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Double-Stranded RNA Induces the Synthesis of Specific Chemokines by Bronchial Epithelial Cells

Departments of Pediatrics and Medicine, University of Wisconsin-Madison Medical School, Madison, Wisconsin

Address correspondence to: James E. Gern, M.D., K4/918 CSC, 600 Highland Avenue, Madison, WI 53792-9988. E-mail: gern{at}medicine.wisc.edu

	Abstract

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Virus-induced secretion of proinflammatory chemokines (e.g., regulated on activation, normal T cells expressed and secreted [RANTES], interleukin [IL]-8) by airway epithelial cells helps to initiate antiviral responses and airway inflammation by enhancing inflammatory cell recruitment. To define mechanisms for virus-induced chemokine secretion, monolayers of nontransformed bronchial epithelial cells were transfected or incubated with polydeoxyinosinic-deoxycytidylic acid (synthetic double-stranded [ds] RNA), rhinovirus dsRNA, or single-stranded RNA (ssRNA), and the secretion of selected chemokines was determined. Transfection or incubation with dsRNA, but not ssRNA, significantly enhanced secretion of RANTES and IL-8, but not eotaxin or macrophage inflammatory protein-1. Mechanistically, dsRNA induced and activated dsRNA-dependent protein kinase (PKR), and activated nuclear factor-B and p38 mitogen-activated protein kinase. Furthermore, the PKR inhibitor 2-aminopurine significantly blocked dsRNA-induced RANTES and IL-8 secretion, whereas the p38 mitogen-activated protein kinase inhibitor SB203580 suppressed dsRNA-induced IL-8, but not RANTES. These findings indicate that dsRNA selectively induce the secretion of chemokines such as IL-8 and RANTES, and implicate dsRNA-sensitive signaling proteins in this process. Moreover, these data suggest that this may be an important mechanism for the selective secretion of chemokines by viruses (e.g., rhinovirus, respiratory syncytial virus, influenza) that synthesize dsRNA during replication.

Abbreviations: activator protein-1, AP-1 • bronchial epithelial, BE • double-stranded, ds • enzyme-linked immunosorbent assay, ELISA • interferon, IFN • interleukin, IL • mitogen-activated protein, MAP • macrophage inflammatory protein, MIP • nuclear factor-B, NF-B • polyacrylamide gel electrophoresis, PAGE • phosphate-buffered saline, PBS • dsRNA-dependent protein kinase, PKR • polydeoxyinosinic-deoxycytidylic acid, poly IC • regulated on activation, normal T cells expressed and secreted, RANTES • rhinovirus, RV • sodium dodecyl sulfate, SDS • single-stranded, ss • tumor necrosis factor-, TNF-

	Introduction

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Rhinoviruses (RV) cause over half of common cold illnesses, and are frequently associated with exacerbations of asthma in children and adults (1, 2). Because RV infect only a small subset of airway epithelial cells, and do not cause appreciable damage to the epithelial cell lining, it is proposed that the immune response to RV infections leads to respiratory symptoms and may promote exacerbations of asthma. Airway epithelial cells are important initiators of the immune response to RV and other respiratory viruses through the secretion of inflammatory cytokines and chemokines. During viral respiratory infections, concentrations of epithelial-derived chemokines such as interleukin (IL)-8 and regulated on activation, normal T cells expressed and secreted (RANTES), which contribute to the recruitment of granulocytes and activated lymphocytes into the airway, correlate with cold symptoms (3, 4) and wheezing (5), and in patients with asthma, increased airway responsiveness (6). Together, these observations suggest that the immune response to respiratory viral infections contributes to the pathogenesis of respiratory symptoms, and this effect may be especially troublesome in patients with chronic respiratory diseases such as asthma, chronic obstructive pulmonary disease, or cystic fibrosis.

This line of reasoning has stimulated research to define signaling pathways that initiate and regulate virus-induced chemokine secretion by epithelial cells. Epithelial cells are the principal host cells for these pathogens, and presumably the process of RV replication triggers signaling pathways that stimulate chemokine secretion. This possibility is supported by studies that show that UV-inactivated virus, which is nonreplicative, generally has been found to be a poor stimulus for IL-8 or RANTES (7–9).

