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Am J Physiol Lung Cell Mol Physiol 287: L1048-L1055, 2004. First published July 23, 2004; doi:10.1152/ajplung.00396.2003
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Exaggerated IL-8 and IL-6 responses to TNF-{alpha} by parainfluenza virus type 4-infected NCI-H292 cells

Thierry Roger,1,2 Paul Bresser,1 Mieke Snoek,2 Koen van der Sluijs,1,2,3 Arjen van den Berg,1,2 Monique Nijhuis,4 Henk M. Jansen,1 and René Lutter1,2

1Department of Pulmonology, 2Laboratory of Experimental Immunology, and 3Department of Experimental Internal Medicine, Academic Medical Center, University of Amsterdam, 1100 DE Amsterdam; and 4Eijkman-Winkler Institute, Department of Virology, University Medical Center, 3508 GA Utrecht, The Netherlands

Submitted 19 November 2003 ; accepted in final form 20 July 2004


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Respiratory viruses induce and potentiate airway inflammation, which is related to the induction of proinflammatory mediators such as interleukin (IL)-8 and IL-6. Here we report on mechanisms implicated in IL-8 and IL-6 production by airway epithelium-like NCI-H292 cells exposed to parainfluenza virus type 4a (PIV-4). PIV-4 readily infected NCI-H292 cells as reflected by intracellular PIV-4 antigen expression. PIV-4 infection triggered a biphasic IL-8 and IL-6 mRNA response. Transient transfection with truncated and mutated promoter constructs identified NF-{kappa}B and activator protein (AP)-1, and CCAAT-enhancer binding protein (C/EBP) as the relevant transcription factors for PIV-4-induced IL-8 and IL-6 gene transcription, respectively. An increase of DNA-binding activities for NF-{kappa}B and C/EBP paralleled the induction of the first and second IL-8 and IL-6 mRNA peaks, whereas the onset of AP-1 paralleled the first IL-8 mRNA peak only. The second mRNA peak, apparently dependent on viral replication, coincided also with a marked reduction of IL-8 and IL-6 mRNA degradation. Importantly, cells at the time of the reduced mRNA degradation displayed an exaggerated IL-8 and IL-6 protein production to a secondary stimulus, as exemplified by steeper dose-response curves to TNF-{alpha}. Thus PIV-4 infection enhances epithelial IL-8 and IL-6 production by transcriptional and posttranscriptional mechanisms. The previously unrecognized phase of reduced IL-8 and IL-6 mRNA degradation and the concurrent amplified epithelial IL-8 and IL-6 responses may play an important role in virus-induced potentiation of airway inflammation.

inflammation; exacerbation; mRNA degradation; airway epithelium

RESPIRATORY VIRAL INFECTIONS usually cause mild to severe respiratory illness, which is paralleled by local recruitment and activation of inflammatory cells (8, 9, 15, 18, 20). This inflammatory response is orchestrated, at least in part, by proinflammatory mediators released by infected airway epithelial cells (3, 6). In addition to this response, respiratory viral infections also potentiate inflammatory responses to a secondary, unrelated proinflammatory stimulus. A clinically relevant example is the virus-induced increased inflammatory response to a bacterial challenge, i.e., bacterial superinfection (4, 7, 25). How a viral infection increases responsiveness to a bacterial challenge is not well understood. In part, this is explained by virus-induced damage to epithelial cells as a consequence of which bacterial adherence is promoted, leading to a more pronounced inflammatory response (17). Another, nonexclusive explanation is that bacteria and viruses show a synergism in the proinflammatory tumor necrosis factor-{alpha} (TNF-{alpha}), interleukin-12 (IL-12) and interferon (IFN)-{gamma} responses (1, 33). In line with this, we recently described how IFN-{gamma} exposure of airway epithelium(-like) cells enhances the proinflammatory IL-8 and IL-6 responses to secondary proinflammatory stimuli like TNF-{alpha} (35).

Levels of IL-8 and IL-6 are increased in airway secretions from individuals with respiratory viral infection (14, 18, 31). Furthermore, the kinetics and magnitude of IL-8 and IL-6 production correlate with respiratory symptoms in influenza infection (18, 31), thus underlining the prominent role of these mediators in virus-induced pathophysiology. Respiratory viruses infect and replicate in airway epithelial cells, which are also an important source of IL-8 and IL-6. Previous studies showed that epithelial IL-8 production is triggered by viral infection, the mechanisms of which have been studied in vitro and in detail for respiratory syncytial virus (RSV; 3, 10–13, 26, 27) and to a lesser extent for rhinovirus (21) and influenza virus A (6). There are no similar studies reported for parainfluenza virus, although these viruses are also considered important respiratory pathogens (2, 34). In the present study we determined the mechanisms for IL-8 and IL-6 induction by parainfluenza virus type 4a (PIV-4). For these studies we employed human lung-derived mucoepidermoid adenocarcinoma NCI-H292 cells, which previously were shown to facilitate replication of PIV-4 without causing marked cytopathic effects (5). Moreover, the regulation of IL-8 and IL-6 production in NCI-H292 cells has been studied in detail (24, 28, 29, 35).

