The molecular mechanisms underlying the initiation of innate and adaptive proallergic Th2-type responses in the airways are not well understood. IL-33 is a new member of the IL-1 family of molecules that is implicated in Th2-type responses. Airway exposure of naive mice to a common environmental aeroallergen, the fungus Alternaria alternata, induces rapid release of IL-33 into the airway lumen, followed by innate Th2-type responses. Biologically active IL-33 is constitutively stored in the nuclei of human airway epithelial cells. Exposing these epithelial cells to A. alternata releases IL-33 extracellularly in vitro. Allergen exposure also induces acute extracellular accumulation of a danger signal, ATP; autocrine ATP sustains increases in intracellular Ca2+ concentration and releases IL-33 through activation of P2 purinergic receptors. Pharmacological inhibitors of purinergic receptors or deficiency in the P2Y2 gene abrogate IL-33 release and Th2-type responses in the Alternaria-induced airway inflammation model in naive mice, emphasizing the essential roles for ATP and the P2Y2 receptor. Thus, ATP and purinergic signaling in the respiratory epithelium are critical sensors for airway exposure to airborne allergens, and they may provide novel opportunities to dampen the hypersensitivity response in Th2-type airway diseases such as asthma.
Th2-type immune responses are important for the control of helminth infection, and they are responsible for the development of asthma and allergic inflammation in mucosal organs (1). In genetically susceptible individuals, asthma can be induced and/or exacerbated by environmental stimuli, such as allergens (e.g., house dust mite, molds, and cockroaches), infection with viruses, and airborne pollutants (2, 3). In addition to the Th2 cell-mediated adaptive immune response, asthma is strongly influenced by the innate immune responses from airway cells, such as epithelial cells, mast cells, NKT cells, and dendritic cells (DCs) (4, 5). However, the immunological and molecular mechanisms linking environmental exposure and activation of innate and adaptive Th2-type immune responses in the airways remain enigmatic.
Infection and tissue injury induce inflammation and immune responses (6). During an infection, microorganisms initiate a series of inflammatory responses through their pathogen-associated molecular pattern molecules. Th2-type immune responses can be induced by an experimental allergen, OVA, contaminated with a low-dose LPS (7) or by an allergen’s structural capacity to stimulate the TLR4 (8). Alternatively, immune cells react to tissue injury by recognizing the molecules that are normally located inside the cell, but are released by injured cells (9). These damage-associated molecular patterns (DAMPs) interact predominantly with host pattern recognition receptors and induce inflammation and immune responses. A classic Th2-type adjuvant, alum, likely uses this injury-mediated mechanism by inducing the release of uric acid (10). However, airborne allergens or environmental pollutants generally do not infect hosts and are unlikely to injure tissues. Thus, when mammals are exposed to allergens or pollutants, do these infection or tissue injury models apply to the development of Th2-type immune responses?
Among the most potent molecules of the innate immune system are the IL-1 family members (11); these cytokines, such as IL-1, IL-18, and IL-33, are evolutionarily ancient and are involved in regulating innate and adaptive immune responses. IL-33 (or IL-1F11) is a ligand of the orphan T1/ST2 receptor (12); it was first described as a NF abundantly expressed in the nucleus of endothelial and epithelial cells (13–16). In vivo systemic administration of IL-33 to mice profoundly alters immunity and inflammation, including lung and gastrointestinal eosinophilia and increased levels of serum IgE and IgA (12). IL-33 is implicated in diseases, for example, asthma (17), anaphylactic responses (18), and cardiovascular and rheumatoid diseases (19). Cells in barrier tissues constitutively express and store IL-33, suggesting its central role in mucosal immunity (19). IL-33 drives production of cytokines and chemokines by Th2 cells, mast cells, basophils, eosinophils, NKT cells, and NK cells (12, 20–29). More recent studies reported that IL-33 can induce proliferation of and Th2 cytokine production by a novel non-T/non-B cell population (30–32). Thus, IL-33 may be important in innate and adaptive Th2-type immune responses. However, the mechanisms for the synthesis, processing, and release of IL-33 remain poorly understood (19). Unlike IL-1β, pro–IL-33 (i.e., full-length 31-kDa protein) has biological activity (33–35), and IL-33 does not require proteolytic maturation by caspase-1 (33, 34). Thus, IL-33 may be an “alarmin” that is released during necrotic cell death and associated with infection or tissue injury (33, 36).
