Rhinovirus-induced IL-1β release from bronchial epithelial cells is independent of functional P2X7 - PubMed (original) (raw)

Rhinovirus-induced IL-1β release from bronchial epithelial cells is independent of functional P2X7

Lei Shi et al. Am J Respir Cell Mol Biol. 2012 Sep.

Abstract

Airway epithelial cell defenses to viral infections are often compromised in disease or injury. Danger molecules, including ATP, are released during infection and contribute to nucleotide receptor-dependent inflammatory responses, largely through P2X(7). Although respiratory epithelium has been shown to express a variety of nucleotide receptors, the functional contribution of P2X(7) to the epithelial cell inflammatory response is unclear. We used human donor bronchial epithelial cells (BECs) and primary brushed epithelium to explore responses upon nucleotide and Toll-like receptor stimulation. P2X(7) messenger RNA and protein were observed in unprimed BECs, whereas inflammatory cytokine stimulation increased both messenger RNA and protein. Functional pore activity characteristic of P2X(7) was observed in BECs, and IL-1β was rapidly released by BECs after Toll-like receptor 3 agonist, polyinosine-polycytidylic acid, priming followed by ATP administration, although no change was observed in IL-18 release. BECs produced more IL-1β after stimulation with polyinosine-polycytidylic acid than LPS, showing a different preferential response than monocytes. In addition, blockade of nucleotide receptors with oxidized ATP significantly increased human rhinovirus (HRV) recovered 24 hours after infection in BECs, whereas 2'-3'-O-(4-benzoylbenzoyl) ATP treatment of brushed epithelial cells and respiratory cell lines nonsignificantly decreased HRV recovery. IL-1β release was detected after HRV infection in both BECs and brushed cells, but BzATP did not significantly increase IL-1β release further. BEC processing of pro-IL-1β to the mature, cleaved, 17-kD form was confirmed by Western blotting. These results support the expression of functional P2X(7) in human lung epithelium, although its role in epithelial pathogen defense is likely independent of IL-1 family cytokine processing.

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Figures

Figure 1.

Figure 1.

Bronchial epithelial cells (BECs) have functional P2X7 pore activity. Fluorescent dye, YO-PRO-1, uptake in BECs after 2′-3′-O-(4-benzoylbenzoyl) ATP (BzATP) stimulation was observed by fluorescent microscopy and counted as a proportion of all cells. Representative images of BEC B45 are shown (A). The percentage of adherent BECs containing YO-PRO-1 was calculated and BzATP-treated cells demonstrated a significant increase of uptake for all three BECs (B_–_D). Unpaired t tests were performed within each group. Scale bar, 50 μm.

Figure 2.

Figure 2.

P2X7 is detected in primary respiratory epithelial cells. BECs cultured with vehicle or TNF-α/IFN-γ for 24 hours (A) and brushed epithelial cells from two donors (B) were probed for P2X7 by Western blotting, and indicate GRB2 (growth factor receptor-bound protein 2) as a loading control. Human embryonic kidney (HEK)-293 cells with vector or P2X7 were used as controls. BECs treated with vehicle (C) or TNF-α/IFN-γ (D) for 48 hours were examined for P2X7 protein. DAPI (4′,6-diamidino-2-phenylindole) and secondary antibody were coincubated before visualization. HEK-293 cells contain an empty vector (E) or express P2X7 (F) for negative and positive controls, respectively. Differential interference contrast and fluorescent signals are combined in the right panels. Scale bar, 10 μm.

Figure 3.

Figure 3.

ATP increases IL-1β release from polyinosine-polycytidylic acid (Poly (I:C))–primed BECs. Supernatants from BEC B45 (n = 4) primed with LPS or Poly (I:C) were collected after stimulation with ATP for 30 minutes and analyzed by ELISA. IL-1β levels were augmented by Poly (I:C) priming, which was further increased with ATP treatment (A). IL-18 did not demonstrate the same pattern of release (B). The limit of detection for both cytokines was 12.5 pg/ml and data are shown as mean ± SD. Pairwise comparisons were Bonferroni corrected. *P < 0.05 compared with vehicle-, ATP-, LPS-, and ATP/LPS-treated cells. **P < 0.05 compared with vehicle.

Figure 4.

Figure 4.

Nucleotide receptor blockade increases HRV recovery after infection, whereas HRV infection increases IL-1 family cytokine release. Cellular HRV levels at 24 hours were affected by nucleotide receptor modulators. Oxidized ATP (oATP) treatment of BECs increased HRV1a compared with both vehicle and BzATP groups (A), whereas brushed epithelial cells trended to a small decrease with BzATP treatment (B). Respiratory cell lines A549 (C) and BEAS-2B (F) transfected with either vector control or P2X7 were also infected with HRV1a for 24 hours. Supernatants of BECs or brushed epithelial cells treated with oATP and/or BzATP and incubated with HRV1a were analyzed for cytokine production. BEC IL-1β measured at 24 hours (D) was increased with HRV infection in all groups compared with vehicle or oATP treated. Epithelial cells from bronchoscopic brushings showed similar results with IL-1β (E), generating more IL-1β in the supernatant at 24 hours compared with vehicle, but not BzATP-treated cells. BEC IL-18 measured at 24 hours (G) was increased in all HRV groups. Pairwise comparisons are Bonferroni corrected. For cytokine measures: *P < 0.05 compared with vehicle-, BzATP-, and oATP-treated cells; **P < 0.05 compared with vehicle- and oATP-treated cells; ***P < 0.05 compared with vehicle. Box plots display median and interquartile range for A–C and F, and bar graphs show mean ± SD for D, E, and G.

Figure 5.

Figure 5.

IL-1β released from BECs is cleaved and demonstrates functional activity. Western blotting of concentrated supernatants from BEC B45 infected for 24 hours with HRV1a and/or stimulated with BzATP is shown with detectable bands similar in size to the positive control 17-kD band from primed THP-1 cells, and is representative of three experiments. Immunoblots with Cell Signaling (Danvers, MA) (A) and Santa Cruz Biotechnology (Santa Cruz, CA) (B) antibodies display preferential detection of the cleaved and pro forms of IL-1β, respectively. Positive control indicates LPS-primed and ATP-treated THP-1 cells. Molecular weight markers are shown on the right. (C) Supernatants from BECs primed with Poly (I:C) or vehicle and treated with ATP were used to culture A549 cells for 24 hours. Increasing concentrations of IL-1 receptor antagonist (IL-1RA) were added and IL-8 was measured by ELISA. *P < 0.05 compared with IL-8 release with no IL-1RA. Data points are the average of technical duplicates.

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