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Originally published online as doi:10.1189/jlb.0806507 on December 8, 2006

Published online before print December 8, 2006
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(Journal of Leukocyte Biology. 2007;81:625-631.)
© 2007 by Society for Leukocyte Biology

Interaction of monocytic cells with respiratory syncytial virus results in activation of NF-{kappa}B and PKC-{alpha}/ß leading to up-regulation of IL-15 gene expression

Jamila Ennaciri, Rasheed Ahmad and José Menezes1

Laboratory of Immunovirology, Sainte-Justine Hospital Research Center, and Department of Microbiology and Immunology, Faculty of Medicine, University of Montreal, Montreal, Quebec, Canada

1 Correspondence: Laboratory of Immunovirology, Sainte-Justine Hospital, 3175 Cote Sainte-Catherine Road, Montreal, QC, Canada H3T 1C5. E-mail: jmenezes{at}justine.umontreal.ca

ABSTRACT

Respiratory syncytial virus (RSV) is a major human respiratory pathogen, particularly for infants. RSV is also a powerful inducer of cytokines, one of which is IL-15, an important immunoregulatory cytokine. IL-15 plays a key role in NK and T cell development and differentiation and also regulates NK cell/macrophage interaction, as well as monocyte/macrophage and granulocyte function. We have shown previously that different viruses up-regulate IL-15 gene expression in human PBMCs. Recently, we found that RSV induces the expression of IL-15 mRNA in the monocytic line THP-1. The signaling pathway involved in such virus-induced up-regulation of IL-15 has not yet been identified. We report here a study describing this mechanism. Because of the involvement of the protein kinase C (PKC) and the transcription factor NF-{kappa}B in the regulation of others cytokines by RSV as well as the involvement of NF-{kappa}B in the transactivation of IL-15, our hypothesis was that RSV induced the expression of IL-15 in THP-1 cells through the PKC and NF-{kappa}B activation. We demonstrate here that RSV-induced up-regulation of IL-15 expression in THP-1 cells involves the phosphorylation of PKC-{alpha}/ß. Further, inhibition of PKC by different specific inhibitors blocks this up-regulation. Using the electromobility shift assay, we show that the activated form of NF-{kappa}B binds to the IL-15 promoter sequence. We further confirm, using an ELISA assay, the involvement of p65 in the transcription of IL-15. This study, demonstrating the ability of RSV to induce IL-15 expression, might explain, at least in part, the exacerbated, inflammatory response triggered by RSV infection.

Key Words: cytokine • monocyte • signaling • infection • phosphorylation

INTRODUCTION

Respiratory syncytial virus (RSV) belongs to the family of Paramyxoviridae and the genus of Pneumovirus [1 ]. RSV is an enveloped, medium-size virus (120–300 nm), consisting of a single-negative strand, nonsegmented RNA [1 ]. RSV is the major cause of serious lower respiratory tract disease in children. It is estimated that 70% of infants hospitalized with bronchiolitis and 25% of infants with pneumonia are infected with RSV [2 ]. The severity of respiratory symptoms in those infants correlates with the presence of high concentrations of inflammatory cytokines in nasopharyngeal secretions [3 ]. RSV infection targets the respiratory epithelium and elicits a local inflammatory response involving cellular infiltration by monocytes and neutrophils and the release of inflammatory mediators from epithelial and activated immune cells. Such mediators include IL-1, TNF-{alpha}, IL-6, IL-8 [4 ], IL-11, MIP-1-{alpha}, RANTES [5 , 6 ], and IL-15 [7 ].