PIV-4 induced a biphasic IL-8 and IL-6 mRNA response in NCI-H292 cells, resulting in the release of IL-8 and IL-6. The second phase in the mRNA response, which appeared dependent on viral replication, concurred with a reduced degradation of IL-8 and IL-6 mRNA. Previous studies indicated that a reduced IL-8 and IL-6 mRNA degradation can result in exaggerated IL-8 and IL-6 responses as reflected by steeper dose-response curves (24, 35). Interestingly, when PIV-4-infected cells during this phase of reduced mRNA degradation were exposed to TNF-{alpha}, cells displayed exaggerated IL-8 and IL-6 responses. This mechanism may contribute to the virus-induced potentiation of inflammatory responses to a secondary, unrelated proinflammatory stimulus.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
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Cell culture. NCI-H292 cells (CRL 1848; ATCC, Rockville, MD), a human lung-derived mucoepidermoid adenocarcinoma cell line, were maintained as described previously (29). Cell cultures were screened on a regular basis for mycoplasma infection using RT-PCR and found negative.

Viral stock. PIV-4 (strain M-25) was obtained from ATCC: VR-279 to prepare our own viral stocks or VR-1378 (PIV-4 passaged in NCI-H292 cells), which was used occasionally instead of our own stocks, giving similar results. To prepare viral stocks, 50–80% confluent NCI-H292 cells in 225-cm2 culture flasks were washed with phosphate-buffered saline (PBS, pH 7.4) and exposed for 5 min to PBS containing 1.5 µg/ml of trypsin. Subsequently medium was removed, and cells were exposed to 5 ml of PBS containing 40 µl of viral stock (VR-279). After 1 h at room temperature, serum-free medium containing 1.5 µg/ml of trypsin was added. After 48 h, medium was removed and cells were washed three times with warm PBS. Three milliliters of culture medium without trypsin and FCS were added, and cells were disrupted by three cycles of freezing and thawing. Cellular debris was removed by low-speed centrifugation in a table centrifuge (3,000 rpm, 10 min). Aliquots of clarified supernatant were stored at –80°C until use. As controls, clarified supernatant from noninfected NCI-H292 cells, as well as supernatant from a PIV-4 stock cleared of viral particles by centrifugation (90 min at 50,000 g), was shown not to induce IL-8 and IL-6 release. PIV-4 stocks were confirmed to induce aggregation of guinea pig erythrocytes.

Infection of NCI-H292 cells with PIV-4 induced a weak cytopathic effect (large multinucleated cells that became less adherent) but only after more than a week of infection. PIV-4 is a labile virus, and thus the potency of viral stocks to induce IL-8 and IL-6 was taken as an internal control to ensure uniformity between experiments.

Experimental setup. NCI-H292 cells were grown to 50–80% confluence. Culture medium was removed, and cells were washed twice with warm PBS. Cells were exposed for 5 min to PBS containing 1.5 µg/ml of trypsin. After removing the trypsin medium, we exposed cells for 1 h at room temperature to PIV-4 (0.1 ml of the viral stock/cm2) in PBS. Then the PIV-4-containing supernatant was replaced by culture medium with FCS, and experiments were carried out as indicated. Culture supernatants were stored at –20°C until IL-6 and/or IL-8 was determined. The cell layers were inspected by light microscopy for morphological aspects. None of the conditions resulted in shedding of adherent cells or morphological changes during the experiment. Each condition was tested in triplicate and experiments were carried out at least three times unless specified otherwise.

For some experiments, NCI-H292 cells were exposed to ultraviolet (UV) irradiated or to heat-killed viral stocks. For UV irradiation, virus on ice was exposed for 30 min at a 5-cm distance from a 254-nm UV source. Virus was heat killed by incubation for 30 min at 56°C.

Detection of PIV-4 antigen. NCI-H292 cells exposed to PIV-4 were scraped with a rubber policeman from the tissue-culture plate and transferred to a microscope slide. After air drying, cells were fixed in acetone at room temperature for 10 min. Aspecific binding sites were blocked with 1% (vol/vol) normal goat serum in PBS. After 10 min, cells were washed twice with PBS and incubated with 1:10 dilution of mouse anti-PIV-4 antibody (MAB877; Chemicon, Veenendaal, The Netherlands) for 30 min at 37°C. After two wash steps with PBS, cells were incubated for 30 min with goat anti-mouse IgG-FITC (Fab')2 diluted 1:50 with PBS. After two wash steps with PBS, cells were examined with a fluorescence microscope.

Assays for IL-8 and IL-6 protein and mRNA. IL-8 and IL-6 protein was quantified by ELISA, and levels of IL-8, IL-6, and GAPDH mRNA were assessed by Northern blots as described (28, 29). Intracellular IL-8 was determined after lysis of cells with 1% (vol/vol) Triton X-100 in PBS (pH 7.2). The lysis buffer did not affect recoveries of IL-8 as determined by ELISA. mRNA stability experiments were performed using the transcription inhibitor actinomycin D (Roche Molecular Biochemicals, Almere, The Netherlands) at a final concentration of 10 µg/ml.

Transfection. NCI-H292 cells at 70% confluence were transfected transiently with 3 µg of chloramphenicol acetyltransferase (CAT) reporter vectors using the calcium-phosphate precipitation method as reported before (28, 29). Forty hours after transfection, medium was removed, and cells were infected with PIV-4 or were sham infected. After 8 h, cells were washed twice with cold PBS, and cytoplasmic proteins were extracted using a hypotonic cell lysis buffer containing Triton X-100. Cell debris was removed by centrifugation. CAT protein was determined with an ELISA (Roche Molecular Biochemicals) and expressed relative to total protein assessed with the bicinchoninic acid protein assay (Pierce, Rockford, IL).

