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. 2000 Sep;74(18):8425-33.
doi: 10.1128/jvi.74.18.8425-8433.2000.

Synergistic upregulation of interleukin-8 secretion from pulmonary epithelial cells by direct and monocyte-dependent effects of respiratory syncytial virus infection

L H Thomas  1 , M I Wickremasinghe, M Sharland, J S Friedland
Affiliations

Synergistic upregulation of interleukin-8 secretion from pulmonary epithelial cells by direct and monocyte-dependent effects of respiratory syncytial virus infection

L H Thomas et al. J Virol. 2000 Sep.

Abstract

Respiratory syncytial virus (RSV) infection is the major cause of severe bronchiolitis in infants. Pathology of this infection is partly due to excessive proinflammatory leukocyte influx mediated by chemokines. Although direct infection of the respiratory epithelium by RSV may induce chemokine secretion, little is known about the role of cytokine networks. We investigated the effects of conditioned medium (CM) from RSV-infected monocytes (RSV-CM) on respiratory epithelial (A549) cell chemokine release. RSV-CM, but not control CM (both at a 1:5 dilution), stimulated interleukin-8 (IL-8) secretion from A549 cells within 2 h, and secretion increased over 72 h to 11,360 +/- 1,090 pg/ml without affecting cell viability. In contrast, RSV-CM had only a small effect on RANTES secretion. RSV-CM interacted with direct RSV infection to synergistically amplify IL-8 secretion from respiratory epithelial cells (levels of secretion at 48 h were as follows: RSV-CM alone, 8,140 +/- 2,160 pg/ml; RSV alone, 12,170 +/- 300 pg/ml; RSV-CM plus RSV, 27,040 +/- 5,260 pg/ml; P < 0.05). RSV-CM induced degradation of IkappaBalpha within 5 min but did not affect IkappaBbeta. RSV-CM activated transient nuclear binding of NF-kappaB within 1 h, while activation of NF-IL6 was delayed until 8 h and was still detectable at 24 h. Promoter-reporter analysis demonstrated that NF-kappaB binding was essential and that NF-IL6 was important for IL-8 promoter activity in RSV-CM-activated cells. Blocking experiments revealed that the effects of RSV-CM depended on monocyte-derived IL-1 but that tumor necrosis factor alpha was not involved in this network. In summary, RSV infection of monocytes results in and amplifies direct RSV-mediated IL-8 secretion from respiratory epithelial cells by an NF-kappaB-dependent, NF-IL6-requiring mechanism.