RV and other single-stranded (ss) RNA viruses synthesize double-stranded RNA (dsRNA) during replication (10), and this is a potent stimulus for innate antiviral responses and the secretion of cytokines such as IL-6, interferon (IFN)-, and IFN-ß (11). These findings led us to hypothesize that dsRNA might also be an important stimulus for the synthesis of chemokines by virus-infected epithelial cells. To test this proposal, experiments were conducted to determine the effects of dsRNA (RV dsRNA, and the synthetic dsRNA analog polydeoxyinosinic-deoxycytidylic acid [poly IC]) on the production of chemokines (IL-8, RANTES, eotaxin, macrophage inflammatory protein [MIP]-1) by nontransformed bronchial epithelial (BE) cells, and to determine effects of dsRNA on the activation of signaling pathways (nuclear factor [NF]-B and p38 mitogen-activated protein [MAP] kinase) that promote chemokine production.

	Materials and Methods

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Airway Epithelial Cells
BE cells were obtained by enzymatic digestion of bronchi trimmed from lungs destined for transplantation, as previously described (7). The experimental protocol was approved by the University of Wisconsin Human Subjects Committee.

Synthesis of RV ss- and dsRNA
A plasmid containing the native RV16 sequence (pRV16.11) was synthesized by Wai Ming Lee and Wensheng Wang (Institute of Molecular Virology, University of Wisconsin-Madison, Madison, WI) (12). A 682-bp segment of the 5' untranslated region (5'UTR) of the RV16 genome was excised (XbaI) from the pRV16.11 plasmid and cloned into the pGEM-3Z vector (Promega, Madison, WI) which contains a T7 and an SP6 promoter (pGEM5'UTR). 5'UTR RNA was synthesized by in vitro transcription from each promoter to generate complementary ssRNA. RV16 RNA was synthesized from the linearized pRV16.11 using T7 RNA polymerase. To remove trace amounts of dsRNA, ssRNA was purified through sequential denaturing and nondenaturing gels, eluted, and purified by phenol/chloroform and ethanol precipitation (13). The RNA was quantitated by comparison with known amounts of a reference standard mRNA after electrophoresis in an agarose gel.

To prepare RV dsRNA, complementary ssRNA from the 5'UTR were hybridized and treated with RNAse A to remove any remaining ssRNA. The RNA was purified and quantitated in the same manner as the ssRNA.

Epithelial Cell Transfection
Nearly confluent monolayers of primary epithelial cells in 6-well tissue culture plates were transfected (Lipofectamine [Life Technologies, Rockville, MD] or Effectene [Qiagen, Santa Clarita, CA]) with 1–1,000 ng/well of RV RNA, poly IC (Sigma Chemical Co, St. Louis), poly U (Sigma), or medium alone. After incubation (4–5 h) with the liposomal suspension (100 µl) of RNA and medium without antibiotics (600 µl), the cells were gently washed and incubated for an additional 24–48 h in fresh medium with antibiotics. Cell supernatant fluids were then collected for the quantitation of RANTES and IL-8 protein.

Measurement of Cytokine Protein and mRNA
Cytokine proteins were measured by enzyme-linked immunosorbent assay (ELISA). RANTES and IL-8 ELISAs were performed as previously described (7), and MIP-1 and eotaxin ELISA kits were purchased from commercial sources (Biosource International, Camarillo, CA), and the assays were conducted according to the manufacturer's protocol. The sensitivity of the assays was 16 pg/ml, and the coefficient of variation was generally < 10%.

Total RNA was isolated from pelleted cells using a phenol/chloroform reagent (Trizol; Life Technologies), and RANTES and G3PDH mRNA were analyzed using semiquantitative RT-PCR, as previously described (7).