Lung, skin, and intestinal epithelial cells that interact with the external environment produce antimicrobial molecules, cytokines, and chemokines that are essential for innate and adaptive immunity (37). In this study, we sought to identify the mechanisms for the innate immune regulation of airway Th2-type responses that are induced by exposure to natural environmental allergens. In humans, an association between fungal exposure, in particular to Alternaria alternata, and asthma is recognized clinically and epidemiologically (38, 39). Severe asthma and life-threatening acute exacerbations of asthma in humans have been associated with increased airborne exposure to Alternaria (40–42). Thus, we used Alternaria to provoke Th2-type immune responses relevant to human diseases. Airway exposure of naive mice to Alternaria induces rapid secretion of IL-33 into the airways and subsequent Th2-type cytokine production. In response to Alternaria allergens, airway epithelial cells translocate nuclear IL-33 and actively release it into the extracellular milieu. ATP-mediated activation of a P2 purinergic receptors and sustained increases in intracellular calcium concentration ([Ca2+]i) are required for this IL-33 secretion in vitro and in vivo. Thus, the airway epithelium responds to noncytolytic “stress” induced by components within our atmospheric environment by releasing ATP, leading to Th2-type inflammatory or possibly homeostatic immune responses.
Materials and Methods
Mice and cells
Animals were bred and maintained under specific pathogen-free conditions; all animal experiments were done with the approval of and in accordance with regulatory guidelines and standards set by the Institutional Animal Care and Use Committee of Mayo Clinic–Rochester. BALB/c, C57BL/6, Rag1−/− (B6.129S7-Rag1tm1Mom/J), P2X7R−/− (B6.129P2-P2rx7tm1Gab/J), and P2Y2R−/− (B6.129P2-P2ry2tm1bhk/J) were from The Jackson Laboratory. Myd88−/− mice on the C57BL/6 background were provided by Dr. Larry Pease (Mayo Clinic–Rochester). ST2−/− (Il1rl1−/−) mice on the BALB/c background (43) were provided by Dr. Andrew N. McKenzie (Medical Research Council Laboratory of Molecular Biology, Cambridge, U.K.). Female mice (7–13 wk old) were used. Normal human bronchial airway epithelial (NHBE) cells were maintained in serum-free bronchial epithelial cell growth medium (BEGM) (both from Lonza); cells were used within three passages.
Reagents and Abs
Escherichia coli A. alternata and Oriental cockroach were from Greer Laboratories and they contained 0.003 μg/mg and 1.4 μg/mg endotoxin, respectively. Rabbit anti-human IL-33 and rabbit anti-human high mobility group box-1 (HMGB1) were from MBL International and Abcam, respectively. Rabbit anti-P2X7R Ab was from Proteintech Group, and rabbit anti-P2Y2
Alternaria- and LPS-induced airway inflammation
Mice were lightly anesthetized with i.p. injection of tribromoethanol (Avertin) and 50 μl PBS or Alternaria extract (50 μg for BALB/c background and 100 μg for C57BL/6 background) or LPS (1 μg) in 50 μl PBS were intranasally administered (44). In some experiments, P2 purinergic receptor antagonists, oATP (4 mM) or suramin (2 mM), were mixed with the Alternaria extract, and this mixture was administered to the airways. Mice were killed by an overdose of pentobarbital. After cannulating the trachea, the lungs were lavaged with HBSS (1.0 ml). The supernatants of bronchoalveolar lavage (BAL) fluids were collected and stored at −20°C for cytokine assays. Whole lungs were homogenized in 1.0 ml PBS. The homogenates were centrifuged at 10,000 × g
Detection of IL-33 by immunohistochemistry and by confocal microscopy
NHBE cells were cultured on Lab-Tek 2 chamber slides (Fisher Scientific). After stimulation with Alternaria extract (50 μg/ml), LPS (1 μg/ml), poly(I:C) (10 μg/ml), ionomycin (1 μM), or thapsigargin (3 μM) for 4 h, the cells were incubated with GolgiPlug (BD Pharmingen) for 30 min at 4°C. In some experiments, NHBE cells were cultured with Alternaria extract plus the calcium chelators EGTA (1 mM) or BAPTA-AM (50 μM). The slides were fixed and permeabilized by Cytofix/Cytoperm reagents (BD Pharmingen) for 20 min at 4°C and then washed with BD Perm/Wash buffer for 30 min at room temperature. Fixed cells were blocked with 5% normal rabbit serum (Sigma-Aldrich) for 1 h and stained overnight with rabbit anti-human IL-33, rabbit anti-HMGB1, or normal rabbit IgG at 4°C. To detect P2 purinergic receptors, cells were stained overnight with anti-P2X7R, anti-P2Y2R, or normal rabbit IgG at 4°C. For immunofluorescence, the cells were incubated with FITC-conjugated goat anti-rabbit IgG for 2 h at room temperature, washed in BD Perm/Wash buffer for 30 min, and mounted in Vectashield mounting medium with DNA-binding dye, DAPI (Vector Laboratories). Fluorescent images were visualized using a confocal microscope (LSM510 confocal microscope), and digital images (512 × 512 pixels, ×800 magnification) were captured by using the KS400 image analysis system (both Carl Zeiss). The threshold for each negative control image was calibrated to a baseline value without positive pixels. All images were processed using the Zeiss LSM image browser.