The series of 5'-deleted IL-6 promoter CAT constructs were obtained from Drs. K. Wong and W. M. Rom (Bellevue Hospital Center, New York, NY) (28). The IL-8 promoter CAT constructs were obtained from Drs. N. Mukaida and K. Matsushima (Cancer Research Institute, Kanazawa, Japan) (29).

Nuclear extract preparation and EMSA. Nuclear extracts were prepared as described (28, 29). Protein recovery was measured with the Bio-Rad Protein Assay kit (Hercules, CA). Four micrograms of nuclear extracts (in 10 µl) were incubated for 15 min at room temperature with 1 µl of radiolabeled oligonucleotidic probes (~0.25 ng, ~25,000 cpm) and 7 µl of a buffer composed of 20 mM HEPES pH 8.0, 50 mM KCl, 0.5 mM EDTA, 1 mM DTT, 0.1% (vol/vol) Nonidet P-40, 0.5 mM PMSF, 1 mg/ml bovine serum albumin, 5% (vol/vol) glycerol, and 2 µl of 1 mg/ml poly(dI-dC)-poly(dI-dC) (Pharmacia, Uppsala, Sweden). Reaction mixtures were separated on 4% nondenaturing polyacrylamide gels at 230 V at room temperature in 0.5x Tris-borate/EDTA electrophoresis buffer. After drying, gels were exposed to X-ray films. Oligonucleotides used in EMSA were the following: NF-{kappa}B, 5'-ATCGTGGAATTTCCTCTGAC-3'; activator protein (AP)-1, 5'-GTGTGATGACTCAGGTTTG-3'; distal CCAAT-enhancer binding protein (C/EBP), 5'-TCACATTGCACAATCTTA-3'; proximal C/EBP, 5'-AAGATTTAT CAAATGTG-3'. Bands were identified by supershift using 1 µg of antibodies against p65 for NF-{kappa}B, c-fos, and c-jun for AP-1 and C/EBP{beta} for C/EBP (Santa Cruz Biotechnology, Santa Cruz, CA).

Statistical analysis. Data were analyzed for statistical significance by Kruskal-Wallis nonparametric ANOVA or a paired t-test, when indicated, and for dose dependency by linear regression. P < 0.05 was considered significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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PIV-4 infects NCI-H292 cells and induces IL-8 and IL-6 production. To ensure that exposure to PIV-4 resulted in infection of NCI-H292 cells, we assessed the expression of PIV-4 antigen with time. At 24 h but not at 1 h after exposure to PIV-4 (1:10 dilution), PIV-4 antigen was detected by immunofluorescence in ~10–30% of NCI-H292 cells (Fig. 1).



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  		Fig. 1. Parainfluenza virus type 4a (PIV-4) antigen in infected NCI-H292 cells. NCI-H292 cells were exposed to PIV-4 (1:10 dilution of viral stock) for 1 (A) and 24 h (B) after which cells were treated as described in MATERIALS AND METHODS.

 
Subsequently, we assessed IL-8 and IL-6 release by NCI-H292 cells exposed to serial dilutions of PIV-4 stock for 24 h (Fig. 2, A and B). Exposure to PIV-4 induced the release of both IL-8 and IL-6 in a dose-dependent manner. Exposure to heat-inactivated PIV-4 (HI PIV) did not increase the release of IL-8 and IL-6 compared with cells not exposed to PIV-4. As NCI-H292 cells contain no intracellular stores of IL-8 (not shown) and IL-6 (24), these findings indicate that exposure to live PIV-4 triggers the epithelial production of IL-8 and IL-6. In subsequent experiments we exposed NCI-H292 cells to a 1:10 dilution of the PIV-4 stock, which resulted in a gradual increased release of IL-8 and IL-6 over 24 h (Fig. 2, C and D). As PIV-4 is a labile virus, we used IL-6 and IL-8 levels induced by PIV-4 exposure as an internal control for reproducible infection with PIV-4.



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  		Fig. 2. Production of IL-8 and IL-6 by NCI-H292 cells exposed to various doses of PIV-4 and with time. NCI-H292 cells were exposed to several dilutions of PIV-4 stock and heat-inactivated PIV-4 (HI, 1:10 dilution tested only) for 24 h (A, B) or exposed to PIV-4 (1:10 dilution) for various periods (C, D) after which levels of IL-8 (A, C) and IL-6 (B, D) in supernatants were determined by ELISA. Linear regression: r2 = 0.95 and 0.88 for IL-8 (A) and IL-6 (B), respectively. Values represent means ± SD of 4 experiments. *P < 0.05 compared with control medium.

 
Biphasic IL-8 and IL-6 mRNA expression upon infection with PIV 4. To assess whether PIV-4-induced IL-8 and IL-6 production was paralleled by increased IL-8 and IL-6 mRNA steady-state levels, we determined the respective mRNA levels in PIV-4-infected cells with time (Fig. 3, A and B). PIV-4 induced a fast but transient increase of IL-8 and IL-6 mRNA levels, peaking at 1 h. Four hours after initial exposure, IL-8 and IL-6 mRNA levels dropped to near basal levels. Subsequently, IL-8 and IL-6 mRNA levels increased transiently, peaking at ~8 h and returning to basal levels between 12 and 24 h after initial exposure. HI PIV before exposure to the cells almost fully abolished IL-8 and IL-6 mRNA induction, in agreement with no release of IL-8 and IL-6 protein by HI PIV (Fig. 2, A and B).