Cell Culture and Preparation of Whole Cell Extracts
Human BE cells were grown to 80% confluence in BEGM medium in 6-well tissue culture plates. After transfection with poly IC or RV RNA in a liposomal suspension (Effectene; Qiagen), cells were washed twice with 3 ml phosphate-buffered saline (PBS) and then scraped along with 1 ml PBS into a microcentrifuge tube. The cells were centrifuged (1,500 RPM, 4°C, 10 min), drained, and then flash frozen in liquid nitrogen and stored at -80°C. After thawing the cells on ice, a 15-µl extract buffer was added to the cell pellet. The extract buffer consisted of 20 mM HEPES (pH 7.9), 350 mM NaCl, 20% glycerol, 1% IGEPAL CA-630, 1 mM MgCl₂, 0.5 mM EDTA, and 0.1 mM EGTA, to which the following were added just before use: 0.5 mM dithiothreitol, 15 µg/ml aprotinin, and 1:100 protease inhibitor cocktail (Sigma P-8340). The cells were incubated on ice for 30 min with gentle mixing every 10 min. The extract was centrifuged (14,000 RPM, 10–15 min) to remove cell particulates, and protein was measured by Bradford assay.

NF-B Electromobility Shift Assay
Activation of NF-B was measured by electromobility shift assay, as per the protocol of Miyamoto and colleagues (14). The NF-B binding reaction was performed in 15 mM Tris pH 7.5, 75 mM NaCl, 1.5 mM EDTA, 1.5 mM dithiothreitol, 7.5% glycerol, 0.3% IGEPAL CA-630, and 20 µg/ml bovine serum albumin with freshly added 50 µg/ml poly IC. After a 20-min incubation on ice, the end-labeled, double-stranded oligo (5'-CTCAACAGAGGGGACTTTCCGAGAGGCAT-3') was added and the reaction was incubated at 25°C for 20 min. The complexes were separated on a 4% native polyacrylamide gel. The gel was dried and exposed to X-ray film.

PKR Western Blot and Kinase Assay
After incubation with poly IC or cytokine activators, BE cells were lysed in buffer containing phosphatase inhibitors, and equal amounts of total protein were subjected to sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoresis (PAGE) and transferred to a nitrocellulose membrane. Western blot analysis was performed using a polyclonal antibody to dsRNA-dependent protein kinase (PKR; Santa Cruz Biotechnology, Santa Cruz, CA) and ECL detection reagent (Amersham Biosciences, Piscataway, NJ).

PKR activity was determined by a kinase assay according to the protocol of Williams and colleagues (15). Briefly, BE cells were incubated with dsRNA or medium alone, washed, and cell lysates were prepared. Protein content was determined using Bradford reagent, and the lysates were adjusted to a constant concentration with lysis buffer. Immunoprecipitation was performed using 50 µg total protein in a total volume of 250 µl along with 1 µl PKR mAb (HC 71/10 ascites fluid diluted 1:10 in PBS/ 0/1% bovine serum albumin; Questcor, Hayward, CA), and 20 µl of protein A/G-agarose beads. After washing twice with each lysis buffer and kinase wash buffer, the kinase reaction was performed by incubation (20 min, 30°C) of the beads in a volume of 40 µl containing 10 mM Tris-Cl pH 7.6, 2 mM magnesium acetate, 50 mM KCl, 7 mM mercaptoethanol, 20% glycerol, 0.83 mM MnCl, 0.8 µM ATP, and 180 µCi/mL (-P³²) ATP. For a positive control, a sample from unstimulated BE cells was treated with 100 ng/ml poly IC. After the reaction was complete, 80 µl of 2x SDS loading buffer was added, the mixture was heated (100°C, 5 min), and samples (60 µl) were resolved on a 10% SDS/PAGE gel, dried, exposed to a phosphor screen, and analyzed using the Storm Imaging System (Amersham Biosciences). Phosphorylation of a distinct protein band at the expected MW of 68 kD was interpreted as evidence of PKR activation.

p38 MAP Kinase Activity
To detect p38 MAP kinase, cells were lysed in SDS gel-loading buffer, and equal volumes of lysate were subjected to SDS/PAGE electrophoresis. After transfer to nitrocellulose, total and activated (phosphorylated) forms of p38 MAP kinase were analyzed by Western blot using specific polyclonal antisera (Cell Signaling Technology, Beverly, MA).

