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The Journal of Immunology, 2001, 167: 2496-2501.
Copyright © 2001 by The American Association of Immunologists

Double-Stranded RNA Regulates IL-4 Expression1

Kelly E. Kehoe*, Melissa A. Brown{dagger} and Farhad Imani2,*

* Division of Clinical Immunology, Department of Medicine, Johns Hopkins University School of Medicine, Asthma and Allergy Center, Baltimore, MD 21224; and {dagger} Department of Pathology, Emory University, Atlanta, GA 30322


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
dsRNA, as genomic fragment, replicative intermediate, or stem and loop structure in cells infected by viruses, can act to signal the immune system of the presence of viral infections. Although most viral infections are associated with strong Th1 immune responses, Th2-type responses have also been observed. In this study, we characterize the effects of dsRNA on the induction of Th2 responses in human lymphocytes. We report that in addition to the well-known Th1-inducing capabilities of dsRNA, treatment of human lymphocytes with low concentrations of dsRNA (0.1–1 µg/ml) leads to the expression of the prototypic Th2 cytokine IL-4. This induction was accompanied with the concentration-dependent activation of NF-{kappa}B and NF-AT2 but not NF-AT1. In addition, dsRNA can directly activate an IL-4 promoter-driven chloramphenicol acetyltransferase reporter gene in transiently transfected Jurkat cells. These results are the first demonstration of a non-TCR-associated activator of NF-AT in human cells and suggest that dsRNA directly influences IL-4 gene expression through its effect on NF-AT activation. Our data provide support for the idea that dsRNA at low concentrations in vivo may induce a Th2-dominant response that is not optimal for protective immunity to the virus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Accumulated data suggest that based on the cytokine expression profiles, functionally immune responses mediated by CD4+ T cells can be roughly divided into at least two phenotypes, Th1 and Th2. Exaggerated polarization of the immune responses toward either phenotype leads to immunological disorders. The production of IFN-{gamma} and IL-12 is generally accepted as indicating Th1-type differentiation, whereas the production of IL-4 and IL-13 signifies a Th2-type differentiation (1, 2, 3, 4). Interestingly, Th1/Th2-differentiated cells not only produce specific cytokines, but the presence of specific cytokines is necessary for the initial steps in the differentiation of naive T cells (Th0) into Th1 or Th2 phenotypes.

Viral infections are generally known as a potent inducer of a Th1 phenotype, as characterized by the expression of IFN-{gamma}. There is increasing evidence to show that infections with several different viruses can induce a Th2 response. For example, infection with respiratory syncytial virus has been shown to elicit a Th2 response as indicated by IL-4 expression (5). Moreover, Reiser et al. (6) reported that, in contrast to healthy individuals in which IL-4 levels are undetectable, hepatitis C-infected individuals had elevated levels of IL-4 and IL-10. Studies by Griffin and colleagues (7) showed that experimental infection of mice with Sindbis virus resulted in an intracerebral expression of IL-4 and IL-10, while Mo et al. (8) reported that infection of mice with parainfluenza virus (Sendai virus) resulted in an increase in both Th1 and Th2 cytokines, including IL-4, IL-10, IL-6, and IFN-{gamma}. In addition, data by Whitmire et al. and Su et al. showed that during primary infection with lymphocytic choriomeningitis virus both Th1 and Th2 responses can be induced as detected by cytokine profile and Th subset studies (9, 10). Finally, a Th2 response may be necessary for down-regulation of proinflammatory antiviral Th1 responses during the decline of viral infections (11, 12).

