The IL-1 family of cytokines has recently been expanded by the discovery of seven new members, IL-1F5–11. All of the agonistic IL-1 family cytokines have been reported to share similar signaling pathways. Therefore, the most recently identified agonistic IL-1 family cytokines, IL-1F6, IL-1F8, IL-1F9, and IL-33, are also believed to be involved in inflammation. Initial studies of these latest IL-1 family cytokines suggested that mRNAs for these molecules were expressed in the lungs and in the epithelium. However, regulation and function of IL-1F5–11 in the airway have not been extensively studied. In the present study, we determined the expression profiles of the latest IL-1 family cytokines in primary human bronchial epithelial cells. This study provides the first demonstration that the proinflammatory cytokines TNF and IL-1β, the Th17 cytokine IL-17, and the TLR3 agonist dsRNA induce the expression of both IL-1F6 and IL-1F9 in airway epithelial cells. We have also shown that fibroblasts are primary target cells for IL-1F9, and that IL-1F9 can induce the production of neutrophil activators, neutrophil chemoattractants, and Th17- and dendritic cell-attracting chemokines in lung fibroblasts.
It has been reported that the combination of TNF and IL-17 synergistically enhances the production of neutrophil chemokines IL-8 and CXCL1 in airway epithelial cells (35
). We have recently reported that the Th2 cytokine IL-4 synergistically enhances dsRNA-dependent expression of the dendritic cell activator TSLP in airway epithelial cells (28
). We therefore tested whether combinations of TNF, IL-17, and dsRNA might further enhance the expression of IL-1F6 and IL-1F9 in airway epithelial cells. We found that the combination of TNF and IL-17 or dsRNA and IL-17 synergistically enhanced the expression of IL-1F6 and IL-1F9 (). In contrast, combinations of cytokines including TNF, IL-1β, IL-17, and other TLR ligands showed minimal effects or only additive effects on expression of IL-1F6 and IL-1F9 (data not shown). This suggests that respiratory viral infections that lead to activation of TLR3 may strongly enhance the expression of IL-1F6 and IL-1F9 in the presence of IL-17–producing cells (Th17 cells) in the airway. Although the other TLR ligands that we tested, including Pam3CSK4, LPS, and flagellin, did not strongly activate epithelial IL-1F6 and IL-1F9; these TLR ligands are well known to activate monocytes, macrophages, and dendritic cells to induce production of TNF and IL-1β, which would be expected to activate epithelial production of these cytokines during bacterial infections in vivo
We demonstrate for the first time the production of IL-1F6 and IL-1F9 protein in human primary cells. Surprisingly, IL-1F6 protein was not detected in culture supernatants of activated NHBE, despite detection of the protein in cell lysates ( and ). In contrast, IL-1F9 protein was detected in both cell lysates and supernatants, although levels of the protein were higher in cell lysates ( and ). As is the case with IL-1β and IL-18, IL-1F6 and IL-1F9 lack signal peptides and may require additional signaling for secretion. The mechanism of IL-1β production and secretion has been studied extensively in many laboratories (see Reference [36
]). The TLR-NF-κB pathway is well known as an initial signal for the large accumulation of pro-IL-1β in the cytosol. A second signal for IL-1 release is the activation of the nucleotide-binding oligomerization domain–leucine-rich repeats containing pyrin domain 3 (NLRP3) inflammasome. Activation of NLRP3 triggers the processing of procaspase-1 into mature active caspase-1, which then catalyzes IL-1β processing and release. ATP is known to induce the activation of NLRP3 and the secretion of mature IL-1β via P2X7 receptor-dependent signaling in monocytes. Secretion of IL-18 also involves a similar mechanism (37
). Most recently, Martin and colleagues have shown that secretion of IL-1F6 in murine macrophages also requires two signals, TLR activation and ATP stimulation, using IL-1F6-transfected bone marrow-derived macrophages (33
).We investigated whether ATP is capable of inducing the secretion of IL-1F6 and IL-1F9 in NHBE but found that it did not affect IL-1F6 and IL-1F9 protein secretion after stimulation with dsRNA, IL-17, and their combination in NHBE ( and data not shown). This suggests that the caspase-1-inflammasome pathway may not be involved in the secretion of IL-1F6 and IL-1F9 in human epithelial cells. Future study is required to identify species and/or cell specific differences in the mechanisms of secretion of IL-1F6 and IL-1F9.