Statistical Analyses
Values for RANTES and IL-8 after activation with dsRNA were compared using two-way ANOVA, and factors considered included RNA dose and the individual experiment number. After screening with ANOVA, multiple comparisons with the control group (mock-transfected cells) were evaluated using Bonferroni's corrections. The data set for effects of inhibitors on RANTES and IL-8 secretion was log₁₀ transformed to approximate a normal distribution, and were then analyzed by paired t test. P values of 0.05 were considered to indicate statistically significant differences.

	Results

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Induction of RANTES and IL-8 Secretion by Transfection of dsRNA
To determine effects of dsRNA on epithelial cells, BE cells were transfected with a liposomal suspension containing 0–1,000 ng/well poly IC (synthetic dsRNA), and supernatant fluids were collected 24 h later and analyzed for secretion of the chemokines RANTES, IL-8, and eotaxin. Transfection of poly IC caused vigorous secretion of RANTES (Figure 1A) , which was maximal at 100–1,000 ng/well. Secretion of IL-8 also tended to be greater in the cells treated with poly IC (Figure 1B), but eotaxin was not induced (data not shown). In addition, transfection with the ssRNA analog poly U, plasmid DNA (data not shown), or mock transfection did not increase secretion of either RANTES or IL-8 (data not shown). Cytopathic effects were observed in cells that were transfected with concentrations of poly IC in excess of 1 µg/well.

View larger version (22K): [in this window] [in a new window]   Figure 1. Effect of dsRNA transfection on RANTES and IL-8 secretion. Monolayers of BE cells in 6-well tissue culture plates were mock-transfected (lipid reagent without RNA), or transfected with 1–1,000 ng/well of either poly IC (filled bars), ds RV RNA (hatched bars), or the ssRNA analog poly U (not pictured). 24 h after transfection, supernatant fluids were analyzed for RANTES (A) and IL-8 (B). The data represent mean values ± SEM for three separate experiments (*P < 0.05).

Effects of dsRNA Incubation on BE Cell Cytokine Secretion
Previous studies have demonstrated that mononuclear cells and fibroblasts can be activated by incubation with dsRNA, and it has recently been demonstrated that dsRNA can induce type-I interferon synthesis in murine cells through a toll-like receptor 3–dependent mechanism (16). Therefore, experiments were conducted to determine the effects of dsRNA incubation on BE cells. Incubation with poly IC (1–100 µg/ml) induced vigorous secretion of RANTES (maximal 7.1 ng/ml at 100 µg/ml versus 0 pg/ml in unstimulated cells, Figure 2A) . Furthermore, incubation with poly IC was a potent stimulus for IL-8 secretion (maximal 19.2 pg/ml versus 0.4 pg/ml in unstimulated cells, Figure 2B). In contrast, poly IC did not have significant effects on the secretion of either MIP-1 or eotaxin (data not shown).

View larger version (17K): [in this window] [in a new window]   Figure 2. Effect of dsRNA incubation on chemokine secretion. Monolayers of BE cells were incubated with poly IC (0–100 µg/ml) for 24 h, and supernatant fluids were analyzed for (A) RANTES and (B) IL-8. The data represent geometric means ± SEM for three experiments (*P < 0.05 versus cells incubated in medium alone).

View larger version (30K): [in this window] [in a new window]   Figure 3. Kinetics of RANTES mRNA and secretion after incubation with poly IC. Monolayers of BE cells were incubated with poly IC or medium alone for 0–8 h, and paired samples were analyzed for the presence of (A) RANTES and GAPDH mRNA, and (B) RANTES protein secretion.