In contrast to the viral-induced Th1 responses, the molecular mechanisms for the viral-induced immune differentiation toward Th2 pathways are not well studied. However, NF-AT and NF-{kappa}B have both been reported to be important for IL-4 production leading to Th2 differentiation (13, 14, 15, 16). Also, we recently reported that treatment of human B cells with dsRNA induced IgE class switching, a known Th2 response (17). dsRNA is present during infection with most viral strains and has been shown to induce Th1 differentiation (18, 19). However, based on our previous data, we speculated that dsRNA could also induce a signal for Th2 differentiation. Here, we provide data to suggest that at low concentrations, dsRNA can induce IL-4 expression this is concomitant with NF-AT2 activation. These data provide a potential molecular mechanism for the association of viral infections with immune differentiation toward a Th2 phenotype.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture conditions and reagents

The human T lymphoma Jurkat cell line at (1 x 105–1 x 106 cells/ml) was grown in RPMI 1640 supplemented with 10% FCS, 0.1 mM nonessential amino acids, and 1 mM sodium pyruvate at 37°C in a 5% CO2 humidified chamber. Freshly prepared human PBMC were isolated by Ficoll-Paque density centrifugation of normal blood (Pharmacia Biotech, Uppsala, Sweden). The synthetic dsRNA, polyinosinic-polycytidilic acid (poly I:C),3 was obtained from Sigma (St. Louis, MO), and each lot is first tested and selected based on activation of dsRNA-activated protein kinase (PKR) in the in vitro kinase assays. All other reagents were of the highest quality available. To obtain conditioned medium, 1 x 107 PBMCs were treated with 1 µg/ml poly I:C. After 24 h, the supernatant was harvested by centrifugation at 1000 x g for 5 min. Fresh PBMCs cells were then grown in the conditioned medium for 24 h before RNA extraction.

RNA extraction, detection of cytokine mRNA, and ELISA

RNA was isolated using the TRIzol total RNA isolation reagent (Life Technologies, Gaithersburg, MD). After reverse transcription, the cDNA was amplified in the presence of 2 µg/ml primers, 100 µM dNTPs, 0.25 U of Taq polymerase (PerkinElmer, Norwalk, CT), 10 mM Tris-HCl, pH 9.0, 50 mM KCl, 1.5 mM MgCl2, and 0.001% gelatin in a final volume of 25 µl. The sequence of primers were as follows: IL-4 forward, 5'-ggg tct cac ctc cca act gc-3'; IL-4 reverse, 5'-cga aca ctt tga ata ttt ctc-3'; IFN-{gamma} forward, 5'-agt tat atc ttg gct ttt ca-3'; IFN-{gamma} reverse, 5'-acc gaa taa tta gtc agc tt-3'; GAPDH forward, 5'-cac agt cca tgc cat cac tg-3'; GAPDH reverse, 5'-tac tcc ttg gag gcc atg tg-3'. Number of PCR cycles was optimized for each primer set. ELISAs were performed using R&D Systems (Minneapolis, MN) high-sensitivity IL-4 and IFN-{gamma} ELISA kits.

EMSA and Ab-mediated supershifts

Cell extracts for EMSA were prepared according to Schreiber et al. (20). EMSAs were performed using [{gamma}-32P] end-labeled NF-{kappa}B (from {kappa} light chain) consensus oligonucleotide (Promega, Madison, WI) and NF-AT consensus oligonucleotide (Santa Cruz Biotechnology, Santa Cruz, CA). The reactions (20 µl) consisted of 2 µl of nuclear extract in buffer containing 20 mM HEPES (pH 7.5), 50 mM KCl, 0.2 mM EDTA, 10% glycerol, 40 µg/ml poly I:C, and 0.5 µl of labeled probe. After 30 min of incubation at 37°C, the protein-DNA complexes were resolved on 4.5% nondenaturing polyacrylamide gel and were visualized by autoradiography of the dried gels. Supershift experiments were performed on dsRNA-treated Jurkat and PBMC extracts. mAbs to NF-AT2 and NF-AT1 (Affinity BioReagents, Boulder, CO) and specific Abs to p50 and p65 subunits of NF-{kappa}B (Santa Cruz Biotechnology) were added at 25 µg/ml final concentrations. The protein content was determined by bicinchoninic acid assay (Pierce, Rockford, IL).

