available sources.
Islet isolation and culture
Islets were isolated from male Sprague-Dawley rats (250 to 300 g; Harlan, Indianapolis, IN) by collagenase digestion as described previously (19). Islets were cultured overnight at 37°C in an atmosphere of 95% air and 5% CO2 in complete CMRL-1066 tissue culture medium (2 mM L-glutamine, 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin) before each experiment. Experiments were initiated by the addition of 5 U/ml IL-1ß, 10 ng/ml TNF, 10 µg/ml LPS, or 1 mM AG, and the islets were incubated at 37°C for the indicated time periods as described in the figure legends. In some experiments, islets were pretreated with murine rIRAP (5 µg/ml) for 30 min before adding cytokines.
Islet lymphoid cell depletion
Resident lymphoid cells were depleted from islets as described previously (18). In brief, isolated islets were cultured for 7 days in complete CMRL-1066 at 24°C in an atmosphere of 95% air and 5% CO2. Islets were removed from the 24°C culture, washed three times with fresh complete CMRL-1066, and then cultured for 3 days at 37°C in complete CMRL-1066. Experiments were conducted as described above for 37°C islets.
Glucose-stimulated insulin secretion
Islets were cultured for 40 h in either complete CMRL-1066, or complete CMRL-1066 containing IL-1ß, TNF, LPS, IRAP, and AG as indicated. Islets were then washed three times with 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% BSA, pH 7.4) containing 3 mM D-glucose. Islets were aliquoted into siliconized 10-mm x 75-mm borosilicate culture tubes (20 islets/200 µl of KRB containing 3 mM D-glucose), and preincubated for 30 min at 37°C with shaking. Insulin secretion was initiated by removing the preincubation KRB (containing 3 mM D-glucose) followed by the addition of 200 µl of KRB containing either 3 mM D-glucose or 20 mM D-glucose. Islets were incubated for 30 min in an atmosphere of 95% air and 5% CO2 at 37°C. After the incubation, the supernatant was removed and analyzed for insulin by RIA (20).
Nitrite determination
Nitrite production was determined as described previously (21) by mixing 50 µl of culture supernatant with 50 µl of Griess reagent and the absorbance at 540 nm was determined. Nitrite concentrations were calculated from a sodium nitrite standard curve.
Western blot analysis
Rat islets (150/400 µl of complete CMRL-1066) were treated with IL-1, TNF, LPS, IRAP, and AG as indicated. The islets were isolated by centrifugation (6000 x g, 3 min), and washed three times with 0.1 M PBS. Islets were lysed by adding 25 µl of SDS sample mix (0.25 M Tris-HCl, 20% ß-mercaptoethanol, and 4% SDS), and 15 µl of distilled H2O, followed by boiling for 4 min, and the addition of 4 µl of loading dye (0.05% bromphenol blue in 80% glycerol). Proteins were separated by SDS gel electrophoresis (22), and transferred to Nitrocell nitrocellulose membranes (Pharmacia Biotech, San Francisco, CA) under semidry transfer conditions. The 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 once with TBST and then incubated for 1.5 h at room temperature with rabbit anti-mouse iNOS (diluted 1:2000) in TBST containing 1% nonfat dry milk. After incubating in the primary antisera, blots were washed four times with TBST (5 min/wash), and then incubated for 1 h at room temperature with horseradish peroxidase-conjugated donkey anti-rabbit secondary Ab (1:7000 dilution). The blots were then washed three times in TBST and once in 0.1 M PBS at room temperature. Rat iNOS was detected by ECL according to product specifications.
Immunoprecipitation of IL-1 and IL-1ß
Rat islets (700) were cultured with or without 10 ng/ml TNF
+ 10 µg/ml LPS for 2.5 h in 1 ml of methionine-deficient medium
(9 parts MEM minus methionine:1 part MEM containing methionine).
[35S]Methionine (500 µCi) was added and the islets were
cultured for an additional 6 h. Islets were isolated by
centrifugation and then 600 ml of lysis buffer (10 mM Tris, 140 mM
NaCl, 1% Nonidet P-40, 1% BSA, 100 µg/ml aprotinin, 100 µg/ml
leupeptin, 1 mM iodoacetamide, and 1 mM PMSF) was added and the islets
were incubated for 1 h at 4°C. Cellular debris was removed by
centrifugation (30 min, 10,000 x g, 4°C), and
IL-1 and IL-1ß were immunoprecipitated by methods previously
described (23). Immunoprecipitations were performed sequentially with
IL-1 precipitated first followed by IL-1ß. The immunoprecipitates
were separated by SDS gel electrophoresis (15% polyacrylamide), and
IL-1 and IL-1ß (precursor and mature forms) were visualized by
fluorography.