Cytokine production and release by NHBE cells
NHBE cells were seeded (3 × 104 cells/well) in a 24-well tissue culture plates and grown until 80% confluence, usually 4 d. These cells were then stimulated with AlternariaAlternaria extract. Total cellular IL-33 was recovered with five freeze/thaw cycles or treating cells with 0.5% Nonidet P-40.
Gene knockdown in NHBE cells
When 30–50% confluent, NHBE cells were transfected with small interfering RNAs (siRNAs) targeting P2X7R or P2Y2R or control RNA at 5 nM using HiPerFect transfection reagent (Qiagen). The transfected cells were grown for 48 h and then stimulated with Alternaria extract (50 μg/ml) for 2 h. Target gene knockdown was verified by examining expression of P2X7R and P2Y27R (Hs00175721_m1), P2Y2
Immunoblot for IL-33
NHBE cells (1 × 107 cells/ml) were lysed with RIPA lysis buffer (Santa Cruz Biotechnology). Culture supernatants from NHBE cells, which were treated with five freeze/thaw cycles (45) or stimulated with Alternaria extract for 2 h, were also obtained. Cell lysates and supernatants were concentrated by lyophilization. The samples were boiled for 10 min and aliquots equivalent to 1.3 × 105
Cell viability assays
Alternaria extract (50 μg/ml) for 2 or 8 h, NHBE cells were incubated with 2 μM calcein AM and 4 μM EthD-1 for 30 min at room temperature. Using fluorescence microscopy, intact (calcein AM positive and EthD-1 negative) and damaged (EthD-1 positive) cells in five randomly chosen fields were counted and expressed as percentage of cells over the total cells (≥500 cells were counted).
Measurement of [Ca2+]i
NHBE cells were seeded at low density on coverslip chamber slides for 48–72 h in BEGM. In some experiments, cells were treated with P2X7R siRNA, P2Y22+]i was measured as the ratio of fluorescence emitted at 510 nm when the cells were alternately excited at 340 and 380 nm. Alternaria extract and P2R antagonists were introduced by single-pass, continuous-flow perfusion. [Ca2+]i
ATP measurements in cell culture media
To measure ATP levels in cell culture supernatants, NHBE cells were treated with medium alone, Alternaria extract (50 μg/ml), or Oriental cockroach extract (50 μg/ml) for periods ≤32 min in a 24-well tissue culture plate. Cell culture media was collected and immediately placed on ice. ATP concentrations were measured with an ATP determination kit (Bioassay Systems) and a luminometer, following the manufacturer’s instructions.
CD4+ T cell cytokine production in response to NHBE cell lysates
Isolated CD4+ T cells were cultured with BM-derived DCs and cell lysates as previously described (29). Briefly, CD4+ T cells isolated from spleens of BALB/c mice were seeded at 1 × 106
All data are reported as the mean ± SEM from the indicated number of replicates. Two-sided differences between two samples were analyzed with the Mann–Whitney U test or Student t test. Multiple comparisons between treatment and control conditions were performed by ANOVA. The p values < 0.05 were considered significant.
