From the Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, Missouri 63104
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The effects of double-stranded RNA
(synthetic polyinosinic-polycytidylic acid; poly(I-C)) on macrophage
expression of inducible nitric-oxide synthase (iNOS), production of
nitric oxide, and release of interleukin-1 (IL-1) were investigated.
Individually, poly(I-C), interferon- (IFN-
), and
lipopolysaccharide (LPS) stimulate nitrite production and iNOS
expression by RAW 264.7 cells. In combination, the effects of poly(I-C) + IFN-
are additive, while poly(I-C) does not further potentiate
LPS-induced nitrite production. These results suggest that poly(I-C)
and LPS may stimulate iNOS expression by similar signaling pathways,
which may be independent of pathways activated by IFN-
. LPS-induced
iNOS expression is associated with the activation of NF-
B. We show
that inhibition of NF-
B by pyrrolidinedithiocarbamate prevents
poly(I-C) + IFN-
-, poly(I-C) + LPS-, and LPS-induced iNOS
expression, nitrite production and I
B degradation by RAW 264.7 cells. The effects of poly(I-C) on iNOS expression appear to be
cell-type specific. Poly(I-C), alone or in combination with IFN-
,
does not stimulate, nor does poly(I-C) potentiate, IL-1-induced nitrite
production by rat insulinoma RINm5F cells. In addition, we show that
the combination of poly(I-C) + IFN-
stimulates iNOS expression,
nitrite production, I
B degradation, and the release of IL-1 by
primary mouse macrophages, and these effects are prevented by
pyrrolidinedithiocarbamate. These findings indicate that
double-stranded RNA, in the presence of IFN-
, is a potent activator
of macrophages, stimulating iNOS expression, nitrite production, and
IL-1 release by a mechanism which requires the activation of
NF-
B.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Nitric oxide (NO)1 is the product of the five-electron oxidation of L-arginine to L-citrulline catalyzed by the enzyme nitric- oxide synthase (NOS) (1). Three isoforms of NOS have been cloned and characterized: endothelial NOS (eNOS or NOSIII), neuronal NOS (nNOS or NOSI), and inducible NOS (iNOS or NOSII) (2-4). Collectively, eNOS and nNOS are known as cNOS because their enzymatic activity is regulated by Ca2+ and because these isoforms are constitutively expressed. Nitric oxide, produced in low levels by cNOS isoforms, functions as a signaling molecule associated with diverse biological processes including the regulation of vascular tone and neuronal signaling (4-6). Nitric oxide, produced in large quantities following induction of iNOS by cytokines or endotoxin, can have cytotoxic or cytostatic effects on target cells (2, 7) and has recently been implicated as an effector molecule that participates in antiviral responses (8, 9).
Viral infection has been shown to stimulate NO production by iNOS in
several cell types including mixed glial cell cultures, lymphocytes and
monocytes/macrophages (10-13). Karupiah et al. (14) have
shown NO production is required for IFN- inhibition of ectromelia,
vaccinia, and herpesvirus replication in mouse macrophages. Also,
Bukrinsky et al. (15) have shown that infection of human
monocytes with human immunodeficiency virus type 1 stimulates iNOS
expression and nitric oxide production. Although viral infection stimulates iNOS expression and nitric oxide production, the mechanism by which viral infection stimulates iNOS expression is unknown.
NF-B appears to play a primary role in the transcriptional
regulation of iNOS gene expression by macrophages (16, 17). In
unstimulated cells, NF-
B is found as an inactive heterodimer of
p50/p65 subunits bound to the NF-
B inhibitor protein, I
B. Upon
stimulation, I
B is phosphorylated on specific serine residues which
targets I
B for degradation in a ubiquitin-dependent
manner (18). The antioxidant inhibitors of NF-
B activation,
pyrrolidinedithiocarbamate (PDTC) and diethyldithiocarbamic acid,
prevent lipopolysaccharide (LPS)- and LPS + IFN-
-induced iNOS
expression and nitrite production by RAW 264.7 cells (17), indicating
that NF-
B participates in LPS- and LPS + IFN-
-induced iNOS
expression.
