Regulation and function of the IL-1 family cytokine IL-1F9 in human bronchial epithelial cells

Abstract

The IL-1 family of cytokines, which now includes 11 members, is well known to participate in inflammation. Although the most recently recognized IL-1 family cytokines (IL-1F5-11) have been shown to be expressed in airway epithelial cells, the regulation of their expression and function in the epithelium has not been extensively studied. We investigated the regulation of IL-1F5-11 in primary normal human bronchial epithelial cells. Messenger (m)RNAs for IL-1F6 and IL-1F9, but not IL-1F5, IL-1F8 or IL-1F10, were significantly up-regulated by TNF, IL-1β, IL-17 and the Toll-like receptor (TLR)3 ligand double-stranded (ds)RNA. mRNAs for IL-1F7 and IL-1F11 (IL-33) were weakly up-regulated by some of the cytokines tested. Notably, mRNAs for IL-1F6 and IL-1F9 were synergistically enhanced by the combination of TNF/IL-17 or dsRNA/IL-17. IL-1F9 protein was detected in the supernatant following stimulation with dsRNA or a combination of dsRNA and IL-17. IL-1F6 protein was detected in the cell lysate but was not detected in the supernatant. We screened for the receptor for IL-1F9 and found that lung fibroblasts expressed this receptor. We found that IL-1F9 activated mitogen-activated protein kinases and the transcription factor NF-κB in primary normal human lung fibroblasts. IL-1F9 also stimulated the expression of the neutrophil chemokines IL-8 and CXCL3 and the Th17 chemokine CCL20 in lung fibroblasts. These results suggest that epithelial activation by TLR3 (e.g., by respiratory viral infection) and exposure to cytokines from Th17 cells (IL-17) and inflammatory cells (TNF) may amplify neutrophilic inflammation in the airway via induction of IL-1F9 and activation of fibroblasts.

Figure 1.

Regulation of newly recognized IL-1 family cytokines in human bronchial epithelial cells. Submerged- primary normal human bronchial epithelial cells (NHBE) were incubated with 100 ng/ml of TNF, IL-1β, IL-4, IL-6, IL-13, IL-17A (IL-17), OSM, IL-29, 10 ng/ml IFN-γ, 1,000 U/ml IFN-β, 1 μg/ml Pam3CSK4, 5 μg/ml dsRNA, 1 μg/ml LPS, 10 ng/ml Flagellin, 1 μg/ml FLS-1, 10 μg/ml R-837 and 2 μg/ml CpG-C as indicated.Messenger (m)RNA was then extracted and analyzed for mRNA expression using real-time PCR (A) 6 hours, (B) 6 hours (no-stimulation), (C) 1 to 48 hours, and (D) 24 hours. (E) NHBE were incubated for 24 hours with 0.05–25 μg/ml double-stranded (ds) RNA or 1–100 ng/ml IL-17. (F) NHBE were preincubated with 0.01% DMSO or 1–10 μg/ml cycloheximide (CHX) for 1 hour and then stimulated with 50 ng/ml IL-17 or 5 μg/ml dsRNA for 24 hours. (G) NHBE were infected with RV16 at a multiplicity of infection (MOI) of 2 and then cultured for 24 hours at 33°C. (H) NHBE were transfected with small interfering (si)RNA against control RNA, RELA or IRF-3 at 5 nM for 48 h, and then stimulated with 5 μg/ml dsRNA for 6 hours. The expression of mRNAs for IL-1F6, IL-1F9 and β-actin (ACTB) was analyzed by real-time PCR. The copy number is expressed as the number of transcripts/ng of total RNA. Results shown are mean ± SEM of three to seven independent experiments; *P < 0.05.

Figure 2.

Effect of the combination of TNF, IL-17, and dsRNA on the expression of IL-1F6 and IL-1F9 in human bronchial epithelial cells. Submerged-NHBE were incubated for 24 hours (A, B, D and E) or 6 to 48 hours (C and F) with 100 ng/ml TNF, 100 ng/ml IL-17, and 5 μg/ml dsRNA or their combination. Expression of mRNAs for IL-1F6 and IL-1F9 was analyzed by real-time PCR. The copy number is expressed as the number of transcripts/ng of total RNA. Results shown are mean ± SEM of six independent experiments; *P < 0.05.

Figure 3.

Detection of IL-1F6 and IL-1F9 protein in human bronchial epithelial cells. Submerged-NHBE were incubated for (A) 48 to 72 hours, (B) 72 hours, or (C) 48 hours with 100 ng/ml of TNF, 100 ng/ml IL-17, and 5 μg/ml dsRNA or their combination. (D) NHBE were stimulated with 5 μg/ml dsRNA and 50 ng/ml IL-17 for 48 hours and treated with 4 mM ATP during the last hour. Protein expression of IL-1F6 and IL-1F9 in supernatant was analyzed by (A, B, and D) ELISA and (C) Western blot, and (C) protein expression in cell lysate was analyzed by Western blot using rat anti-human IL-1F9 mAb or biotinylated goat anti-human IL-1F6 antibody. (A) Results shown are mean ± SEM of four to seven independent experiments. (C) The results are representative of four separate experiments.

Figure 4.

Induction of IL-1F6 and IL-1F9 in differentiated human bronchial epithelial cells. NHBE were differentiated at an air–liquid interface and then stimulated with 50 ng/ml of TNF, 50 ng/ml IL-17, and 2.5 μg/ml dsRNA or their combination for (A) 24 hours and (B and C) 48 hours. (A) The expression of mRNAs for IL-1F6 and IL-1F9 was analyzed by real-time PCR, (B) protein expression of IL-1F9 in supernatant was analyzed by ELISA and (C) protein expression in cell lysate was analyzed by Western blot. Results shown are mean ± SEM of (A) eight and (B) six independent experiments. (C) The results are representative of three separate experiments.

Figure 5.

IL-1F9 induces the expression of cytokines and chemokines via its receptor complex, IL-1RL2 and IL-1RAP in human lung fibroblasts. NHLF were incubated for (A) 3 hours or (B) 24 hours with 10–500 ng/ml of IL-1F9. (C) Primary normal human lung fibroblasts (NHLF) were incubated with 500 ng/ml of heat-inactivated IL-1F9 or polymyxin B (PMB)-treated IL-1F9 for 3 hours. (D, E) NHLF were transfected with small interfering RNA (siRNA) against control RNA, IL1RL2, or IL1RAP at 20 nM for 48 hours and then stimulated with 250 ng/ml IL-1F9 or 1 ng/ml IL-1β for 3 hours. (A, C, D and E) Levels of mRNA expression were determined by real-time PCR and (B) protein concentration was determined by cytometric bead array. Results shown are mean ± SEM of four to five independent experiments; *P < 0.05, **P < 0.005.