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. 2005 Jul 1;175(1):404-12.
doi: 10.4049/jimmunol.175.1.404.

Role of IL-17A, IL-17F, and the IL-17 receptor in regulating growth-related oncogene-alpha and granulocyte colony-stimulating factor in bronchial epithelium: implications for airway inflammation in cystic fibrosis

Florencia McAllister  1 , Adam Henry, James L Kreindler, Patricia J Dubin, Lauren Ulrich, Chad Steele, Jonathan D Finder, Joseph M Pilewski, Beatriz M Carreno, Samuel J Goldman, Jaana Pirhonen, Jay K Kolls
Affiliations

Role of IL-17A, IL-17F, and the IL-17 receptor in regulating growth-related oncogene-alpha and granulocyte colony-stimulating factor in bronchial epithelium: implications for airway inflammation in cystic fibrosis

Florencia McAllister et al. J Immunol. .

Abstract

IL-17R signaling is critical for pulmonary neutrophil recruitment and host defense against Gram-negative bacteria through the coordinated release of G-CSF and CXC chemokine elaboration. In this study, we show that IL-17R is localized to basal airway cells in human lung tissue, and functional IL-17R signaling occurs on the basolateral surface of human bronchial epithelial (HBE) cells. IL-17A and IL-17F were potent inducers of growth-related oncogene-alpha and G-CSF in HBE cells, and significant synergism was observed with TNF-alpha largely due to signaling via TNFRI. The activities of both IL-17A and IL-17F were blocked by a specific anti-IL-17R Ab, but only IL-17A was blocked with a soluble IL-17R, suggesting that cell membrane IL-17R is required for signaling by both IL-17A and IL-17F. Because IL-17A and IL-17F both regulate lung neutrophil recruitment, we measured these molecules as well as the proximal regulator IL-23p19 in the sputum of patients with cystic fibrosis (CF) undergoing pulmonary exacerbation. We found significantly elevated levels of these molecules in the sputum of patients with CF who were colonized with Pseudomonas aeruginosa at the time of pulmonary exacerbation, and the levels declined with therapy directed against P. aeruginosa. IL-23 and the downstream cytokines IL-17A and IL-17F are critical molecules for proinflammatory gene expression in HBE cells and are likely involved in the proinflammatory cytokine network involved with CF pathogenesis.

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Conflict of interest statement

Disclosures: The authors have no financial conflict of interest.

Figures

FIGURE 1
FIGURE 1
A, Dose-dependent elevation of GRO-α, G-CSF, and MCP-1 protein levels by recombinant human IL-17A and IL-17F. Primary HBE cells were treated with different doses of IL-17A and IL-17F, as indicated. Basolateral media were collected 24 h after the treatment, and cytokine concentrations were measured by ELISA. B, Time course study. HBE cells were stimulated with IL-17A and IL-17F (both at 10 ng/ml), and basolateral media were collected after 4, 8, 16, and 24 h for G-CSF, GRO-α, and MCP-1 measuring. Cytokine concentrations are shown in fold changes vs control. Results are expressed as the mean ± SEM of triplicate samples from one representative experiment.
FIGURE 2
FIGURE 2
GRO-α (A) and G-CSF (B) secreted after stimulation with IL-17F and/or TNF-α. primary HBE cells were treated with IL-17F (10 ng/ml) and TNF-α (1 ng/ml). The cytokine mixture (IL-17F + TNF-α) was also preincubated with anti-IL-17 mAb (2 μg/ml), recombinant human IL-17R:Fc (1 μg/ml), or isotype-matched controls. Basolateral media were collected 24 h after the treatment, and cytokine levels were determined by ELISA. Results are expressed as the mean ± SEM of triplicate samples from one representative experiment (*, p < 0.05). C, G-CSF secreted after stimulation with IL-17A (10 ng/ml) and/or TNF-α. Cells were treated as outlined above. Results are expressed as the mean ± SEM of triplicate samples from one representative experiment (*, p < 0.05).
FIGURE 3
FIGURE 3
Effects of blocking IL-17R on GRO-α (A) and G-CSF (B) production by IL-17A and IL-17F. Primary HBE cells were pretreated with IL-17R Ab (2 μg/ml) 30 min before IL-17A and IL-17F treatment (both at 10 ng/ml). Apical and basolateral medium was collected 24 h later. Results are expressed as the mean ± SEM of three separate experiments (*, p < 0.05).
FIGURE 4
FIGURE 4
Detection and localization of the IL-17R. A, Representative immunohistochemical staining for IL-17R in a human lung section with a specific detecting monoclonal anti-IL-17R Ab, showing basolateral localization of the IL-17R. B, G-CSF and GRO-α secretion by primary HBE cells after addition of IL-17A and IL-17F (both at 10 ng/ml) to basolateral or apical surface. Basolateral media were collected after 24 h, and cytokine levels were measured by ELISA. Results are expressed as the mean ± SEM of triplicate samples from one representative experiment (*, p < 0.05).
FIGURE 5
FIGURE 5
Detection and localization of TNFRs, TNF-RI, and TNF-RII. A, Immunohistochemical staining of TNF-RI, TNF-RII, and isotype-matched control. Conventional xy images are shown on upper panels, and x–z axis reconstructions are shown in lower panels. B, G-CSF secretion by primary HBE cells after addition of IL-17F and/or TNF-α (both at 10 ng/ml) to basolateral or apical surface. Basolateral media were collected after 24 h, and G-CSF levels were measured by ELISA. Results are expressed as the mean ± SEM of triplicate samples from one representative experiment (*, p < 0.05). C, Primary HBE cells were pretreated with anti-human TNF-RI and/or recombinant TNF-RII:Fc chimera (0.5 μg/ml) 2 h before IL-17F and/or TNF-α treatment (both at 10 ng/ml). Basolateral media were collected after 24 h, and G-CSF levels were measured by ELISA. Results are expressed as the mean ± SEM of triplicate samples from one representative experiment (*, p < 0.05). D, Primary HBE cells were pretreated with anti-human TNF-RI and/or recombinant TNF-RII:Fc chimera (0.5 μg/ml) 2 h before IL-17A and/or TNF-α treatment (both at 10 ng/ml). Basolateral media were collected after 24 h, and G-CSF levels were measured by ELISA. Results are expressed as the mean ± SEM of triplicate samples from one representative experiment (*, p < 0.05).
FIGURE 6
FIGURE 6
Cytokines levels in sputum from CF patients undergoing pulmonary exacerbation. Panel A: IL-17A and IL-17F concentration in sputum from CF patients at different time points, as indicated, were measured by ELISA. Panel B: IL-17- induced cytokines IL-8 (upper figure) G-CSF, GRO-a, MCP-1, MIP-1b, GM-CSF and IL-1b (lower figure) levels were measured in sputum samples at the same time points, using Luminex cytokine beads and ELISA. Panel C: Western blot of IL-23p19 in the sputum of patients undergoing pulmonary exacerbation of CF.
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