The active component of a viral infection that stimulates antiviral
activities appears to be double-stranded RNA (dsRNA), which accumulates
during the replication of many viruses. The purpose of this study was
to determine if the activation of macrophages by dsRNA results in iNOS
expression, nitric oxide production, and IL-1 release, and if these
events are dependent on NF-B activation. We show that dsRNA (in the
form of poly(I-C)), in combination with IFN-
, is a potent activator
of murine macrophages, stimulating iNOS expression, nitric oxide
production, and IL-1 release. Furthermore, we show that
polyinosinic-polycytidylic acid (poly(I-C))-induced iNOS expression and
IL-1 release are associated with the activation of NF-
B. These
studies provide direct support for double-stranded RNA as one effector
molecule that mediates macrophage activation in an
NF-
B-dependent mechanism.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials and Animals--
Mouse macrophage RAW 264.7 and rat
insulinoma RINm5F cells were obtained from Washington University Tissue
Culture Support Center. RPMI medium 1640 containing 1×
L-glutamine, CMRL-1066 tissue culture medium,
L-glutamine, penicillin, streptomycin, and mouse and rat
recombinant IFN- were from Life Technologies, Inc. Fetal calf serum
was obtained from Hyclone (Logan, UT). Male CD-1 mice (20-24 g) were
purchased from Harlan (Indianapolis, IN). Aminoguanidine (AG)
hemisulfate, LPS (serotype 0111:B4), poly(I-C), and PDTC were from
Sigma. [
-32P]dCTP and enhanced chemiluminescence (ECL)
reagents were purchased from Amersham Corp. NF-
B consensus
oligonucleotide and rabbit anti-I
B-
and I
B-
antiserum were
purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
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, University of Alabama-Birmingham,
AL), respectively. All other reagents were from commercially available
sources.
CD-1 Mouse Peritoneal Macrophage Isolation and Cell
Culture--
Peritoneal macrophages (peritoneal exudate cells, PEC)
were isolated from male CD-1 mice by lavage as described previously (19). Following isolation, the cells were plated at a concentration of
400,000 cells/400 µl 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) in
24-well microtiter plates and incubated for 3 h under an
atmosphere of 95% air and 5% CO2 at 37 °C. Nonadherent
cells were removed by washing (three times with complete CMRL) followed
by treatment of the adherent macrophages with poly(I-C), mouse IFN-,
and LPS as indicated in the figure legends.
Western Blot Analysis--
RAW 264.7 cells or CD-1 mouse PEC
(400,000 cells/400 µl of complete CMRL-1066), were prepared for
Western analysis as described previously (20). Proteins were separated
by electrophoresis on 10% SDS-polyacrylamide gels using standard
conditions (21), and transferred to Nitrocell nitrocellulose membranes
(Pharmacia Biotech Inc.) under semidry transfer conditions. Blots were
blocked in TBST (20 mM Tris, 500 mM NaCl, and
0.1% Tween 20, pH 7.5) containing 5% nonfat dry milk and incubated
for 1.5 h in primary antisera (rabbit anti-mouse iNOS, 1:2000;
rabbit anti-human IB-
, 1:1000; rabbit anti-human I
B-
,
1:1000) containing 1% nonfat dry milk. The blots were then washed four
times with TBST (5 min/wash), and incubated for 45 min at room
temperature with horseradish peroxidase-conjugated donkey anti-rabbit
secondary antibody at a dilution of 1:7000. The blots were washed three
times in TBST and once in 0.1 M phosphate-buffered saline
(pH 7.4) at room temperature. Detection of iNOS and I
B was by ECL
according to manufacturer's specifications (Amersham Corp.).
Northern Blot Analysis--
RAW 264.7 cells (10 × 106 cells/3 ml complete CMRL-1066) were cultured for 6 h at 37 °C with the indicated concentrations of poly(I-C), mouse
IFN-, and/or LPS. 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 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 32P-labeled probes specific
for rat iNOS or cyclophilin (20, 22). The DNA probes were radiolabeled
with [
-32P]dCTP by random priming using the
Prime-a-Gene nick translation system from Promega (Madison, WI). The
iNOS DNA probe corresponds to bases 509-1415 of the rat iNOS coding
region. The cyclophilin probe was used as an internal control for RNA
loading. Hybridization and autoradiography were performed as described
previously (23).