Exposure to Alternaria induces IL-33 release and Th2 cytokine production in mouse airways
To investigate the immunological mechanisms of asthma and Th2-type airway immunity, we used a naive mouse model and administered Alternaria extract (50 μg/dose) into the airways of mice. A bacterial product and TLR4 agonist, namely LPS (1 μg/dose), acted as control. The Alternaria extract contained minimal endotoxin (3 ng/mg, 0.15 ng/dose). Six hours later, Th2 cytokine levels, including IL-5 and IL-13, were increased in Alternaria-exposed lungs of BALB/c mice (Fig. 1A); by 24 h, these cytokine levels decreased. IL-5 and IL-13 levels did not increase in LPS-exposed mice. IL-4 was undetectable in LPS- and Alternaria-exposed BALB/c mice. Interestingly, within 1 h after receiving Alternaria extract, IL-33 levels increased markedly in BAL fluids, preceding the increases in IL-5 and IL-13; LPS did not induce IL-33 in BAL fluids. After receiving LPS, IL-1β levels in BAL fluids increased from 6 to 24 h; Alternaria induced small increases in IL-1β levels. Similarly, in C57BL/6 mice (Fig. 1B), IL-33 levels increased markedly in BAL fluids within 1 h after receiving Alternaria extract; the levels of IL-5 and IL-13 were increased by 6 and 12 h, respectively. Thus, respiratory exposure to the fungus increases airway IL-33 levels that are followed by increases in Th2 cytokines in both BALB/c and C57BL/6 mice; LPS increases IL-1β levels, but not IL-33 or Th2 cytokines.
To examine the role of IL-33 in Th2 cytokine responses, we used mice deficient in the IL-33 receptor T1/ST2 (ST2−/−) (46). Alternaria-induced increases in IL-5 and IL-13 were reduced to baseline levels (i.e., PBS) in ST2−/− mice (Fig. 2A), but increases in IL-6 or IL-33 were not affected. The production of IL-5 and IL-13 in Alternaria-exposed mice was similar in Rag1−/− and wild-type mice (Fig. 2B), suggesting that these Th2-type cytokine responses are independent of adaptive immunity. The production of IL-33 was not significantly affected in Rag1−/− mice. An adaptor protein, MyD88, mediates signal transduction by most TLRs (47); MyD88 is also required for signaling of IL-1 family cytokines, such as IL-1β and IL-33 (48). Exposing MyD88−/− mice to Alternaria extract abolished IL-5 and IL-13 production (Fig. 2C), consistent with the potential involvement of IL-33, but the increase in BAL IL-33 was not affected. Thus, when mice are exposed to Alternaria, the MyD88-independent release of IL-33 likely mediates production of Th2-type cytokines in the airway mucosa through an innate mechanism or mechanisms.
Biologically active full-length IL-33 is localized in nuclei of airway epithelial cells
In resting conditions, epithelial cells in mucosal organs and vascular endothelial cells are probably the dominant cells expressing and storing IL-33 in their nuclei (15, 16). We used immunohistochemistry to verify localization of IL-33 in the nuclei of airway epithelial cells in human nasal tissues, especially in basal layer cells (Fig. 3A). To dissect the roles of epithelial cell-derived IL-33 in Th2-type immune responses, we cultured NHBE cells. By double-staining NHBE cells for IL-33 and DAPI, IL-33 was mainly localized in each nucleus with faint staining in the cytoplasm (Fig. 3B). As a control, we used HMGB1 protein (45), which is a prototypic nuclear alarmin (49). HMGB1 localized in the nuclei of NHBE cells (Fig. 3B). Immunoblot analysis revealed that IL-33 in NHBE cell lysates migrated as a 30- to 31-kDa band (Fig. 3C), suggesting that it is the full-length proform and unprocessed by caspase-1 (33–35). To examine whether this full-length IL-33 is biologically active, we incubated cocultures of CD4+ T cells and DCs with NHBE cell lysates. These lysates induced production of IL-5, IL-13, and IFN-γ by the CD4+ T cells (Fig. 3D). Treating the cells and lysates with anti-ST2 (i.e., IL-33 receptor) Ab or anti–IL-33 Ab abolished IL-5 production (p < 0.01, n = 4); these Abs partially inhibited IL-13 production (p < 0.05, n = 4). In contrast, IFN-γ production was unaffected, implicating roles for non–IL-33 molecules (e.g., HMGB1, uric acid) in the lysates that promote IFN-γ production.
IL-33 is released extracellularly when airway epithelial cells are exposed to allergen extracts, but not to TLR agonists
To examine whether NHBE cells release nuclear IL-33 extracellularly after innate immunological stimulation, we incubated cells with extracts of common airborne allergens, Alternaria and Oriental cockroach (38, 50), or with TLR ligands, including zymosan (TLR2 ligand), poly(I:C) (TLR3 ligand), and LPS (TLR4 ligand). Immunoreactive IL-33 was mainly localized to the nuclei of resting NHBE cells (Fig. 4A). When cells were exposed to Alternaria, IL-33 appeared in the cytoplasm, but HMGB1 remained in the nuclei. Furthermore, unlike Alternaria, poly(I:C) did not induce cytoplasmic localization of IL-33.