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  		Fig. 3. Time course of IL-8 and IL-6 mRNA expression in PIV-4-infected NCI-H292. NCI-H292 cells were exposed to PIV-4 or heat-inactivated (HI PIV, 1:10 dilution). Total RNA was isolated at indicated time points and analyzed by Northern blot for IL-6, IL-8, and GAPDH mRNA (A). Signals obtained after hybridization were quantified using a PhosphorImager. B: IL-8 (left) and IL-6 (right) mRNA levels normalized to GAPDH were expressed relative to that of resting cells [time (t) = 0]. Similar results were obtained in 3 experiments. C: as above, but NCI-H292 cells were exposed to PIV-4 and PIV-4 irradiated with UV for 30 min. Single points are shown for PIV-4 irradiated with UV for 5 and 15 min.

 
UV irradiation disables replication of single-stranded RNA viruses, although this is less efficient in viruses like PIV-4 with a small viral genome. Exposure of NCI-H292 cells to UV-irradiated PIV-4 showed a normal first peak of IL-8 mRNA but consistently reduced the second peak (Fig. 3C). The mean ± SE of the absolute differences between the two curves at time points 4, 6, 8, and 12 h was 3.95 ± 0.9 (P = 0.02), thereby refuting the null hypothesis that there is no difference between the curves. Similar to IL-8 mRNA, the second IL-6 mRNA peak was reduced by UV irradiation of PIV-4, with a significant (P = 0.02) absolute difference (0.29 ± 0.07, mean ± SE).

Together these findings indicate that IL-8 and IL-6 mRNA expression are regulated by two events. The first event is related to infection of NCI-H292 cells by PIV-4, which is lost by heat inactivation of the virus. The second event appears related to viral replication as UV irradiation of PIV-4 markedly reduced the second peak of IL-8 and IL-6 mRNA expression.

Relevant transcription factors for PIV-4-induced IL-8 and IL-6 expression. To identify the transcription factors mediating PIV-4-induced IL-8 and IL-6 gene transcription, NCI-H292 cells were transiently transfected with CAT reporter gene constructs containing various truncated and mutated portions of human IL-8 and IL-6 promoters. Transfected cells were exposed for 8 h either to medium or to PIV-4, after which CAT protein expression was determined in cell lysates. For the IL-8 promoter, the maximal response to PIV-4 was observed with the full-length construct –546-CAT and the deletion construct –133-CAT (Fig. 4A). Further deletion of the AP-1 site in –94({Delta}–70/–51)-CAT still resulted in CAT expression, although at a reduced level. CAT expression was abolished using –85-CAT and when the C/EBP site or the NF-{kappa}B site from –94({Delta}–70/–51)-CAT was mutated. This indicates that a minimal element containing the NF-{kappa}B and the C/EBP sites was required for IL-8 gene transcription. Disruption of the NF-{kappa}B site in –133-CAT revealed that an intact NF-{kappa}B site was essential for transcription. By contrast, mutation in the C/EBP site of –133-CAT had no effect, whereas disruption of the AP-1 site slightly reduced CAT expression by PIV-4. This suggests that AP-1 was also involved in IL-8 gene activation by PIV-4 but that the C/EBP site could replace the AP-1 site when it was missing.



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  		Fig. 4. Transcriptional activity of the IL-8 and IL-6 promoters in response to PIV-4 infection in NCI-H292 cells. NCI-H292 cells were transiently transfected with chloramphenicol acetyltransferase (CAT) expression vectors in which various truncated and mutated (mt) IL-8 (A) and IL-6 promoter (B) regions were cloned. A schematic presentation of the 5'-flanking region of the IL-8 and the IL-6 gene is shown at the top of A and B, respectively. CAT protein expression in transfected cells cultured for 8 h with medium or PIV-4 (1:10 dilution) was assessed by ELISA. CAT values were normalized to total protein. Results represent means ± SD of 3 experiments. AP, activator protein; CRE, cAMP response element; C/EBP, CCAAT-enhancer binding protein.

 
As shown in Fig. 4B for the IL-6 promoter, a maximal response to PIV-4 was obtained with –1,158-CAT and –224-CAT constructs but lost with the –109-CAT construct. This suggests that the AP-1 site (–282/–276) was not involved in PIV-4-mediated IL-6 gene induction. Relevant transcriptional sites were positioned in between positions –224 and –109 from the IL-6 promoter. Disruption of the distal (–153/–145) or the proximal (–83/–75) C/EBP site in the –224-CAT construct dramatically reduced CAT protein recovery, indicating that these two C/EBP sites played a major role in PIV-4-induced IL-6 gene transcription.

PIV-4 induces and maintains nuclear AP-1, C/EBP, and NF-{kappa}B DNA-protein complexes. Band shift assays with nuclear extracts obtained from NCI-H292 cells exposed to PIV-4 were performed to estimate relative levels of the relevant transcription factors (Fig. 5). Exposure to PIV-4 rapidly increased (t = 12 min) NF-{kappa}B and C/EBP DNA-protein complexes (5–10 times) and, to a lesser extent, that of AP-1 (two to three times). Only the levels of NF-{kappa}B DNA-protein complexes appeared to parallel the induction of the second IL-8 mRNA peak, whereas that of AP-1 did not change. The amount of C/EBP DNA-protein complexes was maximal 4 h after exposure to PIV-4. This suggests that both IL-6 mRNA peaks may be directed primarily by an increased transcriptional activity. At 24 h after PIV-4 exposure, in apparent contradiction with the low levels of IL-8 and IL-6 mRNA (c.f. Fig. 3), the DNA-protein complexes were still abundant.