Nuclear Extracts--
RAW 264.7 cells (5 × 106
cells/3 ml complete CMRL) were incubated for 30 min with 100 µg/ml
poly(I-C), 10 µg/ml LPS, and 150 units/ml mouse IFN- as indicated.
Nuclear extracts were prepared essentially as described by Greenlund
et al. (24). In brief, cells were isolated by
centrifugation, washed twice with ice-cold 0.1 M
phosphate-buffered saline, resuspended in 400 µl of buffer I (10 mM HEPES, pH 7.8, 5 mM MgCl2, 10 mM KCl, 1 mM ZnCl2, 0.2 mM EGTA, 1 mM Na3VO4,
10 mM NaF, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 1 µg/ml of each of
the following: leupeptin, antipain, aprotinin, and pepstatin A) and
incubated on ice for 10 min. Cells were lysed by the addition of 50 µl of 10% Nonidet P-40 (1.1% final concentration), and cell nuclei
were isolated by centrifugation (2000 × g, 4 °C, 5 min). The nuclear pellets were resuspended in 60 µl of buffer II (20 mM HEPES, pH 7.8, 5 mM MgCl2, 300 mM NaCl, 1 mM ZnCl2, 0.2 mM EGTA, 25% glycerol, 1 mM
Na3VO4, 10 mM NaF, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 1 µg/ml of each of the following: leupeptin, antipain, aprotinin, and pepstatin A) and incubated for 15 min on ice with occasional mixing. Nuclear debris was removed by centrifugation (13,500 rpm, 4 °C, 15 min), and the nuclear protein extract was used for gel
shift analysis. Protein concentration was determined by the BCA protein
assay according to manufacturer's instructions (Pierce).
Gel Shift Analysis--
Gel shift analysis of nuclear extracts
was performed as described previously (24, 25). The probe consisted of
a double-stranded oligonucleotide containing the consensus binding
sequence for NF-B (5'-AGT TGA GGG GAC TTT CCC AGG C-3'; Santa Cruz)
end-labeled with [
-32P]ATP using T4 polynucleotide
kinase (Promega). Typical binding reactions consisted of 10 µg of
nuclear extract, 0.5 ng of DNA probe, 2 µg/ml poly[d(I-C)]
(Boehringer Mannheim) in a buffer containing 20 mM HEPES
(pH 7.8), 50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, and 5% glycerol and were incubated at 30 °C
for 20 min. Binding reactions were separated on 4% Tris-glycine
nondenaturing polyacrylamide gels in a 2× Tris-glycine (0.05 M Tris-HCl, pH 8.3, 0.38 M glycine, 2 mM EDTA) buffer system (26). The gels were transferred to
Whatman paper, dried, and subjected to autoradiography.
Nitrite and IL-1 Determination-- Nitrite production was determined by mixing 50 µl of culture medium with 50 µl of Griess reagent (27). The absorbance at 540 nm was measured, and nitrite concentrations were calculated from a sodium nitrite standard curve. IL-1 release from mouse PEC was performed using the RINm5F cell bioassay as described by Hill et al. (28).
Densitometry and Image Analysis-- Autoradiograms were scanned into NIH Image version 1.59 using a high performance CCD camera (Cohu, Inc., Brookfield, WI). Densities were determined using NIH Image version 1.59 software. Phosphorimaging analysis of RAW 264.7 cell mRNA accumulation experiments (normalized to cyclophilin mRNA levels) 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.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effects of Poly(I-C), IFN-, and LPS on Nitrite Production and
iNOS mRNA and Protein Expression by RAW 264.7 Cells--
LPS and
IFN-
alone will activate mouse macrophage RAW 264.7 cells, to
express iNOS and produce nitric oxide; however, maximal production of
nitric oxide occurs in response to a combination of LPS + IFN-
. To
determine if dsRNA stimulates macrophage activation, the effects of the
synthetic dsRNA molecule, poly(I-C), alone or in combination with LPS
and IFN-
, on nitrite production by RAW 264.7 cells were examined. As
shown in Fig. 1a, a 24-h
incubation of RAW 264.7 cells with either poly(I-C) (100 µg/ml) or
IFN-
(150 units/ml) stimulates a ~9- and ~6-fold increase in
nitrite production, respectively. While poly(I-C) does not appear to
further increase the level of nitrite produced in response to LPS, in combination with IFN-
, poly(I-C) stimulates an over 20-fold increase in nitrite production by RAW 264.7 cells. The levels of nitrite production in response to poly(I-C) + IFN-
are similar in magnitude to the effects of IFN-
+ LPS on RAW 264.7 cell nitrite production. AG, a selective inhibitor of iNOS (29), completely prevents poly(I-C)-
(data not shown), IFN-
- (data not shown), poly(I-C) + IFN-
-,
poly(I-C) + LPS-, and LPS + IFN-
-induced nitrite production by RAW
264.7 cells.