IL-4 promoter construct transfection and chloramphenicol acetyltransferase (CAT) assays

The reporter plasmid pCATbasic or a plasmid containing the IL-4 promoter construct spanning to -797 nt of murine IL-4 was used in the assays (21). Jurkat cells (2.5 x 106) were transiently transfected with 10 µg of plasmid construct using Lipofectamine (Life Technologies) according to the manufacturer’s recommendation. After 48 h, cells were either left alone or were treated with 1 µg/ml dsRNA or 40 ng/ml calcium ionophore A23187 (Boehringer Mannheim, Mannheim, Germany). The cells were harvested after 24 h of induction, and CAT assays were performed using a CAT enzyme assay system (Promega) and [14C]chloroamphenicol (ICN Pharmaceuticals, Irvine, CA). CAT activity was normalized for protein content using a bicinchoninic acid assay.

IFN treatment and in vitro kinase reactions

To enhance the detection of PKR in the in vitro kinase assays, Jurkat cells were first treated with 100 µm/ml human IFN-{alpha} (Lee Biomolecular, San Diego, CA). After 24 h, cells were washed twice with isotonic buffer containing 20 mM HEPES, pH 7.5, 120 mM KCl, 5 mM magnesium acetate, and 1 mM DTT. Cells were then lysed in buffer containing 20 mM HEPES, 120 mM KCl, 5 mM magnesium acetate, 1 mM benzamidine, 1 mM DTT, and 1% Nonidet P-40.

In vitro kinase reactions were performed in a mixtures containing 20 mM HEPES, pH 7.5, 90 mM KCl, 5 mM magnesium acetate, 1 mM DTT, 100 µM [{gamma}-32P]ATP (sp. act. 1 Ci/mM; Amersham, Arlington Heights, IL), 100 µM ATP (Sigma), and equal amounts of detergent extract prepared from 1 x 106 cells, in a final volume of 25 µl. dsRNA (poly I:C) was added to the reaction mixtures at indicated concentrations, and the mixture was allowed to incubate at 30°C. After 10 min, the reactions were quenched by adding an equal volume of 2x SDS-sample buffer containing 62.5 mM Tris-HCl (pH 6.8), 10% glycerol, 2% SDS, 0.0125% bromophenol blue, and 5% 2-ME. After boiling for 2 min, the reduced, denatured proteins were then subjected to electrophoresis through 10% SDS-PAGE. The labeled proteins were visualized by autoradiography of the dried gels.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
dsRNA treatment can induce IL-4 expression

The mechanisms for the viral induced immune differentiation into Th2 phenotype are not well understood. However, it is well established that the induction of Th2 phenotype is accomplished by secretion of cytokines such as IL-4 and IL-13 (4, 22). Accordingly, we examined the effects of dsRNA on the expression of the potent Th2-inducing cytokine IL-4 in human PBMCs. Freshly prepared human PBMCs were isolated from healthy individuals and were treated with 1 µg/ml of the synthetic dsRNA poly I:C. Total cellular RNA was extracted at indicated times and was subjected to semiquantitative RT-PCR (Fig. 1GoA). IL-4 mRNA was detectable at 6 h after dsRNA treatment, and the maximal induction was reached at 24 h after treatment.