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  		Fig. 5. Nuclear NF-{kappa}B, C/EBP, and AP-1 DNA activities in response to PIV-4 infection. Nuclear extracts were prepared from NCI-H292 cells infected with PIV-4 (1:10 dilution) at indicated time points. EMSAs were performed with oligonucleotides specific for NF-{kappa}B and AP-1 binding sites in the IL-8 promoter and that of the proximal and distal C/EBP binding sites in the IL-6 promoter. The results for the distal C/EBP binding site were similar to those for the proximal C/EBP binding site, the latter of which are shown. Specific retarded complexes (indicated by an arrowhead) were identified by competition with unlabeled specific oligonucleotides (100x and 1x excess, 2 lanes on the right, respectively), but not with an irrelevant oligonucleotide (not shown), and by supershift (not shown). These results are representative of 4–6 experiments.

 
IL-8 and IL-6 mRNA stability in NCI-H292 cells exposed to PIV-4. IL-8 and IL-6 mRNA in NCI-H292 cells are relatively unstable with a half-life of 30–40 min (28, 29). To determine whether the second IL-8 and IL-6 mRNA peak after exposure to PIV-4 resulted from mRNA stabilization, we determined IL-8 and IL-6 mRNA half-lives at various time points after exposure to PIV-4 (Fig. 6). IL-8 and IL-6 mRNA were unstable at 1 and 4 h after exposure to PIV-4, with an estimated half-life for both mRNAs of 30 min. Six hours after exposure to PIV-4, IL-8 and IL-6 mRNA were considerably more stable, with a half-life of >2 and 4 h, respectively. For IL-8 mRNA we also assessed the half-life at 8 and 11 h after viral exposure. Whereas IL-8 mRNA was still more stable at 8 h, at 11 h the half-life was similar to that at 1 h after exposure to PIV-4. Thus the second peak of IL-8 and IL-6 mRNA expression was at least in part due to a reduced IL-8 and IL-6 mRNA degradation.



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  		Fig. 6. Modulation of IL-8 and IL-6 mRNA degradation in NCI-H292 cells infected with PIV-4. NCI-H292 cells were infected with PIV-4 (1:10 dilution). At different times after infection (1, 4, 6, 8, or 11 h), actinomycin D (Act, 10 µg/ml) was added to the cultures and total RNA was recovered after an additional 0, 0.5, 1, 2, or 4 h of incubation. The extracted RNA was subjected to Northern blot analysis. Blots were hybridized with IL-8, IL-6, and GAPDH probes. A typical result for IL-8 is shown in A. Signals obtained after hybridization were quantified using a PhosphorImager. IL-8 and IL-6 mRNA levels normalized to GAPDH were expressed relative to the corresponding level before adding Act (t = 0) and plotted as a function of time (B). Results are representative of 3 experiments.

 
Exaggerated IL-8 and IL-6 responses by PIV-4-infected NCI-H292 cells. We have previously shown that stabilization of IL-6 and IL-8 mRNA resulted in an exaggerated IL-8 and IL-6 production as reflected by steeper dose-response curves (24, 35). Thus we analyzed whether PIV-4-infected NCI-H292 cells, compared with noninfected cells, exhibited an exaggerated IL-8 and IL-6 production to TNF-{alpha} during the phase of mRNA stabilization (5 h after initial exposure). Also, we assessed the responsiveness after 16 h of exposure to PIV-4, at which stage the levels of IL-8 and IL-6 mRNA had returned to basal levels. Figure 7 shows that cells exposed to PIV-4 for 5 h, but not for 16 h, display an exaggerated IL-8 and, less so for IL-6 production, compared with noninfected cells. The virus-induced steeper dose-response curve is particular evident when the curves for PIV-4-infected cells are transposed (dashed curves) to those for the noninfected cells.



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  		Fig. 7. IL-8 and IL-6 production in response to a concentration range of TNF-{alpha} by NCI-H292 cells infected for 5 and 16 h by PIV-4. NCI-H292 cells were exposed to PIV-4 (1:10 dilution, filled symbols) or no virus (open symbols) for 5 h (A and C) and 16 h (B and D) after which cells were exposed for 8 h to TNF-{alpha}. IL-8 (A and B) and IL-6 (C and D) in supernatants were determined by ELISA. Results are expressed as mean ± SD (n = 3) and are representative of 3 experiments. The dashed curves are similar to those for the filled symbols but are corrected for the virus-induced IL-8 and IL-6 production in the absence of TNF-{alpha} and were used for comparison with the response by cells not exposed to virus. *P = 0.02, **P < 0.006, and ***P = 0.051 (2-tailed, paired t-test).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study we show that infection with PIV-4 induced a dose- and time-dependent release of the proinflammatory mediators IL-8 and IL-6, which at the mRNA level is a biphasic response. The initial interaction between live virus and cells rapidly increased DNA binding activities of the relevant transcription factors NF-{kappa}B and AP-1 for IL-8 and C/EBP for IL-6, which coincided with the occurrence of the first mRNA peak. Moreover, the second IL-8 and IL-6 mRNA peaks were paralleled by increased NF-{kappa}B and C/EBP DNA-binding activities, respectively. The second mRNA peak for IL-8 and IL-6, however, also concurred with a reduced IL-8 and IL-6 mRNA degradation. Previous studies have indicated that a reduced IL-8 and IL-6 mRNA degradation can lead to exaggerated IL-8 and IL-6 responses to a secondary stimulus. This appears true also for cells infected with PIV-4, as stimulation of the infected cells during the phase of a reduced mRNA degradation resulted in exaggerated IL-8 and, less so, IL-6 responses. Thus PIV-4 infection may contribute to local IL-8 and IL-6 production by three means. Firstly, the initial interaction between epithelial cells and virus triggers gene transcription. Secondly, in infected cells IL-8 and IL-6 mRNA degradation is reduced, which further enhances the IL-8 and IL-6 response. Thirdly, during the phase of a reduced mRNA degradation, infected epithelial cells display an exaggerated IL-8 and IL-6 response to a secondary stimulus.