Effect of dsRNA on Induction and Activation of PKR
Because dsRNA activation of PKR is required for synthesis of interferons in fibroblasts and mononuclear cells (11), and activation of nitric oxide in epithelial cells (17), experiments were conducted to evaluate the potential role of PKR induction and activation in dsRNA-induced chemokine synthesis. To evaluate the regulation of PKR synthesis in BE cells, cell monolayers were incubated with either poly IC (100 µg/ml), tumor necrosis factor (TNF)- (100 U/ml), IFN- (10 U/ml), or medium alone. Poly IC incubation induced PKR protein after 16–23 h (Figure 4A) . IFN- also induced PKR protein, although to a lesser degree, whereas TNF- had no effect on PKR expression. None of the treatments affected the amount of intracellular actin.

View larger version (52K): [in this window] [in a new window]   Figure 4. Effects of dsRNA on PKR. (A) Monolayers of BE cells were incubated (37°C, 5% CO2, 16–23 h) with poly IC (100 µg/ml), IFN- (10 U/ml), TNF- (100 U/ml), or medium alone, and cell extracts were analyzed by Western blot for PKR and actin proteins. The data shown are representative of three experiments. (B) BE cells were incubated in either medium alone (0) or dsRNA for 2–120 min, and cell extracts were prepared. For a positive control (+), dsRNA was added to one sample after the cell extract was prepared. Cell extracts were then analyzed for PKR kinase activity (autophosphorylation), as indicated by incorporation of radiolabeled ATP. The data shown are representative of three experiments.

Activation of NF-B by dsRNA
Activated PKR can phosphorylate IB and thereby activate NF-B (18), which is an important positive regulator for the transcription of many proinflammatory genes. To determine whether dsRNA activates NF-B in BE cells, cell monolayers were transfected with poly IC (100 ng/ml), or incubated with TNF- (positive control), and 0–24 h later, whole cell extracts were analyzed for NF-B–binding activity. Both dsRNA and TNF- caused an increase in NF-B activation compared with cells incubated in medium alone (Figure 5A) . Binding to the labeled oligonucleotide was competed away by incubation of the cell extract with an excess of unlabeled oligonucleotide containing an NF-B–binding sequence, but not by an irrelevant oligonucleotide (activator protein [AP]-1), confirming the specificity of NF-B binding (Figure 5B).

View larger version (46K): [in this window] [in a new window]   Figure 5. Effect of dsRNA on NF-B activation. (A) BE monolayers were transfected with RV16 dsRNA (100 ng/ml), or incubated with either TNF- (100 U/ml) or medium alone, and cell extracts were prepared at the indicated times. NF-B activation was analyzed by EMSA. (B) To demonstrate specificity of the protein binding, additional samples were transfected with dsRNA for 6 h or TNF- for 1 h. Cell extracts were prepared, and were then incubated with an excess of oligonucleotide with a binding site specific for either NF-B or AP-1. The data shown are representative of three experiments.

View larger version (35K): [in this window] [in a new window]   Figure 6. Effect of poly IC on activation of p38 MAP kinase. BE cells were incubated with poly IC (100 µg/ml) or TNF- (100 U/ml), or medium alone, and cell extracts were prepared at the specified time points. Phosphorylated p38 MAP kinase, total p38 MAP kinase, and actin were quantitated by Western blot. The blot is representative of a total of three experiments.

	Discussion

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dsRNA has been shown to interact with at least two intracellular enzyme systems, PKR and 2'5' oligoadenylate synthase, although there is evidence in murine cells lacking PKR and RNase L that dsRNA can also activate a third as yet unidentified protein that causes downstream activation of MAP kinases (19). Effects of dsRNA on PKR have been best characterized; binding of dsRNA to PKR can cause dimerization, autophosphorylation, and PKR activation (11). Effects of PKR activation are diverse and include inhibition of protein synthesis, which may be an important means to limit viral replication, and downstream activation of NF-B and p38 MAP kinase (18), cytokines (IL-6, IFN-ß), and nitric oxide (17, 19, 20). Although the effects of dsRNA and PKR on cell activation can vary with the type of cell, our experiments demonstrate that dsRNA also activates NF-B and p38 MAP kinase in BE cells. These results are in agreement with those of Erzurum and colleagues, who also found that dsRNA could activate PKR and NF-B in primary cultures of airway epithelial cells (15). Because NF-B activation is a positive regulator of IL-8 and RANTES promoter activity (21, 22), it is likely that dsRNA effects on chemokine synthesis are at least partially due to increased gene transcription, and we are now in the process of conducting additional experiments to test this hypothesis.