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Figures

FIG. 1
FIG. 1
Kinetics of RSV-CM-induced IL-8 from human respiratory epithelial cells. (A) A549 cells were stimulated with C-CM or RSV-CM at a 1:5 dilution. Cell culture supernatants were harvested at 0, 2, 8, 24, 48, and 72 h poststimulation, and IL-8 protein was measured by ELISA. (B) A549 cells were stimulated with C-CM or RSV-CM, and RNA was extracted from lysates taken at 0, 1, 2, 4, 8, and 34 h poststimulation. RNA was analyzed by ethidium bromide staining for 18S and 28S rRNA bands and by Northern blotting for IL-8 and β-actin mRNA. The graph depicts densitometrical analysis of IL-8 mRNA normalized for total mRNA using β-actin. (C) NHBE cells were stimulated with C-CM or RSV-CM as described for panel A. Results are the means ± SEM of three independent experiments (each using CM prepared on a separate occasion).
FIG. 2
FIG. 2
Kinetics of RSV-CM-induced RANTES secretion from A549 cells. Cells were stimulated with C-CM or RSV-CM, and culture supernatants were harvested at specific time points up to 72 h. RANTES protein was measured in the supernatants by ELISA. Results are means ± SEM of three independent experiments (each using CM prepared on a separate occasion).
FIG. 3
FIG. 3
Synergism between RSV-CM and RSV infection of A549 cells. A549 cells were incubated with media alone (control), C-CM, or RSV-CM or were exposed to direct RSV infection (MOI = 0.05) with (RSV + RSV-CM) or without (RSV) RSV-CM. Cell culture supernatants were harvested after 24 or 48 h of culture, and IL-8 was measured by ELISA. Results are means ± SEM of three independent experiments. ∗, P < 0.05 for a comparison of the sum of the two single stimuli with the effect of dual stimulation (paired t test).
FIG. 4
FIG. 4
Kinetics of IκB degradation and resynthesis in CM-stimulated A549 cells. A549 cells were stimulated by C-CM or RSV-CM, and cytoplasmic lysates were prepared at specific time points after stimulation. Lysates were resolved by SDS-PAGE, and Western blotting was performed for IκBα (A) and IκBβ (B).
FIG. 5
FIG. 5
Activation of NF-κB in RSV-CM-stimulated A549 cells. (A) Nuclear extracts were prepared from A549 cells at 0, 1, 2, and 4 h poststimulation with either C-CM or RSV-CM. Equal quantities of nuclear protein were mixed with 32P-labeled, double-stranded oligonucleotides specific for the NF-κB binding sequence and were resolved by PAGE. Bound complexes were visualized by autoradiography. (B) Competition experiments were performed on extracts prepared 1 h poststimulation with RSV-CM. NF-κB binding to labeled probe was competed out with a 10-fold excess of unlabeled NF-κB probe but not with a 10-fold excess of unlabeled, irrelevant (NF-IL6-binding) probe.
FIG. 6
FIG. 6
Activation of NF-IL6 in RSV-CM-stimulated A549 cells. (A) Nuclear extracts were prepared from A549 cells at 0, 2, 4, 8, and 24 h poststimulation with either C-CM or RSV-CM. Equal quantities of nuclear protein were mixed with 32P-labeled, double-stranded oligonucleotides specific for the NF-IL6 binding sequence and were resolved by PAGE. Bound complexes were visualized by autoradiography. (B) Competition experiments were performed on extracts prepared 24 h poststimulation with RSV-CM. NF-IL-6 binding to labeled probe was not competed out with a 10-fold excess of unlabeled NF-κB probe but was completely blocked with a 10-fold excess of unlabeled NF-IL6 probe.
FIG. 7
FIG. 7
RSV-CM-induced IL-8 promoter activity: effect of NF-κB- or NF-IL6-binding region mutations on activation. (A) Schematic representation of the wild-type IL-8 promoter (bases −1370 to +82) inserted upstream of the luciferase gene in pGL2-basic. Lowercase letters, construct variants containing mutations in either the NF-κB or NF-IL6 sequences. (B) A549 cells were grown to 50% confluency and then were cotransfected with the wild-type, NF-κB-mutated, or NF-IL6-mutated IL-8 promoter constructs plus a control plasmid, pRL-TK, which expresses low, constitutive levels of Renilla luciferase. Cells were stimulated the following day with RSV-CM, and lysates were prepared and assayed for luciferase activity. Data are percentages of maximal promoter activity (obtained from RSV-CM-stimulated cells containing the wild-type promoter construct) after normalization of experimental plasmid values with control plasmid luciferase activity. Results are the means ± SEM of three independent experiments (each using CM prepared on a separate occasion).
FIG. 8
FIG. 8
Effect of IL-1Ra or neutralizing anti-TNF-α on RSV-CM-induced IL-8 secretion from human epithelial cells. (A) A549 cells were preincubated for 2 h in the absence or presence of human Il-1Ra (2, 20, or 200 ng/ml) prior to addition of RSV-CM. Alternatively, RSV-CM was incubated for 1 h in the absence or presence of rabbit anti-human neutralizing anti-TNF-α (5, 20, or 50 μg/ml) prior to its addition to A549 cell cultures. RSV-CM was also incubated with rabbit anti-human neutralizing anti-IL-6 at 5, 20, and 50 μg/ml as a negative control. Culture supernatants were harvested after 24 h and analyzed for IL-8 content by ELISA. (B) NHBE cells were incubated in the presence of IL-1Ra (200 ng/ml) as described for panel A. IL-8 in supernatants harvested after 24 h was measured by ELISA. All results are the means ± SEM of three independent experiments.
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