Traditionally, PIV-4 is considered a mild and not common pathogen compared with parainfluenza virus types 1, 2, and 3. However, recent studies employing improved methods for detection of PIV-4 indicate that PIV-4 infections appear more frequently than parainfluenza type 2 infections and are also associated with severe clinical disease (2, 34). An important consideration for using PIV-4 here was that PIV-4 causes weak and late cytopathic effects only. We reasoned that infection with PIV-4, therefore, is less likely to affect microtubular architecture, the modulation of which can trigger IL-8 production by airway epithelial cells (30).

PIV-4, like RSV, belongs to the negative-sense, single-stranded RNA viruses (32). The virus-induced mechanism of IL-8 gene transcription is similar for RSV and PIV-4, despite the use of different cell lines. With both viruses, an early and sustained activation of NF-{kappa}B is a key feature (11–13, 26, and present study). NF-{kappa}B DNA-binding activity was still increased at 24 h after initial exposure to either virus, as were also DNA binding activities for AP-1 and C/EBP, here with PIV-4. Remarkably, despite the activation of relevant transcription factors, IL-8 and IL-6 mRNA levels returned to basal level in PIV-4-infected cells, whereas in RSV-infected cells the IL-8 mRNA level was still increased (10). Although these apparent contradictory results for PIV-4-infected cells remain to be explained, there is considerable evidence now in support of viruses taking over the NF-{kappa}B pathway to promote viral pathogenesis (19). Our results are consistent with this finding and seem to indicate that similar modulation may apply to AP-1 and C/EBP. RSV, like PIV-4, induces a biphasic expression of IL-8 mRNA, although the second phase of IL-8 mRNA expression seems more extended with RSV than with PIV-4 (Ref. 10 and present study). Whether this biphasic regulation is unique to PIV-4 and RSV remains to be determined.

The biphasic mRNA expression over 24 h of PIV-4 infection is not paralleled by a biphasic IL-8 and IL-6 production, but rather by a gradual increase of IL-8 and IL-6 protein over 24 h. As yet we cannot offer a clear explanation for this apparent discrepancy. Preliminary studies, in which we address the question whether at conditions of reduced IL-6 mRNA degradation all IL-6 mRNA molecules are translated, indicate that only a minority of the IL-6 mRNA molecules associate with polysomes, which is indicative of translation (A. van den Berg, J. de Freitas, R. Lutter; unpublished results). This does not exclude the possibility that IL-6 mRNA molecules not associated with polysomes can modulate translation of polysome-associated IL-6 mRNA molecules, but this remains to be investigated.

As described here for PIV-4, there was no modulation of IL-8 mRNA degradation up to 4 h after initial exposure of A549 cells to RSV, that is, during the first IL-8 mRNA peak (10). There have been no studies reported on IL-8 mRNA degradation in RSV-infected cells during the second phase of IL-8 mRNA expression. Interestingly, in a recent study, RSV-induced regulated on activation, normal T cell expressed, and presumably secreted (RANTES) expression in A549 cells was shown to depend on a reduced degradation of RANTES mRNA (22), suggestive of a similar mechanism as described here for IL-8. Moreover, in that study, there was a clear correlation between viral replication and a reduced RANTES mRNA degradation. Whether a similar exaggerated production of RANTES was observed as here for IL-8 and IL-6 is not known, and whether other respiratory viruses also reduce IL-8 and IL-6 mRNA degradation in the infected cells remains to be studied.

The mechanism by which PIV-4 reduces IL-8 and IL-6 mRNA degradation remains to be solved. UV irradiation of PIV-4 reproducibly reduced but not abrogated the second IL-8 and IL-6 mRNA peak, indicative of a role for viral replication. The relative minor effect of UV irradiation may be due to the small genome of PIV-4, which makes it less sensitive to UV irradiation. Other studies in which we employed ribavirin to inhibit PIV-4 replication (not shown) showed a direct effect of ribavirin on IL-8 and IL-6 production, thus precluding its further use. Previous studies have indicated that a reduced overall protein synthesis reduces mRNA degradation (19, 28, 29). This may indeed occur during viral replication, as at least part of the capacity of host cell protein synthesis will be devoted to synthesis of viral proteins.

Overall, this study shows that PIV-4 infection induces production of proinflammatory mediators and emphasizes the critical role of a reduced mRNA degradation in these responses during PIV-4 infection. Clearly, similar analyses have to be performed for other respiratory viruses. These findings, however, provide an explanation for the apparent synergy between a viral infection and another or secondary proinflammatory stimulus on airway inflammation. Patients with a respiratory viral infection are more prone to develop secondary bacterial pneumonia, i.e., bacterial superinfection. Similarly, viral airway infections in patients with asthma or chronic obstructive pulmonary disease can lead to an exacerbation of the disease, which is paralleled by an exaggerated inflammatory response. The exaggerated response by virus-infected epithelial cells reported here may contribute to the enhanced airway inflammation, although other mechanisms are not excluded (1, 17, 33, 35). Further in vivo studies are required to assess which of these mechanisms are most dominant.