|
Role of NF-B in Poly(I-C)-induced iNOS Expression and Nitrite
Production by RAW 264.7 Cells--
Since poly(I-C) does not further
increase LPS-induced nitrite production by RAW cells and nitrite
production in response to poly(I-C) and IFN-
are additive (Fig. 1),
it is likely that the signaling pathway activated by and associated
with poly(I-C)-induced iNOS expression may be similar or identical to
the pathway activated by LPS. One of the signaling molecules that
participates in LPS-induced iNOS expression is the transcriptional
regulator NF-
B. Previous studies have shown that the antioxidant
PDTC, a potent inhibitor of NF-
B activation (30), prevents
LPS-induced iNOS expression by RAW 264.7 cells (17). To determine if
NF-
B participates in poly(I-C)-induced nitrite production and iNOS
expression, RAW 264.7 cells were pretreated for 30 min with 100 µM PDTC. Poly(I-C) (100 µg/ml), IFN-
(150 units/ml),
and LPS (10 µg/ml) were then added, and the cells were cultured for
24 h. As shown in Fig. 2a, PDTC completely prevents
poly(I-C)-, poly(I-C) + IFN-
-, LPS-, and LPS + poly(I-C)-induced
nitrite production by RAW 264.7 cells. Consistent with its inhibitory
effects on nitrite production, PDTC also inhibits poly(I-C)- (data not
shown), poly(I-C) + IFN-
-, LPS-, and LPS + poly(I-C)-induced iNOS
protein expression (Fig. 2b). These findings provide
evidence that poly(I-C) (in the presence or absence of IFN-
)
stimulates iNOS expression by a mechanism that is associated with the
activation of NF-
B.
|
Effects of Poly(I-C) on Nitrite Production by RINm5F
Cells--
The insulinoma RINm5F cell line represents a homogenous
population of pancreatic islet -cells that express iNOS and produce nitric oxide in response to IL-1 (31). In addition, IL-1-induced nitric
oxide production by RINm5F cells appears to require the activation of
NF-
B as PDTC and diethyldithiocarbamic acid inhibit both NF-
B
activation and iNOS expression (25, 32). Recently, we have shown that
IFN-
potentiates IL-1-induced iNOS expression and nitrite production
by RINm5F cells at concentrations of IL-1 that alone do not stimulate
iNOS expression (20). Since poly(I-C)-induced iNOS expression and
nitrite production by RAW 264.7 cells appears to require the activation
of NF-
B, and IL-1-induced iNOS expression by RINm5F cells also
requires NF-
B activation, the effects of poly(I-C), alone and in
combination with IL-1 and IFN-
, on nitrite production by RINm5F
cells were examined (Fig. 3). Treatment
of RINm5F cells with IL-1 stimulates a ~20-fold increase in nitrite production following a 24-h incubation. Alone, poly(I-C) does not
stimulate nitrite production, nor does it further potentiate IL-1-induced nitrite production by RINm5F cells. Individually, neither
IFN-
(150 units/ml) nor 0.1 units/ml IL-1 stimulate nitrite production by RINm5F cells; however, in combination these two cytokines
stimulate the production of nitrite to levels nearly identical to the
levels stimulated by maximal concentrations of IL-1 (1 unit/ml). In
combination with either IFN-
or submaximal concentrations of IL-1
(0.1 unit/ml), poly(I-C) does not stimulate nitrite production by
RINm5F cells. Poly(I-C) also does not further potentiate IL-1
(submaximal or maximal concentrations) + IFN-
-induced nitrite
production (data not shown). These findings suggest that the actions of
poly(I-C) on iNOS expression and nitrite production may be cell-type
specific.