Immunocytochemistry
Islets (150/400 µl of complete CMRL-1066), cultured for 4 h with or without 10 ng/ml TNF + 10 µg/ml LPS, were isolated and dispersed into individual cells as described previously (17). Islet cells were washed three times with 0.1 M PBS (pH 7.4) and diluted to a concentration of 40,000 cells/100 µl. The cells were transferred to Superfrost/Plus microscope slides by cytospin. The slides were fixed in 4% paraformaldehyde for 1 h at 4°C and then blocked for 1 h with 5% BSA (in 0.1 M PBS). The slides were incubated overnight at room temperature with a 1:25 dilution (in 0.1 M PBS containing 1% BSA) of goat anti-rat IL-1ß antiserum (R&D Systems; Minneapolis, MN), followed by a 1-h incubation with a 1:100 dilution of mouse anti-rat macrophage antiserum (ED1; Serotec, Raleigh, NC). The slides were washed three times with 150 µl of PBS and then incubated for 1 h with a 1:200 dilution of FITC-conjugated donkey anti-goat and CY3-conjugated donkey anti-mouse secondary Ab, for IL-1ß and ED1, respectively. Immunofluorescence microscopy was used for the detection of IL-1ß and ED1.
Polymerase chain reaction
Total RNA was isolated from islets using the Qiagen RNeasy RNA
isolation kit. Total RNA (2.5 µg) from each sample was then used to
prepare first-strand cDNA using the Superscript Preamplification System
from Life Technologies. A standard 25-µl PCR reaction containing 2.5
µl from the reverse transcriptase reaction, 200 µM of each dATP,
dCTP, dGTP, dUTP, 50 pmol of each forward and reverse primers, 2 U of
Taq DNA polymerase from Promega (Madison, WI), 1.5 mM
MgCl2, and 2.5 µl of the supplied 10x reaction buffer
was performed. IL-1, IL-1ß, iNOS, and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) primers were designed according to the published
sequences: 1) IL-1 forward primer 5'-CCAACCCAGATCAGCACCTCA-3',
reverse primer 5'-TCTCCTCCCGATGAGTAGGCA-3' (PCR product size =
204 bp); 2) IL-1ß forward primer 5'-CCTGTGGCCTTGGGCCTCAA-3', reverse
primer 5'-GGTGCTGATGTACCAGTTGGG-3' (PCR product size = 204
bp); 3) iNOS forward primer 5'-CCAACCGGAGAAGGGGACGAACT-3', reverse
primer 5'-GGAGGGTGGTGCGGCTGGAC-3' (PCR product size = 297 bp); 4)
GAPDH forward primer 5'-GCTGGGGCTCACCTGAAGGG-3', reverse primer
5'-GGATGACCTTGCCCACAGCC-3' (PCR product size = 343 bp).
Each PCR mixture was overlaid with 1 drop of mineral oil, and incubated in a Hybaid Omnigene thermal cycler using the following profile: an initial denaturation step at 94°C for 5 min, 30 cycles of 94°C for 45 s, 60°C for 45 s, and 72°C for 75 s. The samples were finally incubated at 30°C for 2 min. To each reaction, 5 µl of 6x loading dye (Promega) was added, and then 12 µl from each PCR reaction was run alongside 5 µl of 100-bp ladder (Promega) on a 1.5% agarose gel containing 0.5 µg/ml ethidium bromide. PCR products were visualized with UV light and photographed.
Semiquantitative PCR
The cDNA samples described above were used in semiquantitative
PCR as previously described (24, 25). In brief, each cDNA template was
diluted 1:25 and 1:50 for both IL-1 and IL-1ß reactions, and 1:125
and 1:250 for GAPDH reactions. Amplification of each cDNA species for
each sample was therefore represented by two different concentrations
of initial cDNA template. This would later be used to verify that each
reaction was amplified within the linear range, and that PCR product
yields were proportional to initial concentrations of cDNA template.
From each cDNA dilution, 5 µl was used in a standard 25 µl PCR
reaction as described above. In addition, 2.5 µCi of
[-32P]-labeled dCTP was added. PCR was conducted
according to the standard profile described above with the exception of
reducing the cycle number to 22. A total of 12 µl from each PCR
reaction was then run on a 1.5% agarose gel; the gels were dried and
subjected to phosphorimage analysis using a Molecular Dynamics
PhosphorImager and Molecular Dynamics ImageQuant Software Version 3.3
(Molecular Dynamics, Sunnyvale, CA). The values obtained for each IL-1
PCR reaction were normalized as a percentage of the values obtained for
each GAPDH control. Under the conditions used, the PCR product signal
was proportional to the amount of cDNA subjected to PCR
amplification.