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 MATERIALS AND METHODS
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T. Roger was supported in part by a European Community grant (contract ERBCHBGCT940673) and A. van den Berg by Netherlands Asthma Foundation Grant 99.27.


   ACKNOWLEDGMENTS
 
We are grateful to Drs. N. Mukaida and K. Matsushima (Cancer Research Institute, Kanazawa, Japan) and to Drs. K. Wong and W. M. Rom (Bellevue Hospital Center, New York, NY) for providing the IL-8 and the IL-6 promoter CAT constructs, respectively. Dr. J. E. Echevarria and coworkers (Centro Nacional de Microbiologia, Instituto de Salud Carlos III, Madrid, Spain) are acknowledged for their support with the initial studies. We thank Dr. T. A. Out (Clin. and Lab. Immunol. Unit, AMC) for critical reading of the manuscript.

Present address for T. Roger: Division of Infectious Diseases, Department of Internal Medicine, Centre Hospitalier Universitaire Vaudois, CH-1011 Lausanne, Switzerland.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. Lutter, Dept. of Pulmonology/Lab. of Experimental Immunology, G1-140, Academic Medical Center, Meibergdreef 9, PO Box 22700, 1100 DE Amsterdam, The Netherlands (E-mail: r.lutter{at}amc.uva.nl)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
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  1. Aggarwal BB, Eessalu TE, and Hass PE. Characterization of receptors for human tumour necrosis factor and their regulation by interferon-{gamma}. Nature 318: 665–667, 1985.[CrossRef][Medline]
  2. Aguilar JC, Pérez-Brena MP, Garcia ML, Cruz N, Erdman DE, and Echevarria JE. Detection and identification of human Parainfluenza viruses 1, 2, 3, and 4 in clinical samples of pediatric patients by multiplex reverse transcription-PCR. J Clin Microbiol 38: 1191–1195, 2000.[Abstract/Free Full Text]
  3. Arnold R, Humbert B, Werchau H, Gallati H, and Konig W. Interleukin-8, interleukin-6, and soluble tumor necrosis factor receptor type I release from a human pulmonary epithelial cell line (A549) exposed to respiratory syncytial virus. Immunology 82: 126–133, 1994.[ISI][Medline]
  4. Bale JF, Kern ER, Overall JC, and Glasgow LA. Enhanced susceptibility of mice infected with murine cytomegalovirus to intranasal challenge with Escherichia coli: pathogenesis and altered inflammatory response. J Infect Dis 145: 525–531, 1982.[ISI][Medline]
  5. Castells E, George VG, and Hierholzer JC. NCI-H292 as an alternative cell line for the isolation and propagation of the human paramyxoviruses. Arch Virol 115: 277–288, 1990.[CrossRef][ISI][Medline]
  6. Choi AMK and Jacoby DB. Influenza virus A infection induces interleukin-8 gene expression in human airway epithelial cells. FEBS Lett 309: 327–329, 1992.[CrossRef][ISI][Medline]
  7. Degre M and Glasgow LA. Synergistic effect in viral-bacterial infection. I. Combined infection of the respiratory tract in mice with parainfluenza virus and Haemophilus influenzae. J Infect Dis 118: 449–462, 1968.[ISI][Medline]
  8. De Kluijver J, Grunberg K, Sont JK, Hoogeveen M, van Schadewijk WA, de Klerk EP, Dick CR, van Krieken JH, and Sterk PJ. Rhinovirus infection in nonasthmatic subjects: effects on intrapulmonary airways. Eur Respir J 20: 274–279, 2002.[Abstract/Free Full Text]
  9. Empey DW, Laitinen LA, Jacobs L, Gold WM, and Nadel JA. Mechanisms of bronchial hyperreactivity in normal subjects after upper respiratory tract infection. Am Rev Respir Dis 113: 131–139, 1976.[ISI][Medline]
  10. Fiedler MA, Wernke-Dollries K, and Stark JM. Respiratory syncytial virus increases IL-8 gene expression and protein release in A549 cells. Am J Physiol Lung Cell Mol Physiol 269: L865–L872, 1995.[Abstract/Free Full Text]
  11. Fiedler MA, Wernke-Dollries K, and Stark JM. Mechanism of RSV-induced IL-8 gene expression in A549 cells before viral replication. Am J Physiol Lung Cell Mol Physiol 271: L963–L971, 1996.[Abstract/Free Full Text]
  12. Fiedler MA, Wernke-Dollries K, and Stark JM. Inhibition of viral replication reverses respiratory syncytial virus-induced NF-{kappa}B activation and interleukin-8 gene expression in A549 cells. J Virol 70: 9079–9082, 1996.[Abstract]
  13. Garofalo R, Sabry M, Jamaluddin M, Yu RK, Casola A, Ogra PL, and Brasier AR. Transcriptional activation of the interleukin-8 gene by respiratory syncytial virus infection in alveolar epithelial cells: nuclear translocation of the RelA transcription factor as a mechanism producing airway mucosal inflammation. J Virol 70: 8773–8781, 1996.[Abstract]
