Double-stranded RNA-induced Inducible Nitric-oxide Synthase Expression and Interleukin-1 Release by Murine Macrophages Requires NF-κB Activation*

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-γ (IFN-γ), and lipopolysaccharide (LPS) stimulate nitrite production and iNOS expression by RAW 264.7 cells. In combination, the effects of poly(I-C) + IFN-γ 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-γ. LPS-induced iNOS expression is associated with the activation of NF-κB. We show that inhibition of NF-κB by pyrrolidinedithiocarbamate prevents poly(I-C) + IFN-γ-, poly(I-C) + LPS-, and LPS-induced iNOS expression, nitrite production and IκB 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-γ, 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-γ stimulates iNOS expression, nitrite production, IκB 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-γ, is a potent activator of macrophages, stimulating iNOS expression, nitrite production, and IL-1 release by a mechanism which requires the activation of NF-κB.

cloned and characterized: endothelial NOS (eNOS or NOSIII), neuronal NOS (nNOS or NOSI), and inducible NOS (iNOS or NOSII) (2)(3)(4). Collectively, eNOS and nNOS are known as cNOS because their enzymatic activity is regulated by Ca 2ϩ 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, IB. Upon stimulation, IB is phosphorylated on specific serine residues which targets IB 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 ϩ IFN-␥-induced iNOS expression and nitrite production by RAW 264.7 cells (17), indicating that NF-B participates in LPS-and LPS ϩ 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 medi-ates 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 1ϫ 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. [␣-32 P]dCTP and enhanced chemiluminescence (ECL) reagents were purchased from Amersham Corp. NF-B consensus oligonucleotide and rabbit anti-IB-␣ and IB-␤ 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 l complete CMRL-1066 (CMRL-1066 containing 2 mM L-glutamine, 10% heat-inactivated fetal calf serum, 100 units/ml penicillin, and 100 g/ml streptomycin) in 24-well microtiter plates and incubated for 3 h under an atmosphere of 95% air and 5% CO 2 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.
RAW 264.7 and RINm5F cells were removed from growth flasks by treatment with 0.05% trypsin, 0.02% EDTA at 37°C. Cells were washed two times with complete CMRL-1066, plated at a concentration of 400,000 cells/400 l of complete CMRL-1066 in 24-well microtiter plates, and cultured at 37°C for 2-3 h prior to treatment. Experiments were initiated by the addition of poly(I-C), mouse IFN-␥, and/or LPS, and the cells were cultured as indicated at 37°C. In some experiments, cells were pretreated for 30 min with 100 M PDTC prior to stimulation with poly(I-C), IFN-␥, or LPS.
Western Blot Analysis-RAW 264.7 cells or CD-1 mouse PEC (400,000 cells/400 l 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 IB-␤, 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 IB was by ECL according to manufacturer's specifications (Amersham Corp.).
Northern Blot Analysis-RAW 264.7 cells (10 ϫ 10 6 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 g) 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 20ϫ 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 32 P-labeled probes specific for rat iNOS or cyclophilin (20,22). The DNA probes were radiolabeled with [␣-32 P]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).
Nitrite and IL-1 Determination-Nitrite production was determined by mixing 50 l of culture medium with 50 l 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.

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 ϩ 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 g/ml) or IFN-␥ (150 units/ml) stimulates a ϳ9and ϳ6-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) ϩ IFN-␥ are similar in magnitude to the effects of IFN-␥ ϩ LPS on RAW 264.7 cell nitrite production. AG, a selective inhibitor of iNOS (29), com-pletely prevents poly(I-C)-(data not shown), IFN-␥-(data not shown), poly(I-C) ϩ IFN-␥-, poly(I-C) ϩ LPS-, and LPS ϩ IFN-␥-induced nitrite production by RAW 264.7 cells.