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  		FIGURE 1. dsRNA treatment can induce IL-4 expression. A, To determine the effects of dsRNA on IL-4 expression, human PBMCs from healthy volunteers were treated with 1 µg/ml poly I:C, and, after indicated times posttreatment, total cellular RNA was extracted and was subjected to RT-PCR with specific primers to IL-4 (n = 2). To collect the PCR products at the linear range, the number of PCR cycles was optimized for each primer set. B, To determine the optimal concentration for the dsRNA-induced IL-4 expression, human PBMCs were treated with increasing concentrations of poly(I:C). After 24 h, RT-PCR was performed on total cellular RNA (n = 4). C, To examine whether the dsRNA-induced IL-4 expression was attributable to secretion of other mediators, isolated PBMCs were treated with dsRNA, and after 24 h the supernatants were harvested. The cells were then either grown in the conditioned supernatants from mock treatment (lane A), treatment at 1 µg/ml (lane B) poly I:C, or were directly treated with 1 mg/ml poly I:C (lane C) or 5 µg/ml PHA (lane D). After 24 h, total cellular RNA was isolated, and RT-PCR was performed using specific primers to human IL-4 (n = 2). D, To test for secretion of IL-4 after dsRNA treatment, human PBMCs were isolated from healthy volunteers and were either treated with 1 µg/ml poly I:C (left) or were treated with poly I:C and 5 µg/ml PHA (right). After 24 h, culture supernatants were harvested and were subjected to IL-4-specific ELISA. ELISAs were performed with a high-sensitivity IL-4 kit (R&D Systems). The median cytokine production +SD, expressed in picograms per milliliter, of three experiments is shown (n =2).

 
To determine the optimal dsRNA concentration for IL-4 induction, PBMCs were treated with increasing concentrations of dsRNA. After 24 h, we performed RT-PCR on total cellular RNA using primers to IL-4, IFN-{gamma}, and GAPDH. The data revealed that dsRNA treatment of PBMCs resulted in a concentration-dependent increase in the mRNA levels of IL-4 (Fig. 1GoB, left and right). The maximal cytokine mRNA induction was achieved at 1–5 µg/ml dsRNA. Treatment of cells with high concentrations of dsRNA (>10 µg/ml) was not as effective in the induction of IL-4 mRNA (Fig. 1GoB). The level of IFN-{gamma} mRNA did not increase after dsRNA treatment at low concentration. However, high concentrations of dsRNA (>10 µg/ml) preferentially increased the expression of IFN-{gamma} mRNA (Fig. 1GoB).

To test whether the dsRNA-induced IL-4 expression was mediated by secretion of other mediators, we first treated PBMCs with 1 µg/ml dsRNA, and after 24 h the medium was harvested; the conditioned medium was then used to treat fresh PBMCs. The data from RT-PCR showed that treatment of cells with conditioned medium did not induce IL-4 expression, suggesting that the effect of dsRNA is not through other intermediate steps (Fig. 1GoC).

Specific ELISA on culture supernatants from dsRNA-treated (1 µg/ml, optimal concentration) PBMCs revealed that ~8 pg/107 cells of IL-4 was detected in the medium (Fig. 1GoD). We hypothesized that the low level of IL-4 production in PBMCs may be due to the fact that circulating lymphocytes are at the resting state. To determine whether activation could increase dsRNA-potentiated cytokine production, PBMCs were stimulated with 5 µg/ml PHA before dsRNA treatment. The data showed that PHA alone resulted in the production of 20 pg/ml IL-4, whereas addition of dsRNA to the PHA-stimulated PBMCs enhanced the IL-4 expression to 50 pg/ml (Fig. 1GoD, right). Treatment of PBMCs with 1 µg/ml poly I:C did not lead to any increase in level of secreted IFN-{gamma} (data not shown).

dsRNA treatment can activate the IL-4 promoter

To further characterize the dsRNA induction of IL-4 transcription, we performed promoter activation studies using CAT reporter assays. Jurkat T cells were transiently transfected with IL-4 promoter constructs spanning to -797 nt in the murine IL-4 promoter containing multiple NF-AT sites. After 48 h, cells were either mock treated or were treated with 1 µg/ml dsRNA or 40 ng/ml ionophore A23187. After 24 h of induction, cells were harvested, and equal amounts of cell extracts were subjected to CAT assays. The data showed that dsRNA treatment could induce an ~6-fold increase in IL-4 promoter activity. Promoter activation increased by 8-fold after treatment with A23187 (Fig. 2Go).