Statistical analysis and figure preparation
Statistical comparisons were made between groups using a one-way analysis of variance. Significant differences between groups were determined by Scheffe’s F-test pocthoc analysis. For figure preparation, ethidium bromide-stained agarose gels and Western blot autoradiograms were scanned into National Institutes of Health Image version 1.59 using a COHU high performance CCD camera (Brookfield, WI). The images were then imported into Canvas 3.5 (Deneba Software, Miami, FL) for the preparation of figures.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To determine whether the source of IL-1 released in islets in
response to TNF + LPS is resident islet lymphoid cells, the
effects of TNF + LPS on nitric oxide production and insulin
secretion by rat islets either freshly isolated or cultured for 7 days
at 24°C have been evaluated. Treatment of freshly isolated rat islets
with IL-1 results in a potent inhibition of insulin secretion and a
2.5-fold increase in nitrite production (Fig. 1
A). The inhibitory effects of
IL-1ß on insulin secretion and stimulatory effects on nitrite
production are prevented by the IL-1R antagonist protein (IRAP). We
have previously shown that ß cells are the islet cellular source of
iNOS in response to IL-1 (17). Treatment of freshly isolated rat islets
with TNF + LPS also results in a potent inhibition of insulin
secretion and a twofold increase in the production of nitrite. IRAP
completely prevents the inhibitory effects of TNF + LPS on
glucose-stimulated insulin secretion and significantly attenuates
nitrite formation by isolated islets. The selective inhibitor of iNOS,
AG (12), also prevents TNF + LPS-induced inhibition of insulin
secretion and nitrite formation by rat islets, providing evidence that
nitric oxide mediates the inhibitory effects of TNF + LPS on
insulin secretion. These findings are consistent with our previous
studies showing that TNF + LPS-induced inhibition of
glucose-stimulated insulin secretion is mediated by the release of IL-1
within islets, followed by IL-1-induced iNOS expression and nitric
oxide production by ß cells (17).
|
B, TNF + LPS fails
to induce the production of nitrite or to inhibit glucose-stimulated
insulin secretion by rat islets cultured for 7 days at 24°C. These
findings indicate that lymphoid cell depletion prevents TNF +
LPS-induced inhibition of insulin secretion. Although this culture
condition prevents TNF + LPS-induced inhibition of insulin
secretion, the inhibitory effects of IL-1 on glucose-stimulated insulin
secretion and the stimulatory effects of IL-1 on nitrite formation were
not modified. IL-1 stimulates a greater than 2-fold increase in nitrite
production by both freshly isolated islets, and islets cultured for 7
days at 24°C (
75 pmol nitrite/islet for both culture conditions).
Also, 20 mM glucose induced a 6.6-fold increase in the release of
insulin (3 mM glucose, 1.86 ng/20 islets, 30 min; 20 mM glucose, 12.3
ng/20 islet, 30 min) from freshly isolated rat islets, and a 4.2-fold
increase by islets cultured for 7 days at 24°C (3 mM glucose, 1.96
ng/20 islets, 30 min; 20 mM glucose, 8.0 ng/20 islet, 30 min). These
findings indicate that a 7-day culture at 24°C does not impair islet
function, and does not reduce ß cell production of nitric oxide in
response to IL-1. A 7-day culture at 24°C prevents TNF + LPS-induced iNOS expression by rat islets
The effects of TNF + LPS and IL-1 on iNOS expression by
freshly isolated rat islets, and islets cultured for 7 days at 24°C
are shown in Figure 2. Treatment of
freshly isolated islets with TNF + LPS results in the expression
of iNOS at the mRNA (Fig. 2A) and protein level (Fig. 2B) as determined by RT-PCR and Western blot analysis,
respectively. IRAP completely prevents TNF + LPS-induced iNOS
protein expression by freshly isolated rat islets (Fig. 4). This
finding is consistent with the ability of IRAP to prevent TNF +
LPS-induced inhibition of insulin secretion and nitrite production by
freshly isolated islets as shown in Figure 1A. Conversely,
TNF + LPS fails to stimulate the expression of iNOS at either the
mRNA or protein level by rat islets depleted of resident tissue
lymphoid cells (Fig. 2, A and B). The lack of
iNOS expression is consistent with the inability of TNF + LPS to
stimulate nitrite formation or to inhibit insulin secretion by rat
islets cultured for 7 days at 24°C (Fig. 1B). These
findings indicate that lymphoid cell depletion prevents TNF +
LPS-induced iNOS expression by rat islets.
|
|
, IL-1
induces the expression of iNOS mRNA and protein by either freshly
isolated islets or islets cultured for 7 days at 24°C. These results
are consistent with the inhibitory effects of IL-1 on insulin secretion
and IL-1-induced nitrite production by rat islets under both
conditions, and indicate that culture conditions used to deplete
resident macrophages do not modify the ability of IL-1 to induce the
expression of iNOS by ß cells. Colocalization of IL-1ß and macrophage surface markers
Immunocytochemistry was used to identify the islet cellular source
of IL-1. For these experiments, islets were cultured for 4 h with
TNF + LPS, dispersed into individual cells, fixed on slides, and
then stained for IL-1ß and the macrophage surface marker ED1. As
shown in Figure 3, TNF + LPS
stimulates the expression of IL-1ß in a limited number of islet cells
(green fluorescence) as evidenced by the punctate green fluorescence in
the two indicated cells (arrows in Fig. 3a). IL-1ß
expression appears to colocalize with cells that express the macrophage
surface marker ED1 (Fig. 3b, red fluorescence). Importantly,
not every ED1-positive macrophage stains positive for IL-1ß
(approximately 50 to 60% stain for both IL-1ß and ED1); however,
IL-1ß was not detected in any islet cell type other than ED1-positive
macrophages. In addition, IL-1ß expression was not detected in
untreated islet endocrine or nonendocrine cells although a similar
number of ED1-positive cells were detected, as compared with TNF +
LPS-treated islets cells (data not shown). For controls,
cross-reactivity of secondary antisera and nonspecific primary antisera
of the same species was not observed. These findings provide direct
evidence that ED1-positive macrophages are the islet cellular source of
IL-1ß in response to TNF + LPS.