IL-15 plays a key role in innate immunity through multifunctional aspects including NK cell development, proliferation, cytotoxicity, and regulation of monocytes/macrophages and neutrophils [8 ]. IL-15 also acts as a link between the innate and the adaptive immune system. It shares many biological properties with IL-2, including its ability to stimulate CD4+, CD8+, and {gamma}{delta} T cell proliferation, as well as B cell proliferation and differentiation [9 10 11 ]. IL-15 mRNA is expressed constitutively by a wide variety of cells and tissues such as skeletal muscle, placenta, kidney, lung, heart, epithelial, and fibroblast cells lines, synovial cells, and monocytes/macrophages [10 11 12 ]. In monocytes/macrophages, IL-15 mRNA expression is up-regulated by exogenous stimuli such as IFN-{gamma} and LPS or by bacteria, protozoa, and virus infection [13 14 15 16 17 ]. However, IL-15 protein expression is regulated highly and tightly, not only at the transcriptional level but also at the translational and intracellular trafficking levels [8 ]. It is interesting that IL-15 protein was found to be produced only by a limited number of cells, including activated monocytes/macrophages, epithelial cells [10 11 12 ], and in some conditions of chronic inflammation, such as rheumatoid arthritis (RA), in the synovial fluids and synovial membranes of patients with active RA [18 ], by alveolar macrophages in sarcoidosis [19 ] and by PBMCs in inflammatory bowel disease [8 , 20 ]. Conversely, there are many reports supporting the role of IL-15 in host defense against various microorganisms such as HIV [21 , 22 ], HCV [23 ], and human herpes virus 7 [24 ]. Moreover, previous studies from our laboratory have demonstrated that different viruses, including influenza, HSV-1, EBV, vesicular stomatitis virus, reovirus, Sendai, and RSV, up-regulate IL-15 gene expression in human PBMCs [7 ]. Our recent results demonstrated that RSV also induces the expression of IL-15 mRNA in the monocytic THP-1 cells. The signaling pathway involved in RSV-induced up-regulation of IL-15 has not been identified yet. Here, we report a study using RSV-treated THP-1 cells describing this mechanism. Because of the involvement of the protein kinase C (PKC) pathway [25 26 27 ] and the transcription factor NF-{kappa}B in the regulation of some other cytokines by RSV [28 29 30 31 32 ] and the involvement of NF-{kappa}B in the transactivation of IL-15 [33 ], our hypothesis was that RSV induced IL-15 expression in THP-1 cells through the PKC and NF-{kappa}B activation. PKC is a large superfamily of serine/threonine (S/T) kinases, which mediate essential cellular signals required for activation, proliferation, differentiation, and survival. There are at least 10 PKC isotypes, which are subdivided into three classes, based on primary structure and biochemical properties [34 ]. These include classical PKC isotypes (cPKC), novel PKC isotypes, and atypical PKC isotypes. The cPKC isotypes include PKC-{alpha}, -ßI, -ßII, and -{gamma}. NF-{kappa}B defines a family of dimeric transcription factors composed of combinations of members of the Rel/NF-{kappa}B family such as p50, p52, p65 (RelA), c-Rel, and RelB [35 , 36 ]. The predominant species is the p50–p65 complex, which is retained in the cytoplasm by its inhibitor I{kappa}B-{alpha}. Particular cell stimuli, such as mitogens, cytokines, and viruses, cause proteolytic degradation of the I{kappa}B-{alpha} subunits in the cytoplasm leading to subsequent translocation of NF-{kappa}B proteins into the nucleus and activation of various genes [37 , 38 ]. In this study, we found that RSV up-regulates the IL-15 mRNA expression in the monocytic THP-1 cells, which is detectable as early as 4 h postinfection (p.i.). We demonstrate that this IL-15 gene induction does not require RSV replication and that it involves the phosphorylation of cPKC-{alpha}/ß and the activation of NF-{kappa}B p65.

MATERIALS AND METHODS

Reagents
Bisindolylmaleimide, PKC inhibitor peptide 19-31, Ro-31-8220, and staurosporine (stauro) were purchased from Calbiochem (La Jolla, CA, USA). FBS was from Gibco (Grand Island, NY, USA). The FITC-conjugated anti-IL-15 mouse mAb was purchased from R&D Systems Technology (Minneapolis, MN, USA). FITC-conjugated mouse IgG1 antibody was purchased from BD Biosciences (Mississauga, ON, Canada). Rabbit antiphospho-PKC-{alpha}/ß, phospho-PKC-µ, and phospho-PKC-{zeta} antibodies and goat antirabbit IgG HRP-linked antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). ß-Actin antibody was purchased from Sigma Chemical Co. (St. Louis, MO, USA). The ECL kit was purchased from Amersham Biosciences (Baie d’Urfé, QC, Canada), the protease inhibitor cocktail of mammalian cells was from Sigma Chemical Co. (Oakville, ON, Canada), the Cytofix/Cytoperm kit was from BD Biosciences, the PKC kinase activity assay kit (nonradioactive) was from Stressgen Bioreagents Corp. (Victoria, BC, Canada), and the ELISA kit transAm NF-{kappa}B family was from Active Motif (Carlsbad, CA, USA). The gel shift assay system was purchased from Promega (Madison, WI, USA).