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  		FIGURE 2. dsRNA treatment induces IL-4 promoter activation. Because dsRNA induced IL-4 expression in PBMCs, we tested whether dsRNA treatment could directly activate IL-4 promoter in Jurkat T cells. The cells were transiently transfected with a pCAT plasmid construct carrying IL-4 promoter (nt -797). After 48 h, cells were left alone (lane A), were treated with poly I:C at 1 µg/ml (lane B), or were treated with 40 ng/ml ionophore A21387 (lane C). Equal amounts of cell extracts were subjected to CAT assays. The relative increase in CAT activity ±SD of three separate experiments are shown.

 
dsRNA treatment activates NF-AT2 and NF-{kappa}B

It is well accepted that activation of NF-AT and NF-{kappa}B are necessary for IL-4 expression and subsequent induction of Th2 differentiation (14, 15, 23). Because the reporter gene studies showed that dsRNA could activate the IL-4 promoter containing the NF-AT binding sites, we performed EMSA experiments to determine whether dsRNA could activate these NFs.

Freshly prepared PBMCs and human T cell line Jurkat were treated with increasing concentrations of dsRNA. After 2 and 24 h, determined to be maximal for NF-{kappa}B and NF-AT activation, respectively, cells were harvested and whole-cell extracts were prepared and used in mobility shift assays with probes corresponding to NF-AT and NF-{kappa}B. Our data revealed that when human PBMCs and human Jurkat T cells were treated with dsRNA, both NF-AT and NF-{kappa}B were activated in a concentration-dependent fashion (Figs. 3Go, A and B, and 4). Consistent with the effects on cytokine expression, the concentration response curve was bell shaped such that at high concentrations (above 10 µg/ml) dsRNA treatment was not effective in activation of either NFs.



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  		FIGURE 3. dsRNA treatment induces NF-AT activation. To determine whether dsRNA treatment could activate NF-AT, we treated human PBMCs with increasing concentrations of poly I:C. After 24 h (determined to be optimal for NF-AT activation), whole-cell extracts were prepared. EMSAs were performed on extracts prepared from dsRNA-treated human PBMCs (A) and Jurkat cells (B) with [{gamma}-32P] end-labeled consensus oligonucleotide specific for NF-AT subunits. After 30 min of incubation at 30°C, the protein-DNA complexes were resolved on 4.5% nondenaturing polyacrylamide gel and were visualized by autoradiography of the dried gels (n = 4). B, Competition and supershift experiment were performed on extracts prepared from dsRNA-treated PBMCs. mAbs to NF-AT2 (NF-ATc1) and NF-AT1 (NF-ATp) were purchased from Affinity BioReagents and were used at 25 µg/ml final concentrations. After a 10-min incubation at 30°C, the specific mAb were added and the mixture was allowed to incubate for an additional 20 min, and the complexes there were resolved by electrophoresis through a 4% nondenaturing polyacrylamide gel (n = 3).

 
To detect which specific NF-{kappa}B and NF-AT subunits were activated by dsRNA, we performed Ab-mediated supershift assays on extracts prepared at 2 and 24 h, respectively. Supershifts identified the NF-AT2, a critical molecule for Th2 differentiation (24), to be activated by dsRNA (Fig. 3GoC). However, specific Ab to NF-AT1 did not cause a supershift. Ab-mediated supershifts detecting NF-{kappa}B subunits showed that both p50-containing and p65-containing complexes were activated by dsRNA treatment (Fig. 4GoB)