|
Two isoforms of IL-1 have been identified, IL-1 and IL-1ß
(26). RT-PCR was used to identify the isoform of IL-1, and the time
course of IL-1 expression by rat islets treated with TNF + LPS. As
shown in Figure 4A, TNF +
LPS stimulates the expression of both IL-1 and IL-1ß mRNA by rat
islets. IL-1 expression is absent in untreated islets, while
TNF + LPS-induced IL-1 mRNA expression is detectable following
a 1-, 2-, 4-, and 8-h incubation. IL-1 mRNA expression appears to be
significantly reduced following an 8-h incubation with TNF + LPS.
TNF + LPS stimulates the expression of significantly higher levels
of IL-1ß mRNA than IL-1 mRNA levels following 1-, 2-, 4-, and 8-h
exposures. The levels of IL-1 and IL-1ß mRNA accumulation in
response to TNF + LPS have been quantitated by semiquantitative
RT-PCR and are shown in Figure 4B. TNF + LPS-induced
IL-1ß mRNA accumulates to levels that are over fourfold higher than
the levels of IL-1. IL-1 and IL-1ß mRNA expression appears to
require the presence of resident islet macrophages. As shown in Figure 4C, a 4-h incubation with TNF + LPS fails to stimulate
IL-1ß mRNA expression in islets cultured for 7 days at 24°C.
Similar results were obtained for IL-1 (data not shown). The lack of
IL-1 and IL-1ß mRNA expression by islets cultured for 7 days at
24°C is consistent with the inability of TNF + LPS to stimulate
iNOS expression and nitrite production, or to inhibit insulin secretion
under similar culture conditions.
To determine whether the expression of IL-1 at the mRNA level
correlates with the synthesis of this cytokine, the effects of TNF
+ LPS on IL-1 and IL-1ß protein expression were examined by
immunoprecipitation. For these experiments, islets were pretreated for
2.5 h with TNF + LPS, [35S]methionine was
added, and the islets were cultured for an additional 6 h. The
precursor and mature forms of both IL-1 and IL-1ß were then
immunoprecipitated using hamster anti-mouse IL-1 and IL-1ß
antisera as described previously (23, 27). Similar to the accumulation
of mRNA, IL-1ß appears to be the major isoform of IL-1 synthesized in
islets in response to TNF + LPS (Fig. 4D). We were
unable to detect the expression of IL-1 (precursor or mature form),
or the mature form of IL-1ß. To confirm that the intraislet
production of IL-1ß stimulates iNOS expression, the effects of
IL-1 and IL-1ß neutralizing antisera on TNF + LPS-induced
nitrite production by rat islets were examined. Neutralization of
IL-1ß results in nearly complete inhibition of TNF + LPS-induced
nitrite production by rat islets (Fig. 4E). Incubation of
rat islets for 40 h with hamster anti-IL-1 antisera also
reduced TNF + LPS-induced nitrite production; however, the level
of neutralization was much less than the effects of hamster
anti-IL-1ß-specific antiserum. In addition, irrelevant hamster
antiserum did not modulate TNF + LPS-induced nitrite production by
rat islets (data not shown). These findings suggest that IL-1ß is the
predominant isoform of IL-1 that is expressed in islets and that
mediates the actions of TNF + LPS on iNOS expression and nitrite
production.