Viral preparation and treatment
The human long strain of RSV (A) was produced in human lung epithelial cells A549. Briefly, A549 cells at 75% confluency were infected with RSV (supernatant from A549-infected cells) and incubated at 37°C in DMEM (Gibco) supplemented with FBS 2% until the cytopathic effect (CPE) in infected cell monolayer reached 70%. Cells were then harvested and centrifuged. Cell-free culture supernatants were prepared, and the pellet was frozen and thawed three times and centrifuged for 10 min at 2000 rpm, and the resulting supernatant was added to the first one. All the supernatants were filtered through a 0.45-µm pore-size filter. Viral particles were harvested by differential centrifugation for 1 h 30 min at 18,000 rpm. Virus stocks were resuspended in RPMI, aliquoted, and stored at –80°C. Viral titer was measured by CPE, and the titer, expressed as the 50% tissue culture infectious dose (TCID50), was determined by evaluating the quantity of virus that will produce obvious CPE in 50% of the cell culture plates infected. The virus stock had a titer of 107 TCID50/ml. The mock control was prepared from RSV-nontreated A549 culture supernatant. Virus and mock control stocks were aliquoted and stored at –80°C. Heat-inactivated virus was generated by incubating the purified virus in a 56°C water bath during 30 min. To obtain replication-deficient virus (UV-inactivated), RSV was exposed at 4°C in a 100-mm2 Petri dish to a 254-nm UV lamp at a distance of 23 cm for 2 h and gentle agitation of the dish every 20 min. All virus treatment tests were performed using RSV at a multiplicity of infection (MOI) of 1 TCID50/cell.

Cell culture and treatments
For all the experiments in this study, the human monocytic cell line THP-1 (2x106) was cultured in RPMI-1640 medium (Gibco) supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg amphotericin B, and 2 mM L-glutamine at 37°C and 5% CO2 in a 5-ml polystyrene tube (Falcon 352058, Becton Dickinson Labware, Franklin Lakes, NJ, USA). The manufacturers certified the media and all reagents as endotoxin-free. PKC inhibitors (bisindolylmaleimide 5 µM; PKC inhibitor peptide 19-31, 0.75 µM; Ro-31-8220, 10 µM; and stauro, 1 µM) were added 2 h prior to the addition of the virus. THP-1 cells were then incubated in 1 ml RPMI with RSV for different times p.i., and mixing was at every 15 min for the first 2 h of treatment.

Determination of IL-15 mRNA expression by RT-PCR
To determine whether RSV infection and different treatments of THP-1 had any effect on the level of IL-15 gene transcripts, we used a semiquantitative RT-PCR assay. Cells were mock-treated and RSV-infected, and PKC inhibitors were treated or not or treated with UV-inactivated and heat-inactivated RSV. Total RNA was extracted from cells at different times p.i. using the RNeasy mini kit (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions. RT-PCR was performed using the One Step RT-PCR kit (Qiagen) with 0.5 µg RNA per reaction and gene-specific primer pairs. The following thermal cycler conditions were used: RT at 50°C for 30 min; RT inactivation step at 95°C for 15 min; 35 cycles of denaturation at 94°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 1 min; and final extension at 72°C for 10 min. The gene-specific primer pairs used were IL-15 (fwd): 5'-GGATTTACCGTGGCTTTGAGTAATGAG-3' and IL-15 (rev): 5'-GAATCAATTGCAATCAAGAAGTG-3' for IL-15, which produces two bands at 550 bp and 640 bp [39 ], and 18S RNA (fwd): 5'-TGCATGTCTAAGTACGCACGGCC-3' and 18S RNA (rev): 5'-GATAGGGCAGACGTTCGAATGGG-3' for the 18S ribosomal subunit, used as control, which produces a 310-bp band. PCR products were run on a 2% agarose gel, which was soaked in Tris-acetate-EDTA buffer containing 0.5 µg ethidium bromide for 15 min, followed by soaking in water for 10 min, and then visualized under UV light using the Alpha imager system (Alpha Innotech, San Leandro, CA, USA). Relative gene expression was determined by band density comparison using AlphaEase software (Alpha Innotech).