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  		FIGURE 4. NF-{kappa}B and PKR are activated in T cells after dsRNA treatment. To test for the activation of NF-{kappa}B after dsRNA treatment, human Jurkat T cells were treated with increasing concentrations of poly I:C and after 2 h were harvested and whole-cell extracts were prepared. A, EMSA was performed using specific probe to NF-{kappa}B binding site, and after 30 min of incubation at 37°C the complexes were resolved by electrophoresis through a 4% nondenaturing polyacrylamide gel (n = 3). B, To identify the NF-{kappa}B subunits, we performed Ab-mediated supershift assays. After 10 min of incubation at 37°C, specific Abs to p50 and p65 subunits of NF-{kappa}B were added at a 25 mg/ml final concentration. The reaction was allowed to incubate for an additional 20 min, and then the complexes were resolved as in A (n = 3). C, To determine whether PKR is present in Jurkat cells, we performed in vitro kinase assays detecting PKR autophosphorylation. To increase the intracellular levels of PKR, Jurkat cells were pretreated with 100 U/ml IFN-{alpha}. After 24 h, cytoplasmic extracts were prepared, and in vitro kinase reactions were performed on equal amounts of protein (5 x 105 cell equivalence) in the presence of increasing concentrations of dsRNA and [{gamma}-32P]ATP. The proteins were resolved on a 10% SDS-PAGE, and the phosphorylated polypeptides were visualized by autoradiography of the dried gel.

 
One possible explanation for the observed biphasic activation of these NFs by dsRNA could be the presence of a dsRNA-regulated intermediate step. It is known that serine/threonine protein kinases are required for NF-AT and NF-{kappa}B activation (25, 26). A unique dsRNA-activated protein kinase has been described, namely PKR. This kinase is known to activate NF-{kappa}B by phosphorylating I{kappa}B (27). This kinase is activated at low concentrations (0.01–1 µg/ml) of dsRNA but does not respond to high dsRNA concentrations (3–10 µg/ml) (28). To test for the presence and dsRNA activation of PKR in human T cells, we performed in vitro kinase assays. First, to enhance the visualization of PKR in the assays, the level of inactive endogenous PKR was up-regulated by treating the cells with 100 µg/ml IFN-{alpha}. After 24 h, the cells were lysed in buffer containing Nonidet P-40, and phosphorylation of dsRNA-induced autophosphorylation of PKR was tested in in vitro kinase assays. PKR autophosphorylation was detected after the addition of low concentrations of dsRNA, and, as expected, high concentrations of dsRNA were not as effective (Fig. 4GoC). This biphasic activation is likely due to the fact that PKR requires dimerization on the same strand of dsRNA; excess dsRNA prevents this required intramolecular interaction (29, 30).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Protective immunity to most infectious agents requires a balance between Th1/Th2 immune responses. However, an exaggerated or unregulated Th1 response can lead to autoimmune disorders, whereas a polarized Th2 response contributes to the manifestation of allergic reactions (31, 32). A balanced response is due, in part, to the cross-regulatory properties of the cytokines produced in each type of Th response. For example, Th2 cytokines such as IL-4 promote the development of Th2 cells but can interfere with Th1 cell development and inhibit the effects of proinflammatory cytokines (11, 12). In contrast, the Th1-associated cytokine IFN-{gamma} inhibits the proliferation of Th2 cells and block Th2 effector function such as B cell activation (33, 34).

A wealth of information has accumulated regarding the induction of Th1 immune responses by viral infections. dsRNA as genomic fragment, replicative intermediate, or stem and loop structure is present during the life cycle of many viruses and can serves as a signal for the presence of viral infections. Several studies have implicated dsRNA in the induction of Th1-type immune responses. Finkelman and colleagues (18) showed that injection of mice with poly I:C at 200 µg/mouse resulted in an increase in Th1 cytokines and a concomitant decrease in IgE responses. A recent report by Cella et al. (19) has shown that poly I:C at 20 µg/ml was capable of inducing a Th1 differentiation in dendritic cells. Poly I:C treatment has been reported to accelerate the development of Th1 autoimmune disorders such as experimental autoimmune encephalomyelitis, adjuvant arthritis, and type I diabetes presumably through its enhancement of Th1 responses (35, 36, 37).