Nitric oxide is not required for intraislet IL-1 release by resident macrophages
It has recently been shown that nitric oxide production is
required for the release of biologically active IL-1 from peritoneal
exudate cells (PEC) and RAW 264.7 macrophages (23). Nitric oxide
appears to participate in IL-1 release, in part, by stimulating the
accumulation of cGMP. In Figure 5, A and
B, we have evaluated the role
of nitric oxide in the release of IL-1 by resident islet lymphoid
cells. Treatment of rat islets with TNF + LPS stimulates a
threefold increase in nitrite production and high levels of iNOS
protein expression. IRAP completely prevents iNOS expression, and
significantly attenuates, but does not completely prevent, nitrite
formation by rat islets. These findings are consistent with results
shown in Figures 1 and 2, and demonstrate that TNF + LPS
stimulates iNOS expression and nitrite production by rat islets by a
mechanism that involves the intraislet release of IL-1 and the
subsequent expression and production of nitric oxide by ß cells. The
iNOS inhibitor AG completely prevents TNF + LPS-induced nitrite
production by rat islets; however, AG does not inhibit iNOS expression
under these conditions. In addition, AG does not inhibit TNF +
LPS-induced IL-1 or IL-1ß mRNA expression, nor does it prevent the
inhibitory actions of IRAP on TNF + LPS-induced iNOS protein
expression (data not shown). These findings indicate that nitric oxide
production is not required for the release of biologically active IL-1
by resident islet macrophages.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and IL-1ß mRNA, and that
IL-1ß mRNA accumulates to levels that are approximately fourfold
higher than the accumulation of IL-1 mRNA by rat islets. To
investigate the cellular source of IL-1 in islets treated with TNF
+ LPS, we have used culture conditions previously shown to deplete
islets of resident lymphoid cells. Incubation of rat islets for 7 days
at 24°C results in the loss of over 98% of islet class II-positive
lymphoid cells (18). Islets contain approximately 0.5% resident
macrophages (10 to 15/islet), and this cell type is believed to be the
cell type lost under these culturing conditions (18). Treatment of
freshly isolated rat islets with TNF + LPS results in the
expression of iNOS, production of nitric oxide, and a potent inhibition
of glucose-stimulated insulin secretion. IRAP, which competes with IL-1
for receptor binding, prevents TNF + LPS-induced iNOS expression,
markedly attenuates nitrite production, and prevents the inhibitory
effects on glucose-stimulated insulin secretion. Depletion of islet
lymphoid cells by culturing islets for 7 days at 24°C also completely
prevents TNF + LPS-induced iNOS expression, nitrite formation, and
the inhibitory effects on glucose-stimulated insulin secretion. In
addition, TNF + LPS fails to stimulate the expression of IL-1
and IL-1ß in islets cultured for 7 days at 24°C.
We believe that the resident macrophage is the islet cellular source of
IL-1 in response to TNF + LPS; however, islets contain a number of
nonendocrine cells that may also be potential sources of this cytokine.
These nonendocrine cells include endothelial cells (which comprise
10% of islet cells, 150/islet) (28), and a limited number of
fibroblasts and dendritic cells. Fibroblasts and endothelial cells do
not appear to contribute to the intraislet production of IL-1 in
response to TNF + LPS. Fibroblasts are normally found associated
with the islet capsule, a structure that is lost during isolation, and
islet microcapillaries (endothelial cells) appear to form rounded
structures that are extruded from isolated islets following a 48-h
culture (Dr. Susan Bonner-Weir; Joslin Diabetes Center, Harvard Medical
School, Boston, MA; personal communication). In this regard, TNF +
LPS stimulates iNOS and IL-1 expression and nitrite production to
nearly identical levels by islets either freshly isolated, or islets
cultured for 48 h before stimulation with TNF + LPS (data not
shown). Immunocytochemical colocalization of IL-1ß and the macrophage
surface marker, ED1, was used to provide direct support for resident
macrophages as the islet cellular source of IL-1. We show that TNF
+ LPS stimulates IL-1ß expression in approximately 50% of islet
ED1-positive macrophages, and that IL-1 is not detected in other islet
endocrine or nonendocrine cells. These results strongly support
resident macrophages as the islet cellular source of IL-1 in response
to TNF + LPS.
It is somewhat surprising that IL-1ß is the primary isoform of IL-1
that mediates the inhibitory actions of TNF + LPS on islet
function. IL-1ß comprises the majority of secreted IL-1, whereas
IL-1 is commonly believed to be a membrane-associated isoform that
mediates its actions in a localized fashion. It was originally believed
that the isoform of IL-1 expressed in islets under conditions
associated with islet macrophage activation would be IL-1, because
target ß cells are in close proximity to resident macrophages in the
microenvironment of islets. In addition, an increase in IL-1 mRNA
expression has been detected in leukocytes purified from islets
isolated from nonobese diabetic (NOD) mice (25). Although the
identification of IL-1 mRNA expression by leukocytes purified from
islets isolated from recently diagnosed diabetic NOD mice does not
appear to be consistent with our identification of IL-1ß as the
predominant isoform of IL-1 expressed by resident macrophages, it is
important to emphasize that the aforementioned study primarily has
identified cytokine mRNA expression by invading inflammatory
leukocytes, and not cytokine mRNA expression by resident islet
macrophages. Cytokine expression by resident islet macrophages would be
expected to occur very early in the development of diabetes, if this
cell type participates in the initial stages of ß cell damage.
Further support for IL-1ß as a potential early mediator of ß cell
damage includes recent findings by Caileau et al. (29), which show that
neutralizing Abs specific for IL-1ß prevent cyclophosphamide-induced
diabetes in the NOD mouse.
It has been difficult to determine the cellular mechanisms associated
with macrophage release of IL-1 because both IL-1 and IL-1ß are
synthesized as precursor proteins that do not contain signal sequences,
and neither isoform appears to be glycosylated (26). These findings
support the view that both isoforms of IL-1 are not secreted through
the Golgi apparatus as is the case for most secreted proteins. Two
potential mechanisms have been proposed for macrophage IL-1 release.