  14. Grünberg K, Timmers MC, Smits HH, de Klerk EPA, Dick EC, Spaan WJM, Hiemstra PS, and Sterk PJ. Effect of experimental rhinovirus 16 colds on airway hyperresponsiveness to histamine and interleukin-8 in nasal lavage in asthmatic subjects in vivo. Clin Exp Allergy 27: 36–45, 1997.[Medline]
  15. Gwaltney JM. Rhinovirus infection of the normal human airway. Am J Respir Crit Care Med 152: S36–S39, 1995.[ISI][Medline]
  16. Hall WJ, Hall CB, and Speers DM. Respiratory syncytial virus infection in adults: clinical, virologic, and serial pulmonary function subsets. Ann Intern Med 88: 203–207, 1978.[ISI][Medline]
  17. Hament JM, Kimpen JL, Fleer A, and Wolfs TF. Respiratory viral infection predisposing for bacterial disease: a concise review. FEMS Immunol Med Microbiol 26: 189–195, 1999.[CrossRef][ISI][Medline]
  18. Hayden FG, Fritz RS, Lobo MC, Alvord WG, Strober W, and Straus SE. Local and systemic cytokine responses during experimental human influenza A virus infection. J Clin Invest 101: 643–649, 1998.[ISI][Medline]
  19. Hiscott J, Kwon H, and Génin P. Hostile takeovers: viral appropriation of the NF-{kappa}B pathway. J Clin Invest 107: 143–151, 2001.[CrossRef][ISI][Medline]
  20. Johnston SL. Natural and experimental rhinovirus infections of the lower respiratory tract. Am J Respir Crit Care Med 152: S46–S52, 1995.[ISI][Medline]
  21. Johnston SL, Papi A, Bates PJ, Mastronarde JG, Monick MM, and Hunninghake GW. Low grade rhinovirus infection induces a prolonged release of IL-8 in pulmonary epithelium. J Immunol 160: 6172–6181, 1998.[Abstract/Free Full Text]
  22. Koga T, Sardina E, Tidwell RM, Pelletier M, Look DC, and Holtzman MJ. Virus-inducible expression of a host chemokine gene relies on replication-linked mRNA stabilization. Proc Natl Acad Sci USA 96: 5680–5685, 1999.[Abstract/Free Full Text]
  23. Lindquist SW, Darnule A, Istas A, and Demmler G. Parainfluenza virus type 4 infections in pediatric patients. Pediatr Infect Dis J 16: 34–38, 1997.[CrossRef][ISI][Medline]
  24. Lutter R, Loman S, Snoek M, Roger T, Out TA, and Jansen HM. IL-6 protein production by airway epithelial(-like) cells disabled in IL-6 mRNA degradation. Cytokine 12: 1275–1279, 2000.[CrossRef][ISI][Medline]
  25. MacDonald KL, Osterholm MT, Hedberg CW, Schrock CG, Peterson GF, Jentzen JM, Leonard SA, and Schlievert PM. Toxic shock syndrome. A newly recognized complication of influenza and influenza-like illness. JAMA 257: 1053–1058, 1987.[Abstract]
  26. Mastronarde JG, Monick MM, Mukaida N, Matsushima K, and Hunninghake GW. Activator protein-1 is the preferred transcription factor for cooperative interaction with nuclear factor-{kappa}B in respiratory syncytial virus-induced interleukin-8 gene expression in airway epithelium. J Infect Dis 177: 1275–1281, 1998.[ISI][Medline]
  27. Noah TL and Becker S. Respiratory syncytial virus-induced cytokine production by a human bronchial epithelial cell line. Am J Physiol Lung Cell Mol Physiol 265: L472–L478, 1993.[Abstract/Free Full Text]
  28. Roger T, Out TA, Jansen HM, and Lutter R. Superinduction of interleukin-6 mRNA in lung epithelial H292 cells depends on transiently increased C/EBP activity and durable increased mRNA stability. Biochim Biophys Acta 1398: 275–284, 1998.[Medline]
  29. Roger T, Out TA, Mukaida N, Matsushima K, Jansen HM, and Lutter R. Enhanced AP-1 and NF-{kappa}B activities and stability of interleukin-8 (IL-8) transcripts are implicated in IL-8 mRNA superinduction in lung epithelial H292 cells. Biochem J 330: 429–435, 1998.
  30. Shibata Y, Nakamura H, Kato S, and Tomoike H. Cellular detachment and deformation induce IL-8 gene expression in human bronchial epithelial cells. J Immunol 156: 772–777, 1996.[Abstract]
  31. Skoner DP, Gentile DA, Paterl A, and Doyle WJ. Evidence for cytokine mediation of disease expression in adults experimentally infected with influenza A virus. J Infect Dis 180: 10–14, 1999.[CrossRef][ISI][Medline]
  32. Taussig J. Processes in Pathology and Microbiology Infections (2nd ed.). Oxford, UK: Blackwell Scientific, 1984, sect. 3, p. 215–375.
  33. Tsujimoto M, Yip YK, and Vilcek J. Interferon-{gamma} enhances expression of cellular receptors for tumor necrosis factor. J Immunol 136: 2441–2444, 1986.[Abstract]
  34. Vainiopää R and Hyypiä T. Biology of parainfluenza viruses. Clin Microbiol Rev 7: 265–275, 1994.[Abstract/Free Full Text]
  35. Van Wissen M, Snoek M, Smids B, Jansen HM, and Lutter R. IFN-gamma amplifies IL-6 and IL-8 responses by airway epithelial-like cells via indoleamine 2,3-dioxygenase. J Immunol 169: 7039–7044, 2002.[Abstract/Free Full Text]



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