|
Activation of Peritoneal Macrophages by Poly(I-C)--
In contrast
to RAW 264.7 cells, resident mouse macrophages require a combination of
two signals (e.g. IFN- + LPS) for iNOS expression and
nitric oxide production. The effects of dsRNA on primary macrophage
activation was examined by incubating mouse PEC with poly(I-C) in the
presence or absence of IFN-
and LPS. Alone, neither poly(I-C),
IFN-
, nor LPS stimulate nitrite production or iNOS expression by
mouse PEC (Figs. 4, a and
b, respectively). However, in combination, poly(I-C) + IFN-
stimulate the expression of iNOS and the production of nitrite
to levels comparable to the effects of IFN-
+ LPS. The production of
nitrite in response to poly(I-C) + IFN-
, or LPS + IFN-
is
completely prevented by the iNOS inhibitor AG. Importantly, the
combination of poly(I-C) + LPS does not stimulate iNOS expression or
nitrite production by PEC. This finding is consistent with the
inability of poly(I-C) to potentiate LPS-induced nitrite production by
RAW 264.7 cells. These findings indicate that a combination of
poly(I-C) and IFN-
activates primary macrophages stimulating iNOS
expression and nitrite production.
|
Poly(I-C) + IFN- Stimulate IL-1 Release by Primary Macrophages
in an NF-
B-dependent Manner--
Macrophage activation
is characterized by the release of high levels of the cytokine IL-1.
Using the RINm5F cell bioassay (28) we have examined the effects of
poly(I-C) on IL-1 release by PEC, and whether poly(I-C)-induced IL-1
release requires NF-
B activation. Alone, poly(I-C) (100 µg/ml),
IFN-
(150 units/ml), or LPS (10 µg/ml) did not stimulate IL-1
release by PEC following a 24-h incubation (Fig.
5a). However, the combinations
of poly(I-C) + IFN-
and LPS + IFN-
stimulate the release of IL-1
to levels that are over 10-fold higher than that of control PEC.
Preincubation of mouse PEC for 30 min with 100 µM PDTC
completely inhibits poly(I-C) + IFN-
-induced IL-1 release,
implicating a role for NF-
B activation in IL-1 release by PEC.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
One cellular response to viral infection is the expression of iNOS
and the increased production of nitric oxide (8, 9). The mechanism by
which viral infection stimulates iNOS expression is unknown. The active
component of viral infection appears to be dsRNA, which accumulates
during replication of many viruses (33). It was first demonstrated in
1971 that treatment with dsRNA (poly(I-C)) renders macrophages
cytotoxic to target cells in a manner similar to the actions of
endotoxin and lipid A (34). dsRNA-induced inhibition of protein
translation and expression of type I interferons are associated with
the antiviral activity in infected cells (33, 35). In the current
study, the effects of dsRNA on macrophage activation and the mechanism
by which dsRNA activates macrophages have been examined. Treatment of
RAW 264.7 cells with poly(I-C) stimulates iNOS expression and nitrite
production. IFN- potentiates poly(I-C)-induced iNOS expression and
nitrite production, while LPS does not further potentiate
poly(I-C)-induced nitrite production by RAW 264.7 cells. Alone,
poly(I-C) does not stimulate iNOS expression by primary macrophages
(PEC); however, in combination with IFN-
, poly(I-C) stimulates iNOS
expression and high levels of nitrite production. In addition,
poly(I-C) + IFN-
stimulate IL-1 release by mouse PEC. Similar to the
inability of LPS to enhance poly(I-C)-induced nitrite production by RAW 264.7 cells, poly(I-C), in combination with LPS, does not stimulate iNOS expression, nitrite production, or IL-1 release by mouse PEC.