Macrophage death, either by apoptosis or necrosis, is known to result
in the release of IL-1 (27, 30). Also, Hill et al. (23) have shown that
nitric oxide production is required for IL-1 release by a macrophage
cell line, RAW 264.7, and by primary mouse peritoneal macrophages
(PEC). In these studies, iNOS inhibitors (AG and NMMA) were shown to
completely prevent LPS and LPS + IFN--induced release of
biologically active IL-1 by PEC and RAW 264.7 cells, respectively (23).
Nitric oxide appears to participate in IL-1 release by activating
guanylate cyclase and stimulating cGMP accumulation (23). If nitric
oxide production is required for cytokine release by resident
macrophages, then AG, the selective iNOS inhibitor, should prevent
TNF + LPS-induced iNOS expression and nitrite production by rat
islets. However, AG does not inhibit TNF + LPS-induced iNOS
expression, but completely prevents nitrite production by rat islets.
These findings provide evidence to suggest that nitric oxide is not
required for cytokine release by resident islet macrophages. It is
possible that AG does not completely inhibit TNF + LPS-induced
nitric oxide production, and that low levels of nitric oxide may be
sufficient to stimulate cGMP accumulation and macrophage release of
IL-1 under these conditions. Although this interpretation is possible,
it is not likely. We have previously shown that AG completely prevents
IL-1-induced cGMP accumulation by rat islets (12), and LPS-induced cGMP
accumulation by RAW 264.7 cells (31) under conditions similar to those
used in our study. Interestingly, we have recently shown that resident
islet macrophage damage results in the release of sufficient levels of
IL-1 to stimulate iNOS expression by ß cells in the presence of
IFN- (32). Taken together, these findings suggest that cellular
damage or death is one mechanism associated with the release of IL-1 by
resident islet macrophages.
It is also interesting that the primary isoform of IL-1 released by
peritoneal macrophages and RAW 264.7 cells in the study by Hill et al.
(23) was IL-1, while TNF + LPS stimulates the expression and
release of IL-1ß by resident islet macrophages. This difference
suggests that: 1) the primary isoform of IL-1 released by peritoneal
macrophages differs from the isoform released by resident tissue
macrophages (IL-1 vs IL-1ß, respectively); and 2) that IL-1
release may be nitric oxide dependent while IL-1ß is released by
mechanisms that are independent of the production of nitric oxide.
It has been proposed that resident islet macrophage activation may be
one triggering or initiation event that leads to the development of
autoimmune diabetes (1). In this hypothesis, activation of islet
lymphoid cells (macrophages) mediates the initial destruction of ß
cells and this stimulates the subsequent induction of an
immune-mediated process for the eventual destruction of remaining ß
cells. Lacy and Finke (18) first demonstrated that depletion of islet
lymphoid cells prevents islet destruction stimulated by high
concentrations of IFN- (1000 U/ml). Although we have not directly
examined the effects of high concentrations of IFN- on intraislet
IL-1 release, our studies provide mechanistic information on how the
activation of resident macrophages can directly modulate ß cell
function. Activation of resident macrophages results in the expression
and release of IL-1, and IL-1 accumulates in islets to levels that are
sufficient to stimulate the expression of iNOS and the production of
nitric oxide by ß cells. Under these conditions, nitric oxide
produced by ß cells impairs islet oxidative metabolism resulting in
the inhibition of glucose-stimulated insulin secretion and ß cell
damage. It has been proposed that the initiation of autoimmune diabetes
is associated with a chemical or viral agent that targets and
stimulates the initial destruction of ß cells (33, 34, 35). Our studies
support a role for the resident islet macrophage as the target of a
chemical or viral agent. Chemical or viral activation of resident islet
macrophages would result in the release of IL-1 in the microenvironment
of the islet, followed by IL-1-induced ß cell dysfunction and
destruction in a nitric oxide-dependent manner. ß cell death would
then result in the release and presentation of neoantigens and ß
cell-specific Ags by macrophages (or dendritic cells) leading to
classical T cell-dependent destruction of ß cells and the development
of autoimmune diabetes.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. John A. Corbett, Saint Louis University School of Medicine, Department of Biochemistry and Molecular Biology, 1402 South Grand Boulevard, Saint Louis, MO 63104. E-mail address:
3 Abbreviations used in this paper: iNOS, inducible nitric oxide synthase; AG, aminoguanidine; IRAP, IL-1 receptor antagonist protein; NMMA, NG-monomethyl-L-arginine; KRB, Krebs-Ringer bicarbonate buffer; TBST, Tris-buffered saline and Tween-20; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT-PCR, reverse transcriptase-polymer chain reaction; NOD, nonobese diabetic; PEC, peritoneal macrophages; ECL, enhanced chemiluminescence; HRP, horseradish peroxidase; cGMP, cyclic GMP.