Viral infections are often associated with the induction of Th2 responses as well. However, it is not known whether dsRNA plays a role in this event. In this report, we examine the effect of dsRNA on Th2-type cytokine expression in T lymphocytes. We demonstrate that at low concentrations dsRNA can induce IL-4 mRNA. In contrast, at high concentrations, dsRNA treatment is not as effective in the induction of IL-4 mRNA but it can induce an increase in the level of IFN-{gamma} mRNA. This is consistent with other reports showing that dsRNA could induce a Th1 response. It is important to note that there is variability among donors in their responses to dsRNA; however, treatment with low concentrations of dsRNA induces a Th2 cytokine profile. IL-4 protein production is also enhanced in cells treated with low doses of poly I:C. The induction of IL-4 by dsRNA is not through secretion of other mediators because treatment of PBMCs with conditioned medium did not induce IL-4 mRNA. This induction in IL-4 gene activity is associated with a dose-dependent increase in NF-AT2 and NF-{kappa}B activation. Our kinetic studies showed that the maximal level of NF-AT2 activation was reached at 24 h after dsRNA treatment, whereas NF-{kappa}B activation was maximally detected at 2–4 h after treatment (data not shown). At this point, we do not know the significance of this sequential dsRNA-induced activation, but we speculate that this cascade of NF activation may be relevant to Th2 differentiation.

Because it is known that serine/threonine protein kinases are required for NF-AT and NF-{kappa}B activation (25, 26), we speculated that an intermediate dsRNA-regulated step may provide an explanation for the observed dichotomous response to dsRNA. It is known that a unique protein kinase, namely PKR, is specifically regulated by dsRNA and can activate NF-{kappa}B (27). This kinase is activated at low concentrations (0.01–1 µg/ml) and it is inhibited at high dsRNA concentrations (above 3–10 µg/ml) (28). Based on previous observations and our data, we speculate that the dichotomy in the poly I:C-induced Th1/Th2 differentiation may be mediated by the well-established biphasic dsRNA-dependent activation of PKR (28). Additional experiments are underway to characterize the exact role of PKR in this observation.

Our results are consistent with the idea that low concentrations of dsRNA produced during a viral infection induce a Th2 response, thus reducing Th1-associated inflammatory responses. A recent report by Sobel et al. (38) supports this hypothesis. They showed that in contrast to their previous observation that poly I:C treatment at 5 µg/g body weight could induce diabetes in BioBreeding rats, treatment of these animals with low-dose poly I:C at 0.05 µg/g body weight could prevent diabetes (38). One explanation for the prevention of diabetes by low-dose poly I:C is that at low concentrations dsRNA can induce a Th2 phenotype, therefore preventing the Th1-associated induction of diabetes.

It is clear that the development of specific immune responses toward an Ag is mediated by the presence of secreted cytokines. However, the source of the cytokine necessary for the initial steps of the immune differentiation into Th2 pathways is not clear. Based on our data, it is tempting to speculate that during viral infections the presence of dsRNA can provide a signal for the induction of Th2 cytokine production. This may be beneficial for viral pathogenesis (39). Alternatively, during decline of viral infections and viral clearance, a Th2 response may be necessary to down-regulate inflammation and help resolve the anti-viral Th1 responses (11, 12). It is also possible that dsRNA-induced Th2 cytokine production during viral infections may cause a Th2-type differentiation toward a bystander Ag, leading to an increased chance of an atopic response. Additional experiments are necessary to determine the exact nature of dsRNA-induced signals necessary for both Th1 and Th2 phenotypes. This information may lead to improved methods of immune modulation in autoimmune disorders and to control of viral pathogenesis.


   Acknowledgments
 
We gratefully acknowledge Drs. Bradley Undem and David Proud (Johns Hopkins University) for critical review of the manuscript.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant AI44696 (to F.I.). Back

2 Address correspondence and reprint requests to Dr. Farhad Imani, Division of Clinical Immunology, Department of Medicine, Johns Hopkins University School of Medicine, Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224. E-mail address: fimani{at}mail.jhmi.edu Back

3 Abbreviations used in this paper: poly I:C, polyinosinic-polycytidilic acid; PKR, dsRNA-activated protein kinase; CAT, chloramphenicol acetyltransferase. Back

Received for publication November 8, 2000. Accepted for publication June 19, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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