PKC kinase activity assay
For testing of PKC involvement, a sandwich ELISA was used to measure PKC kinase activity. Cells were RSV-treated at different time intervals (30 min, 1 h, 1 h 30 min, and 2 h), and then the cell lysates were prepared according to the manufacturer’s guidelines. Cytoplasmic fractions were analyzed by ELISA. Briefly, 4 µg proteins were added to each well of a 96 plate precoated with substrate peptide for PKC. The reaction was initiated by adding 10 µl ATP, and then the plate was incubated for 90 min at 30°C. The reaction was stopped by emptying contents of each well and then adding 0.04 µg rabbit polyclonal antibody specific for phosphorylated PKC substrate; the plate was then incubated at room temperature for 60 min. After washing, 0.02 µg HRP-labeled secondary antibody was added at room temperature for 30 min. Tetramethylbenzidine (TMB) substrate was then added, and the plate was read at 450 nm.

Western blot analysis
After RSV infection, THP-1 cells were lysed with radioimmunoprecipitation assay lysis buffer (Nonidet P-40 20%; Na deoxycholate 10%; SDS 20%; 1 M Tris-HCl, pH 7.5; 0.5 M EDTA; 0.5 M NaF; one pill mammalian cells protease inhibitor cocktail), and the protein concentration was measured using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). The proteins were denatured and reduced by heating the samples at 95°C for 5 min. Equal amount of protein concentration was resolved on polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Millipore, Nepean, ON, Canada) by electroblotting. After the transfer, the membrane was blocked for 3 h in PBS with 0.1% Tween-20 (PBS-T) with 5% milk. Membranes were then incubated overnight at 4°C with primary rabbit antiphospho-PKC-{alpha}/ß II (Thr638/641), antiphospho-PKC-µ (Ser744/748), or antiphospho-PKC-{zeta} (Thr410/403) antibody diluted in PBS-T with 3% BSA or with the monoclonal anti-ß-actin diluted in PBS-T with 5% milk. After washing, the membranes were incubated for 1 h at room temperature with HRP-linked secondary antibody. Immunoreactive bands were visualized using the ECL Western blot detection kit system.

Determination of the involvement of NF-{kappa}B in IL-15 expression by EMSA
Cells were treated or not (control) with PKC inhibitors for 2 h and then treated or not with RSV. After 1 h incubation, cells were lysed, and nuclear extracts were prepared. The double-stranded oligonucleotide containing the NF-{kappa}B consensus sequence (5'-AGT TGA GGG GAC TTT CCC AGG C-3' and 3'-TCA ACT CCC CTG AAA GGG TCC G-5') or the AP-1 consensus sequence (5'-CGC TTG ATG AGT CAG CCG GAA-3' and 3'-GCG AAC TAC TCA GTC GGC CTT -5') was end-labeled with [{gamma}-P32] ATP by T4 polynucleotide kinase. The reaction consisted of 2 µl nuclear cell extracts; 7 µg was incubated with 20,000 cpm-labeled probe for 20 min at room temperature in binding buffer containing poly (dI·dC). The DNA–protein complexes were resolved on a 4% nondenaturing polyacrylamide gel in 0.5x Tris-boric acid-EDTA buffer at room temperature and were visualized by autoradiography of the dried gels. For competition assays, unlabeled competitor and noncompetitor oligonucleotides were added simultaneously with the probe (and under the same conditions as the positive and negative control) for 20 min before the addition of P32-labeled oligonucleotide.