The concentration-dependent effects of poly(I-C) on nitrite production by RAW 264.7 cells are shown in Fig. 1b. Alone, poly(I-C) stimulates the production of nitrite by RAW 264.7 cells at concentrations from 10 to 100 g/ml. The maximal effect of poly(I-C) on nitrite production by RAW 264.7 cells is observed at concentrations of 50 -100 g/ml. IFN-␥ alone also stimulates nitrite production by RAW 264.7 cells, and this effect is potentiated in a concentration-dependent manner by poly(I-C). Poly(I-C) ϩ IFN-␥ stimulates an over 20-fold increase in nitrite production, which is maximal at 50 -100 g/ml poly(I-C) ϩ 150 units/ml IFN-␥. These findings indicate that alone, poly(I-C) is capable of stimulating RAW 264.7 cell production of nitric oxide; however, the levels are significantly enhanced in the presence of IFN-␥. In addition, these findings indicate that poly(I-C) does not further enhance the levels of nitrite produced in response to LPS.
The effects of poly(I-C), in the presence or absence of IFN-␥ and LPS, on iNOS mRNA accumulation in RAW 264.7 cells was examined by Northern blot analysis. Individually, poly(I-C) and IFN-␥ stimulate the expression of barely detectable levels of iNOS mRNA following a 6-h exposure (ϳ7-and ϳ9-fold increase above control as determined by phosphorimaging analysis, respectively; Fig. 1c). However, the combination of poly(I-C) ϩ IFN-␥ stimulates a ϳ90-fold increase in iNOS mRNA accumulation. Similarly to nitrite production shown in Fig. 1a, poly(I-C) does not further increase the levels of iNOS mRNA accumulation in response to LPS (ϳ22-fold above control for both). Also, iNOS mRNA accumulation in response to poly(I-C) ϩ IFN-␥ is only slightly (ϳ3-fold) less than that accumulated in response to LPS ϩ IFN-␥.
The effects of poly(I-C), alone or in combination with LPS and IFN-␥, on iNOS protein expression (Fig. 1d) are similar to the effects of these agents on iNOS mRNA accumulation. RAW 264.7 cells express low levels of iNOS in response to poly(I-C) or IFN-␥; however, iNOS expression is drastically increased in response to the combination of poly(I-C) ϩ IFN-␥. Poly(I-C) also appears to increase the level of iNOS expression in response to LPS; however, the increase in iNOS protein expression is less than ϳ2-fold (as determined by densitometric analysis) and appears to occur in only ϳ40 -50% of experiments performed under these conditions (data not shown). Taken together, the results in Fig. 1 demonstrate that poly(I-C) stimulates iNOS expression and nitrite production, and that IFN-␥ potentiates the effects of poly(I-C) on 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.  Consistent with its inhibitory effects on nitrite production, PDTC also inhibits poly(I-C)-(data not shown), poly(I-C) ϩ IFN-␥-, LPS-, and LPS ϩ 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.
To determine if dsRNA activates NF-B, we have evaluated the effects of poly(I-C) on NF-B nuclear localization by gel shift analysis. As shown in Fig. 2c To further examine the role of poly(I-C) in NF-B activation, the effects of poly(I-C), alone and in combination with LPS and IFN-␥, on IB degradation was examined. As shown in Fig. 2d, treatment of RAW 264.7 cells for 30 min with 100 g/ml poly(I-C) results in the degradation of greater than 50% of IB-␣. IB-␣ degradation is prevented by treatment of RAW 264.7 cells for 30 min with PDTC prior to the addition of poly(I-C). In combination, poly(I-C) ϩ IFN-␥ stimulate IB-␣ degradation to levels similar in magnitude to the individual effects of poly(I-C), LPS, and LPS ϩ poly(I-C), and this effect is prevented by PDTC. Also, PDTC inhibits LPS-and LPS ϩ poly(I-C)-induced IB-␣ degradation. We have also examined the effects of poly(I-C), LPS, and IFN-␥, alone and in combination, on IB-␤ degradation and have obtained nearly identical results to those shown in Fig. 2d for IB-␣ (data not shown). These findings indicate that poly(I-C) stimulates NF-B nuclear localization by a mechanism that is associated with the degradation of IB.