Poly(I-C) appears to stimulate iNOS expression by a mechanism similar
to that induced by LPS because the effects of LPS and poly(I-C) are not
additive, while RAW 264.7 cell expression of iNOS in response to
poly(I-C) and IFN- are additive. LPS-induced iNOS expression by RAW
264.7 cells requires the activation of NF-
B (17). Our studies show
that poly(I-C)- and poly(I-C) + IFN-
-induced iNOS expression,
nitrite production, and I
B degradation by RAW 264.7 cells is
prevented by the NF-
B inhibitor PDTC. In addition, PDTC inhibits
poly(I-C) + IFN-
-induced iNOS expression, nitrite production (data
not shown), I
B degradation and IL-1 release by PEC. These findings
indicate that poly(I-C)-induced I
B degradation and NF-
B nuclear
localization participate in poly(I-C)- and poly(I-C) + IFN-
-induced
iNOS expression by RAW 264.7 cells and mouse PEC. I
B is
phosphorylated on specific serine residues, and it is this
phosphorylation that appears to direct proteolytic degradation of I
B
(18). The dsRNA-dependent protein kinase is one potential
candidate that may phosphorylate I
B in response to poly(I-C). dsRNA
(poly(I-C) and viral dsRNA) is known to activate
dsRNA-dependent protein kinase by autophosphorylation (36),
and in vitro phosphorylation studies have shown that I
B is one substrate for this kinase (37). We are currently examining the
effects of poly(I-C) on dsRNA-dependent protein kinase
autophosphorylation in both RAW 264.7 cells and PEC.
The effects of poly(I-C) on iNOS expression appear to be cell-type
specific. While poly(I-C), alone and in combination with IFN-,
stimulates iNOS expression by RAW 264.7 cells, poly(I-C), alone or in
combination with IFN-
, does not induce nor does it enhance
IL-1-induced nitrite production by the pancreatic beta cell line
RINm5F. Previous studies have shown that IL-1 stimulates high levels of
iNOS expression and nitric oxide production by RINm5F cells, and the
actions of IL-1 are potentiated by IFN-
(20, 31). IL-1-induced iNOS
expression by RINm5F cells is associated with I
B
degradation2 and NF-
B
nuclear localization (25, 32). Poly(I-C) stimulates I
B degradation
in RINm5F cells (data not shown); however, poly(I-C) does not stimulate
iNOS expression or nitrite production. These results indicate that
other transcriptional regulators in addition to NF-
B are required
for IL-1-induced iNOS expression by RINm5F cells.
The antiviral response generated in infected cells includes the
expression of type 1 interferons, and the inhibition of protein synthesis due to the phosphorylation of elongation factor 2 (33, 35). A number of studies have implicated a dsRNA intermediate as the
activator of the antiviral response in infected cells (34, 38, 39).
Also, viral infection stimulates iNOS expression by target cells, and
nitric oxide appears to participate in the inhibition of viral
replication (8-15). In this study, evidence is presented which
implicates NF-
B as one transcriptional regulator that is activated
and participates in the antiviral response triggered by treatment of
macrophages with dsRNA. In addition, IL-1, released following dsRNA
treatment (in the presence of IFN-
), may also play a primary role in
the antiviral activities of macrophages.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Colleen Kelly for expert technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported in part by research grants from Alteon Inc. and The Tobacco Research Council, and by National Institutes of Health Grant DK-52194.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a Career Development Award from the Juvenile Diabetes
Foundation International. To whom correspondence should be addressed:
Dept. of Biochemistry and Molecular Biology, Saint Louis University
School of Medicine, 1402 South Grand Blvd., Saint Louis, MO 63104. Tel.: 314-577-8131; Fax: 314-577-8156; E-mail: corbettj{at}wpogate.slu.edu.
1 The abbreviations used are: NO, nitric oxide; NOS, nitric-oxide synthase; iNOS, inducible nitric oxide synthase; IL, interleukin; IFN, interferon; AG, aminoguanidine; poly(I-C), polyinosinic-polycytidylic acid; LPS, lipopolysaccharide; PEC, peritoneal exudate cell; PDTC, pyrrolidinedithiocarbamate; ds, double-stranded.
2 J. A. Corbett, unpublished observation.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|