TransAM NF-{kappa}B Family
THP-1 cells were first treated or not with PKC inhibitors: bisindolylmaleimide I, Ro-31-8220, and PKC inhibitor peptide 19-31 for 2 h and then infected with RSV at 1 MOI or mock-treated for 6 h. Cells were then harvested, and nuclear extracts were prepared following the manufacturer’s instructions. The nuclear proteins were prepared for the ELISA assay. Briefly, 20 µg nuclear proteins were added to each well of a 96-well plate precoated with immobilized oligonucleotide containing the NF-{kappa}B consensus site (5-GGGACTTTCC-3), and the reaction was incubated for 1 h at room temperature. Then, the primary antibodies used to detect NF-{kappa}B recognize an epitope on p50 or p65, which is accessible only when NF-{kappa}B is activated and bound to its target DNA. The plate was incubated for 1 h at room temperature, and the secondary HRP-conjugated antibody was then added to each well, and the plate was incubated again for 1 h. TMB substrate was added, and the absorbance was read on a spectrophotometer within 5 min at 450 nm with a reference wavelength of 655 nm.

Statistical analysis
Statistical analysis was performed using Prism (GraphPad Software, San Diego, CA, USA). The difference between cells treated with or without PKC inhibitors and RSV or mock-treated was evaluated using one-way ANOVA test. P values <0.0001 were statistically significant.

RESULTS

Kinetic of IL-15 mRNA induction by RSV in THP-1 cells
THP-1 cells constitutively express low levels of IL-15, and upon stimulation with RSV, there was a remarkable up-regulation of IL-15. To determine the kinetic of IL-15 mRNA expression, THP-1 cells were infected at different time-points (i.e., 2, 4, 7, 24 h). THP-1 cells were then lysed, and the RNA was extracted as described in Materials and Methods. The mRNA levels were assessed by RT-PCR; indeed, IL-15 mRNA levels increased with incubation time and reached a maximum at 24 h p.i. (Fig. 1a and 1b ). A preliminary experiment had indicated that there was no further increase after 24 h p.i. (data not shown).


Figure 1
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  		Figure 1. Time course of RSV-induced IL-15 mRNA in THP-1 cells, which were exposed to RSV for different time periods (2, 4, 7, and 24 h). Cells were then lysed, and total RNA was extracted, measured, reverse-transcribed, and amplified by PCR. (a) RT-PCR of IL-15 mRNA (upper panel) and 18S RNA (lower panel) levels in RSV-treated cells for the indicated times between 2 h and 24 h is shown. Compared with its expression in mock-treated cells, IL-15 mRNA increases gradually, peaking at 24 h in RSV-treated THP-1. (b) Histograms showing the increase (fold) of IL-15 mRNA levels in THP-1 cells infected with RSV. Data shown are means from three separate experiments (n=3, P<0.0001) and are statistically significant.

 
IL-15 induction does not require RSV replication
To determine whether the IL-15 gene induction by RSV depends on virus infectivity or simply on virus-cell surface interaction, THP-1 cells were treated for 7 h with infectious virus at a MOI of 1, in parallel with heat-inactivated as well as UV-inactivated RSV. Mock-treated cells were used as a control. IL-15 mRNA levels were found increased significantly in cells treated with viral preparations, regardless of their infectivity, as compared with the mock-treated control cells (Fig. 2 ).


Figure 2
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  		Figure 2. UV- and heat-inactivated RSV-induced IL-15 mRNA. Induction of IL-15 mRNA by inactivated RSV treatment. RT-PCR of IL-15 mRNA (upper panel) and 18S RNA (lower panel) levels in UV- and heat-inactivated RSV is shown. Infectious and inactivated [UV and heat (HI)] RSV increased IL-15 mRNA expression in THP-1 cells compared with mock-treated cells. This is representative of three independent experiments.