The experiments using pharmacologic inhibitors suggest that dsRNA activation of PKR is necessary for synthesis and secretion of both RANTES and IL-8. The observation that the p38 MAP kinase inhibitor SB203580 blocks secretion of IL-8 but not RANTES provides evidence that there are different signaling pathways involved in the upregulation of these two chemokines by dsRNA. One possible explanation for this effect is that AP-1, which can be induced by p38 MAP kinase (23), is an important positive regulator of IL-8, but not RANTES, promoter activity. Interpretation of these results must be tempered by the inherent limitations of using pharmacologic inhibitors, although SB203580 and 2-aminopurine are considered to be specific inhibitors at the concentrations used in these experiments (24, 24).

Either transfection or incubation with dsRNA induced secretion of RANTES and IL-8 in BE cells, although some differences were noted. Although incubation with dsRNA induced greater chemokine production compared with RNA transfection, direct comparisons cannot be made due to the marked differences between these two techniques. For example, nontransformed BE cells have a relatively low transfection efficiency (2–5% in control experiments using a constitutively-expressed ß-galactosidase reporter plasmid, data not shown), and the amount of dsRNA that is introduced into the cells is unknown. However, there was a difference in the pattern of chemokine secretion for dsRNA incubation versus transfection, as incubation of BE cells with dsRNA was a more potent inducer of IL-8 secretion. Although the reason for this is speculative at the present time, it has recently been shown that dsRNA can activate cells via toll-like receptor-3 (16), and as a consequence, it is possible that dsRNA incubation and transfection activate different signaling pathways. This would imply that there are separate but overlapping innate immune mechanisms to recognize intracellular and extracellular dsRNA.

Although dsRNA is a potent stimulus for the production of IL-8 and RANTES, it is likely that there are other mechanisms that contribute to the upregulation of chemokine synthesis during viral infections. For example, although monoclonal antibodies which block the binding of major group RV to intercellular adhesion molecule-1 effectively inhibit RANTES production, virus-induced secretion of IL-8 is reduced, but not eliminated; ultraviolet irradiation of virus, which inhibits RNA replication, has similar effects (7–9). Oxidative stress also appears to contribute to virus-induced chemokine synthesis (25), and this may be independent of binding to surface receptors (26). Whether there is a relationship between dsRNA and oxidative stress has not been explored. Finally, viral infections enhance the production of MIP-1 (27), and because our data indicate that dsRNA does not induce this chemokine in vitro, it is likely that other mechanisms are responsible for this effect.

In summary, these findings indicate that dsRNA is a potent and specific stimulus for the activation of RANTES and IL-8 synthesis and secretion, and this may be a general mechanism through which the replication of ssRNA viruses (e.g., RV, influenza virus, respiratory syncytial virus) induce the production of these chemokines. Through the effects of RANTES, a potent chemoattractant for activated T cells and eosinophils, and IL-8, a principal chemoattractant for neutrophils, dsRNA could potentially have dual effects during respiratory infections. T cells and granulocytes recruited to the airways are likely to contribute to antiviral activity. On the other hand, increased cellular inflammation could also add to airway obstruction and dysfunction, leading to symptoms in the upper and lower airway. Further definition of the regulation and function of chemokine secretion during viral infections, as well as the development of specific chemokine inhibitors, will help to clarify the roles of these mediators in the pathogenesis of virus-induced respiratory symptoms and exacerbations of asthma.

	Acknowledgments

 
This research was funded by NIH grants R01 HL60993 and R01 HL61879.

Received in original form May 2, 2002

Received in final form November 22, 2002

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