Received for publication August 8, 1997. Accepted for publication November 24, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
via an L-arginine-dependent nitric oxide generation mechanism. FEBS. Lett. 276:42.[Medline]
increases the sensitivity of islets of Langerhans for iNOS expression induced by IL-1. J. Biol. Chem. 272:13653.This article has been cited by other articles:
|
|
 |
|
  K. J. Hughes, K. T. Chambers, G. P. Meares, and J. A. Corbett Nitric oxides mediates a shift from early necrosis to late apoptosis in cytokine-treated {beta}-cells that is associated with irreversible DNA damage Am J Physiol Endocrinol Metab, November 1, 2009; 297(5): E1187 - E1196. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Boni-Schnetzler, J. Thorne, G. Parnaud, L. Marselli, J. A. Ehses, J. Kerr-Conte, F. Pattou, P. A. Halban, G. C. Weir, and M. Y. Donath Increased Interleukin (IL)-1{beta} Messenger Ribonucleic Acid Expression in {beta}-Cells of Individuals with Type 2 Diabetes and Regulation of IL-1{beta} in Human Islets by Glucose and Autostimulation J. Clin. Endocrinol. Metab., October 1, 2008; 93(10): 4065 - 4074. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. T. Chambers, J. A. Unverferth, S. M. Weber, R. C. Wek, F. Urano, and J. A. Corbett The Role of Nitric Oxide and the Unfolded Protein Response in Cytokine-Induced -Cell Death Diabetes, January 1, 2008; 57(1): 124 - 132. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. M. Barbe-Tuana, D. Klein, H. Ichii, D. M. Berman, L. Coffey, N. S. Kenyon, C. Ricordi, and R. L. Pastori CD40-CD40 Ligand Interaction Activates Proinflammatory Pathways in Pancreatic Islets Diabetes, September 1, 2006; 55(9): 2437 - 2445. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. K. Mohammad, M. Morran, B. Slotterbeck, D. W. Leaman, Y. Sun, H. v. Grafenstein, S.-C. Hong, and M. F. McInerney Dysregulated Toll-like receptor expression and signaling in bone marrow-derived macrophages at the onset of diabetes in the non-obese diabetic mouse Int. Immunol., July 1, 2006; 18(7): 1101 - 1113. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. S. Narang and R. I. Mahato Biological and biomaterial approaches for improved islet transplantation. Pharmacol. Rev., June 1, 2006; 58(2): 194 - 243. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. D McKenzie, N. L Dudek, L. Mariana, M. M. Chong, J. A Trapani, T. W. Kay, and H. E Thomas Perforin and Fas induced by IFN{gamma} and TNF{alpha} mediate beta cell death by OT-I CTL Int. Immunol., June 1, 2006; 18(6): 837 - 846. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Moran, M. A. Moxley, R. M. L. Buller, and J. A. Corbett Encephalomyocarditis Virus Induces PKR-Independent Mitogen-Activated Protein Kinase Activation in Macrophages J. Virol., August 15, 2005; 79(16): 10226 - 10236. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Moran, R. M. L. Buller, J. McHowat, J. Turk, M. Wohltmann, R. W. Gross, and J. A. Corbett Genetic and Pharmacologic Evidence That Calcium-independent Phospholipase A2{beta} Regulates Virus-induced Inducible Nitric-oxide Synthase Expression by Macrophages J. Biol. Chem., July 29, 2005; 280(30): 28162 - 28168. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. M. W. Chong, Y. Chen, R. Darwiche, N. L. Dudek, W. Irawaty, P. Santamaria, J. Allison, T. W. H. Kay, and H. E. Thomas Suppressor of Cytokine Signaling-1 Overexpression Protects Pancreatic {beta} Cells from CD8+ T Cell-Mediated Autoimmune Destruction J. Immunol., May 1, 2004; 172(9): 5714 - 5721. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. E. Thomas, W. Irawaty, R. Darwiche, T. C. Brodnicki, P. Santamaria, J. Allison, and T. W.H. Kay IL-1 Receptor Deficiency Slows Progression to Diabetes in the NOD Mouse Diabetes, January 1, 2004; 53(1): 113 - 121. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Gurlo and H. v. Grafenstein Antigen-independent cross-talk between macrophages and CD8+ T cells facilitates their cooperation during target destruction Int. Immunol., September 1, 2003; 15(9): 1063 - 1071. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Cao, Z. Gao, C. E. Robert, S. Greene, G. Xu, W. Xu, E. Bell, D. Campbell, Y. Zhu, R. Young, et al. Pancreatic-Derived Factor (FAM3B), a Novel Islet Cytokine, Induces Apoptosis of Insulin-Secreting {beta}-Cells Diabetes, September 1, 2003; 52(9): 2296 - 2303. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. B. Maggi Jr., J. M. Moran, R. M. L. Buller, and J. A. Corbett ERK Activation Is Required for Double-stranded RNA- and Virus-induced Interleukin-1 Expression by Macrophages J. Biol. Chem., May 2, 2003; 278(19): 16683 - 16689. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Riachy, B. Vandewalle, J. Kerr Conte, E. Moerman, P. Sacchetti, B. Lukowiak, V. Gmyr, T. Bouckenooghe, M. Dubois, and F. Pattou 1,25-Dihydroxyvitamin D3 Protects RINm5F and Human Islet Cells against Cytokine-Induced Apoptosis: Implication of the Antiapoptotic Protein A20 Endocrinology, December 1, 2002; 143(12): 4809 - 4819. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. M. W. Chong, H. E. Thomas, and T. W. H. Kay Suppressor of Cytokine Signaling-1 Regulates the Sensitivity of Pancreatic beta Cells to Tumor Necrosis Factor J. Biol. Chem., July 26, 2002; 277(31): 27945 - 27952. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. E. Thomas, R. Darwiche, J. A. Corbett, and T. W.H. Kay Interleukin-1 Plus {gamma}-Interferon-Induced Pancreatic {beta}-Cell Dysfunction Is Mediated by {beta}-Cell Nitric Oxide Production Diabetes, February 1, 2002; 51(2): 311 - 316. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. A. Blair, L. B. Maggi Jr., A. L. Scarim, and J. A. Corbett Role of Interferon Regulatory Factor-1 in Double-stranded RNA-induced iNOS Expression by Mouse Islets J. Biol. Chem., January 4, 2002; 277(1): 359 - 365. [Abstract] [Full Text]
|
|
|
|
|
|
D. Liu, M. Darville, and D. L. Eizirik Double-Stranded Ribonucleic Acid (RNA) Induces {beta}-Cell Fas Messenger RNA Expression and Increases Cytokine-Induced {beta}-Cell Apoptosis Endocrinology, June 1, 2001; 142(6): 2593 - 2599. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. A. Blair, M. R. Heitmeier, A. L. Scarim, L. B. Maggi Jr., and J. A. Corbett Double-Stranded RNA--Dependent Protein Kinase Is Not Required for Double-Stranded RNA--Induced Nitric Oxide Synthase Expression or Nuclear Factor-{kappa}B Activation by Islets Diabetes, February 1, 2001; 50(2): 283 - 290. [Abstract] [Full Text]
|
|
|
|
 |
|
 D. J. Figueroa, J. F. Hess, B. Ky, S. D. Brown, V. Sandig, A. Hermanowski–Vosatka, R. C. J. Twells, J. A. Todd, and C. P. Austin Expression of the Type I Diabetes-associated Gene LRP5 in Macrophages, Vitamin A System Cells, and the Islets of Langerhans Suggests Multiple Potential Roles in Diabetes J. Histochem. Cytochem., October 1, 2000; 48(10): 1357 - 1368. [Abstract] [Full Text]
|
|
|
|
|
|
T. Gurlo, K. Kawamura, and H. von Grafenstein Role of Inflammatory Infiltrate in Activation and Effector Function of Cloned Islet Reactive Nonobese Diabetic CD8+ T Cells: Involvement of a Nitric Oxide-Dependent Pathway J. Immunol., December 1, 1999; 163(11): 5770 - 5780. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. T. Grey, M. B. Arvelo, W. Hasenkamp, F. H. Bach, and C. Ferran A20 Inhibits Cytokine-Induced Apoptosis and Nuclear Factor {kappa}B-Dependent Gene Activation in Islets J. Exp. Med., October 18, 1999; 190(8): 1135 - 1146. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. R. Heitmeier, A. L. Scarim, and J. A. Corbett Prolonged STAT1 Activation Is Associated with Interferon-gamma Priming for Interleukin-1-induced Inducible Nitric-oxide Synthase Expression by Islets of Langerhans J. Biol. Chem., October 8, 1999; 274(41): 29266 - 29273. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. R. Heitmeier, A. L. Scarim, and J. A. Corbett Double-stranded RNA Inhibits beta -Cell Function and Induces Islet Damage by Stimulating beta -Cell Production of Nitric Oxide J. Biol. Chem., April 30, 1999; 274(18): 12531 - 12536. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. M. Bradley, V. C. Asensio, L.-K. Schioetz, J. Harbertson, T. Krahl, G. Patstone, N. Woolf, I. L. Campbell, and N. Sarvetnick Islet-Specific Th1, But Not Th2, Cells Secrete Multiple Chemokines and Promote Rapid Induction of Autoimmune Diabetes J. Immunol., March 1, 1999; 162(5): 2511 - 2520. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Giannoukakis, W. A. Rudert, M. Trucco, and P. D. Robbins Protection of Human Islets from the Effects of Interleukin-1beta by Adenoviral Gene Transfer of an Ikappa B Repressor J. Biol. Chem., November 17, 2000; 275(47): 36509 - 36513. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. R. Heitmeier, M. Arnush, A. L. Scarim, and J. A. Corbett Pancreatic beta -Cell Damage Mediated by beta -Cell Production of Interleukin-1. A NOVEL MECHANISM FOR VIRUS-INDUCED DIABETES J. Biol. Chem., March 30, 2001; 276(14): 11151 - 11158. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) are as
indicated.