 
Inhibition of PKC reduces the expression of IL-15 induced by RSV
PKC is a large superfamily of S/T kinases, which play an important role in signal transduction. To examine the role of S/T kinases in the RSV-induced up-regulation of IL-15, THP-1 cells were preincubated with stauro (a broad-spectrum inhibitor of S/T kinases) for 2 h and then stimulated with RSV for 7 h. Stauro greatly reduced RSV induction of IL-15 expression. As stauro has the ability to block a number of S/T kinases, and different studies support the role of PKC involvement in the induction of some cytokines by RSV in epithelial cell lines, we performed experiments to determine the effect of two specific inhibitors for PKC. THP-1 cells were preincubated with two different inhibitors, i.e., Ro-31-8220 and PKC inhibitor peptide 19-31, for 2 h and then stimulated with RSV for 7 h. IL-15 mRNA levels were then compared with those of cells stimulated with RSV only (control). The presence of PKC inhibitor peptide 19-31 and RO-31-8220 decreased the expression of the IL-15 gene significantly (Fig. 3 ). This result suggests that PKC plays an important role in IL-15 signaling by RSV in THP-1 cells.


Figure 3
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  		Figure 3. Inhibition of S/T kinases reduces RSV induction of IL-15 expression. RT-PCR of IL-15 mRNA (upper panel) and 18S RNA (lower panel) levels in cells treated or not by PKC inhibitors for 2 h and then treated with RSV is shown. IL-15 mRNA decreased significantly in cells treated by PKC inhibitors compared with cells treated simply by RSV or mock. Data are representative of three independent experiments.

 
RSV induced PKC-{alpha}/ß phosphorylation in THP-1 cells
The results from the above experiments using PKC inhibitors clearly indicated that the activation of PKC is required for IL-15 gene expression. We then tried to examine by PKC kinase activity assay the phosphorylation and the activation of PKC. We found that RSV induces the phosphorylation of PKC from 30 min p.i. to 2 h (Fig. 4a ). This result is consistent with studies published previously, demonstrating the activation of PKC isotype {zeta} within 10 min of infection in epithelial A549 cells [26 ]. Furthermore, we assessed by Western blot the involvement and the phosphorylation of the following isoforms of PKC: PKC-{alpha}/ß, PKC-µ, and PKC-{zeta}. These isozymes were selected based on their potential role in RSV infection reported previously [26 ]. In this context, THP-1 cells were incubated with RSV at a MOI of 1 for different time periods (30 min, 1 h, 2 h) p.i., and then the cells were harvested and the samples processed for the detection of phosphorylated PKC-{alpha}/ß, PKC-µ, or PKC-{zeta}. The results showed that RSV induced the phosphorylation of PKC-{alpha}/ß in the first hour of infection, which was then followed by a decrease in phosphorylation by 2 h p.i. (Fig. 4b) . No significant activation was observed for the PKC-µ and PKC-{zeta} by RSV treatment comparatively with the control cells (data not shown). Taken together, these results also suggest that RSV-mediated activation of PKC-{alpha}/ß occurs in the early stages of RSV infection and does not involve PKC-µ or PKC-{zeta}.


Figure 4
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  		Figure 4. RSV induces the phosphorylation of PKC. (a) Results of the PKC kinase activity measured in cell cytoplasmic extracts using ELISA assay between 30 min and 2 h post-RSV treatment of THP-1 cells. Data shown are means from duplicate experiments. (b) Phosphorylation of PKC-{alpha}/ß post-RSV treatment. Western blot of phosphorylated PKC-{alpha}/ß in mock- and RSV-treated cells for the indicated times between 30 min and 2 h is shown. This is representative of three independent experiments.