A high concentration of IL-1F9 protein was detected in epithelial supernatants, especially after stimulation with dsRNA and IL-17. We next investigated the potential function of IL-1F9 in the airway. We found IL-1RL2 was expressed on epithelial cells and fibroblasts. Although airway epithelial cells expressed IL-1RL2 and responded to IL-1F9, the response was much weaker than in fibroblasts (data not shown). The fact that airway epithelial cells released IL-1F9 protein to the basolateral side of the monolayer (), suggested a potential role for epithelial cell–derived IL-1F9 in the activation of subepithelial cells such as fibroblasts. To this effect we found that IL-1F9 induced the activation of several MAPKs and transcription factors, NF-κB and CREB, in primary human lung fibroblasts (Figure E2). This suggests that epithelial cells may interact with lung fibroblasts via production of IL-1F9 and that the signaling pathway by IL-1F9 in fibroblasts is similar to the signaling pathways used by other IL-1 family cytokines.
Although recombinant IL-1F9 activated lung fibroblasts, it required high concentrations to induce a significant amount of IL-8 production (). To exclude the possibility that this function was the result of contamination by endotoxin or other cytokines, we performed a knock-down experiment for the IL-1F9 receptor. SiRNA against IL-1RL2, which is a specific receptor for IL-1F9, significantly suppressed IL-1F9-dependent IL-8 expression but it did not inhibit IL-1β-dependent IL-8 expression (). In contrast, siRNA against IL-1RAP, which is a shared receptor for IL-1F9 and IL-1β, significantly suppressed both IL-1F9-dependent and IL-1β-dependent IL-8 expression in fibroblasts (). In addition, heat-inactivated IL-1F9 did not activate the induction of IL-8 and PMB did not affect IL-1F9-dependent IL-8 expression in NHLF (). These data suggest that IL-1F9 activates fibroblasts via an IL-1F9 receptor-mediated mechanism. Although the concentration of IL-1F9 required for the response was higher (10
–(500 ng/ml) than that required for IL-1β (), it is in agreement with recent studies showing that recombinant IL-1F9-dependent activation of the NCI/ADR-RES cell line requires 500 ng/ml (9
), and recombinant IL-1F8-dependent activation of synovial fibroblasts requires 5 μg/ml (10
). In the present study, we showed that epithelial cells were able to produce more than 1 ng/ml of IL-1F9 (2.7–16.7 ng/ml). Because the concentration of IL-1F9 in culture supernatants is diluted, the concentration of IL-1F9 in the proximity of the epithelial cells must be significantly higher. Thus, it is possible that production of IL-1F9 from airway epithelial cells is sufficient to activate fibroblasts in the airway, especially myofibroblasts in the epithelial trophic unit immediately adjacent to the lamina reticularis (38
We found that IL-17 enhanced the production of IL-1F9 in the presence of TNF or dsRNA in airway epithelial cells (). Th17 and IL-17 are well known to mediate immunity to extracellular bacteria and initiate neutrophilic inflammation (35
). IL-1F8, which shares the receptor complex with IL-1F9, has been reported to induce production of the neutrophil chemokine IL-8 in human synovial fibroblasts (10
). Therefore, we tested whether IL-1F9 also contributes to neutrophilic inflammation. We found that IL-1F9 induced neutrophil chemokines, IL-8 and CXCL3, and the neutrophil and progenitor cell activator, G-CSF, in lung fibroblasts (). Interestingly, IL-1F9 was also able to induce fibroblasts to produce the chemokine CCL20, which is known as a dendritic cell chemokine and is now also accepted to be a Th17-attracting chemokine. These results suggest that the production of IL-1F9 by epithelial cells and the activation of fibroblasts by IL-1F9 involve feed forward mechanisms to amplify Th17 inflammation and neutrophilic inflammation in the airway. Very recently, Ramadas and colleagues have reported that IL-1F9 was elevated in the lungs of allergen-challenged mice, and intratracheal administration of IL-1F9 induced airway hyperresponsiveness, chemokine production, and neutrophil influx in the lungs of mice (25
). Their data supports that IL-1F9 contributes to the neutrophilic inflammation in the airway. The expression of IL-1F9 in airway epithelial cells from patients with neutrophilic airway inflammatory diseases is therefore worthy of investigation.
In summary, in this study we report that expression of IL-1F9 was induced in airway epithelial cells by stimulation with inflammatory cytokines including IL-17, the prototypic Th17 cytokine, and the TLR3 ligand dsRNA and that these two stimuli acted synergistically to induce expression of the protein. IL-1F9 in turn induced the production of neutrophil-activating and neutrophil-recruiting cytokines and chemokines in lung fibroblasts. Our findings suggest that infection of the airways with respiratory viruses and bacteria along with the recruitment of Th17 cells may greatly amplify neutrophilic inflammation in the airways via the production of IL-1F9 and the activation of fibroblasts.