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-1induced 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 ϳ20-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 Nitrite production was determined on the culture supernatants (a), and expression of iNOS (b) was determined by Western blot analysis of the RAW 264.7 cells as described under "Experimental Procedures." c, RAW 264.7 cells (5 ϫ 10 6 cells/3 ml of complete CMRL) were incubated for 30 min with 100 g/ml poly(I-C), 10 g/ml LPS, and 150 units/ml mouse IFN-␥ as indicated. Nuclear extracts were isolated, and gel shift analysis was performed as described under "Experimental Procedures." d, RAW 264.7 cells (400,000 cells/400 l of complete CMRL) were pretreated for 30 min with 100 M PDTC followed by incubation with 100 g/ml poly(I-C), 10 g/ml LPS, and 150 units/ml mouse IFN-␥ as indicated. IB-␣ degradation was determined by Western blot analysis as described under "Experimental Procedures." Results for nitrite are the average Ϯ S.E. of three independent experiments, and iNOS expression, NF-B gel shift analysis, and IB-␣ degradation are representative of three independent experiments. 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) ϩ 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-␥ ϩ 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) ϩ IFN-␥ stimulate the expression of iNOS and the production of nitrite to levels comparable to the effects of IFN-␥ ϩ LPS. The production of nitrite in response to poly(I-C) ϩ IFN-␥, or LPS ϩ IFN-␥ is completely prevented by the iNOS inhibitor AG. Importantly, the combination of poly(I-C) ϩ LPS does not stimulate iNOS expression or nitrite production by PEC. This finding is consistent with the inability of poly(I-C) to potentiate LPSinduced 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) ϩ 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 g/ml), IFN-␥ (150 units/ml), or LPS (10 g/ml) did not stimulate IL-1 release by PEC following a 24-h incubation (Fig. 5a). However, the combinations of poly(I-C) ϩ IFN-␥ and LPS ϩ 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 M PDTC completely inhibits poly(I-C) ϩ IFN-␥-induced IL-1 release, implicating a role for NF-B activation in IL-1 release by PEC.
To further examine the role of poly(I-C) on NF-B activation in mouse PEC, the effects of poly(I-C), alone and in combination with LPS and IFN-␥, on IB-␣ degradation was examined. Similarly to RAW 264.7 cells, poly(I-C), LPS, poly(I-C) ϩ IFN-␥ and LPS ϩ poly(I-C) induce the degradation of greater than 50% of IB-␣, and these effects are prevented by PDTC (Fig.  5b). PDTC also prevents poly(I-C) ϩ IFN-␥-and LPS ϩ IFN-␥-induced nitrite formation by CD-1 mouse PEC (data not shown). These findings implicate IB degradation and NF-B activation in poly(I-C) ϩ IFN-␥-induced nitrite production and IL-1 release by mouse macrophages. 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) ϩ 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) ϩ IFN-␥-induced iNOS expression, nitrite production, and IB degradation by RAW 264.7 cells is prevented by the NF-B inhibitor PDTC. In addition, PDTC inhibits poly(I-C) ϩ IFN-␥-induced iNOS expression, nitrite production (data not shown), IB degradation and IL-1 release by PEC. These findings indicate that poly(I-C)-induced IB degradation and NF-B nuclear localization participate in poly(I-C)-and poly(I-C) ϩ IFN-␥-induced iNOS expression by RAW 264.7 cells and mouse PEC. IB is phosphorylated on specific serine residues, and it is this phosphorylation that appears to direct proteolytic degradation of IB (18). The dsRNAdependent protein kinase is one potential candidate that may phosphorylate IB 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 IB 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 IB degradation 2 and NF-B nuclear localization (25,32). Poly(I-C) stimulates IB 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.