 
Effect of PKC inhibitors on NF-{kappa}B activation in RSV-induced IL-15 up-regulation
The NF-{kappa}B transcription factor plays a central role in innate immunity through its transactivation of several cytokines. As the IL-15 promoter is regulated in part by a NF-{kappa}B and AP-1 responsive sequence, the role of PKC was tested in NF-{kappa}B and AP-1 activation. THP-1 cells were preincubated or not with PKC inhibitors: bisindolylmaleimide, PKC inhibitor 19-31, and Ro-31-8220 for 2 h and then were mock- or RSV-treated for 1 h. Nuclear cell extracts were harvested for EMSA to detect NF-{kappa}B and AP-1 binding. We found that PKC inhibitors decreased the RSV-induced DNA binding of NF-{kappa}B. In contrast, in cells not treated with PKC inhibitors, RSV significantly increased the binding of NF-{kappa}B to the IL-15 promoter compared with the mock-treated THP-1 cells (Fig. 5 ). No significant activation was observed for the AP-1 transcription factor by RSV treatment comparatively with the control cells (data not shown). Sequence specificity of the DNA binding is shown by competition with unlabeled oligonucleotides in the EMSA. This result indicates that RSV up-regulates the IL-15 gene through NF-{kappa}B and that this mechanism depends on PKC signaling.


Figure 5
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  		Figure 5. Effect of RSV treatment on DNA binding activity of NF-{kappa}B. Gel mobility shift DNA binding assay: nuclear extracts from mock- or RSV-treated cells, with or without PKC inhibitors [bisindolylmaleimide (Bisind), PKC inhibitor 19-31, and Ro-31-8220], incubated in DNA-binding buffer and 32P-labeled NF-{kappa}B duplex oligonucleotide. After incubation, the complexes were fractionated by 6% nondenaturing PAGE. The results show long, autoradiographic exposure, demonstrating the shifted bands from nonspecific and specific competitors.

 
RSV induces IL-15 gene expression via the p65 member of the NF-{kappa}B family in THP-1 cells
Following the latter observation, we decided to determine which member of the NF-{kappa}B family is involved in this RSV-induced IL-15 expression. For this, THP-1 cells were preincubated or not with PKC inhibitors for 2 h and then treated with the mock or RSV at 1 MOI for 6 h. The nuclear proteins were prepared for the ELISA assay as described in Materials and Methods. We found that RSV activated the p65 subunit over 6 h and that this activation was decreased significantly in the presence of PKC inhibitors (Fig. 6 ). This indicates clearly that RSV-induced IL-15 expression is dependent on activation of PKC- and NF-{kappa}B p65. No significant activation was observed for the p50 by RSV treatment comparatively with the control cells (data not shown).


Figure 6
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  		Figure 6. Effect of RSV and treatments with PKC inhibitors on the NF-{kappa}B unit p65 activation. ELISA assay measuring p65 unit activation in nuclear extracts from mock and RSV-treated cells. RSV activates the p65 member, which reaches a maximum at 6 h post-treatment. PKC inhibitors reduced p65 member activation in RSV-treated cells. Data shown are means from duplicate experiments.

 
DISCUSSION

RSV is a major cause of lower respiratory tract infection in young infants. RSV has two major immunogenic determinants: the fusion (F) and the attachment (G) glycoproteins, both of which are associated with the envelope of the virus [1 ]. These two glycosylated surface proteins also play a key role in infectivity [40 ]. The G protein mediates attachment of the virus to target epithelial cells in the host, whereas the F protein facilitates fusion of the virus with the cellular lipid membrane, permitting insertion of the RNA into the host cell [41 ]. RSV infection targets the respiratory epithelium and elicits an inflammatory response characterized by infiltration of mononuclear cells, which can also be infected [42 , 43 ], and neutrophils [44 ]. IL-15 is reported to activate innate immunity in an autocrine pathway [45 , 46 ] and a paracrine pathway through the activation of NK and T cells [8 ]. IL-15 stimulates neutrophils and monocytes to secret inflammatory cytokines and to phagocytize pathogens. It is also associated with serious disorders such as inflammatory and autoimmune diseases [8 ] and has been demonstrated to be involved and expressed in many infectious diseases such as AIDS and adult T cell leukemia [16 ]. In this context, it is important to understand the regulation of IL-15 expression by RSV.

In this study, we examined this mechanism using cells of the monocytic line THP-1, and we show here that the IL-15 gene, up-regulated by RSV in these cells, is detectable as early as 4 h post-treatment. This up-regulation persists, however, for many hours. We also show that the IL-15 gene is up-regulated as a result of cell surface interaction with virus particles from infectious and inactivated preparations (i.e., UV- and heat-inactivated) comparatively with the mock-treated cells. These data indicate that RSV replication is not necessary for the induction of IL-15 in THP-1 cells. However, heat inactivation of RSV, which alters its envelope glycoproteins, induces slightly less IL-15 expression comparatively with the infectious viral particles. This result suggests that RSV envelope glycoproteins (G and F) may play a potential role in IL-15 mRNA up-regulation.

Treatment of THP-1 cells by the stauro, which is a broad-spectrum inhibitor of the S/T kinases, and by PKC inhibitors (PKC inhibitor 19-31 and Ro-31-8220) prior to infection with RSV significantly reduces the level of IL-15 mRNA comparatively with cells infected by RSV or mock-treated. This suggests that PKC activation plays a fundamental role in RSV up-regulation of IL-15 and even in its constitutive expression in the monocytic THP-1 cells. In this regard, we became interested in examining the signaling pathway involved in such virus-induced up-regulation of IL-15.

Furthermore, San-Juan-Vergara et al. [25 ] have demonstrated that PKC-{alpha} plays an important role in the early process of RSV infection in normal human bronchial epithelial cells, such that blocking of PKC-{alpha} alters RSV infection significantly. Thus, to examine the involvement of the PKC activity in IL-15 signaling, we performed a PKC ELISA assay to measure the phosphorylation of PKC in RSV-infected cells. We found that PKC is activated 1 h post-treatment, and this activation persists for 2 h. Furthermore, we found by Western blot, that RSV induced PKC-{alpha}/ß phosphorylation at 1 h of treatment, which is consistent with the viral attachment to the cell membrane and the first events of IL-15 signaling. However, Monick et al. [26 ] have found that in A549 cells, RSV caused two separate peaks of ERK activity: an early (10–30 min), consistent with viral binding and matched by activation of PKC-{zeta}, and a late (24–48 h), consistent with activities of Raf-1 and the PKC isoforms ß1, {delta}, {epsilon}, and µ [26 ]. In our study, IL-15 mRNA was up-regulated from 4 h and persisted for 24 h.

In addition, our data show that RSV initiates a signaling cascade that leads to the activation of different components, which ultimately up-regulate the transcription factor NF-{kappa}B, leading to the activation of the IL-15 gene; treatment of THP-1 cells with PKC inhibitors reduces this activation, which indicates that RSV-induced NF-{kappa}B activation is a PKC-dependent mechanism. NF-{kappa}B is an inducible transcriptional activator implicated in the regulation of many cytokines and immunomodulatory mediators following viral infection. RSV induces several cytokines and chemokines such as IL-1ß, IL-6, IL-8, IL-11, TNF-{alpha}, RANTES, and ICAM-1 via the NF-{kappa}B site [32 ].

Previous work by Garofalo et al. [47 ] showed that proteolysis of I{kappa}B subunits, mainly I{kappa}B{alpha}, is temporally linked to p65 DNA-binding activation in RSV-infected lung epithelial cells. We evaluated, by an ELISA assay, the activation of p65 in RSV-infected cells, treated or not by PKC inhibitors. We found that RSV activates p65 in RSV-infected cells and that activation is less enhanced in cells infected by RSV and treated by PKC inhibitors. p65 activation persisted up to 6 h of treatment, which is consisting with results from other laboratories [27 ].

In conclusion, our results demonstrate for the first time that RSV induces the expression of the proinflammatory cytokine IL-15 in the human monocytic THP-1 cells. Our data also show that this up-regulation of RSV-induced IL-15 expression occurs via activation of the PKC-{alpha}/ß pathway involving the transcription factor NF-{kappa}B p65 and that this activation persists for many hours. Furthermore, the ability of RSV to induce IL-15 expression might explain, at least in part, the exacerbated, inflammatory response triggered by RSV infection.

ACKNOWLEDGEMENTS

We thank Dr. Vincent Wellman for the kind gift of RSV and Dr. Rkia Dardari for the critical review of the manuscript.

Received August 8, 2006; revised October 12, 2006; accepted November 8, 2006.

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