When NHBE cells were stimulated with Alternaria extract, IL-33 was released rapidly and reached a plateau by 2 h (Fig. 4B); in contrast, IL-6 production did not reach a plateau by 8 h. NHBE cells incubated with medium for 2 h released detectable IL-33 (Fig. 4C). When cells were incubated with Alternaria or Oriental cockroach extracts, IL-33 levels increased in the supernatants (p < 0.01); in contrast, exposure to various TLR ligands did not induce IL-33 release. When NHBE cells were incubated with zymosan or poly(I:C) for 8 h, they produced high IL-6 levels (p < 0.01); when cells were incubated with Alternaria, Oriental cockroach, or LPS, they produced low levels of IL-6 (p < 0.05). The IL-33 in the NHBE cell supernatants migrated as a 30- to 31-kDa band (Fig. 4D), suggesting that IL-33 is released as a noncleaved, full-length protein. Thus, IL-33 and IL-6 are released from NHBE cells by allergens and TLR ligands, respectively.
IL-33 has been considered an alarmin that is released during cellular injury or necrotic cell death (33, 36). To address whether injury was responsible for extracellularly released IL-33 induced by Alternaria, we used indicators of cell membrane integrity, including LDH activity in cell-free supernatants and staining with the membrane-impermeable nucleic acid dye, EthD-1. When NHBE cells were exposed to medium or to Alternaria extract for 2 h, LDH activity was undetectable in the supernatants (Fig. 4E); when the cells were treated with freeze/thaw cycles or were exposed to a detergent, Nonidet P-40, LDH release was robust. No differences were observed in the permeability to EthD-1 between the cells incubated with medium alone or those incubated with Alternaria extract for ≤8 h (Fig. 4F). About 90% of NHBE cells were also alive with medium alone or Alternaria extract, as judged by conversion of calcein AM to fluorescent calcein. Overall, when NHBE cells are exposed to Alternaria, stored IL-33 was rapidly and actively released without apparent cellular injury.
Allergen exposure increases [Ca2+]i through extracellular release of ATP
We next addressed how airway epithelial cells might “sense” the allergens and release stored IL-33. Under stress conditions, many plant and animal cells release ATP via lytic or nonlytic mechanisms, and extracellular ATP can be a danger signal and a mediator of inflammation (51). When we exposed NHBE cells to fresh medium, within 60 s after exchange of medium we detected ATP in the cell supernatants (Fig. 5A). ATP levels returned to baseline levels within 4 min, likely because of ecto-ATPase activity (52). Importantly, when we added allergen extracts (e.g., Alternaria and Oriental cockroach) to NHBE cells, the peak extracellular ATP levels were ∼2.5-fold higher than with medium alone, and these elevated ATP levels were sustained over a longer time course.
Most of the NHBE cells exposed to Alternaria extract showed a rapid and robust increase in [Ca2+]i (Fig. 5B). [Ca2+]i increases started simultaneously in nuclear and perinuclear regions (panel 3) and expanded to the entire cell (panel 5) by 333 s. By 300 s the [Ca2+]i gradually increased with a peak response between 400 and 600 s (Fig. 5C). When extracellular calcium was chelated with EGTA, the Alternaria-induced [Ca2+]i response was abolished, suggesting that Ca2+ influx mainly mediates the response. Addition of UTP to EGTA-treated, Alternaria-exposed NHBE cells induced a rapid and sharp [Ca2+]i response, suggesting that the pathways to release Ca2+ from intracellular sources are intact. At 400 s, the Alternaria-induced [Ca2+]i in NHBE cells was reproducibly elevated compared with basal levels of [Ca2+]i (p < 0.01).
We next addressed the link between increases in extracellular ATP, activation of P2 purinergic receptors, and the regulation of [Ca2+]i. P2 receptors mediate the effects of extracellular ATP on cell communication (53) and are comprised of G protein-coupled P2Y receptors that regulate Ca2+ release from internal stores (54, 55) and ion channel forming P2X receptors that function as nonselective cation channels (56). We investigated the roles of P2 purinergic receptors by pretreating NHBE cells with two wide-spectrum antagonists, namely oATP (57) and PPADS (58). Both inhibitors suppressed the Alternaria-induced [Ca2+]i response (p < 0.01) (Fig. 6A); these agents did not affect the basal [Ca2+]i response. After several P2XR and P2YR family members were evaluated through various antagonists, we focused on P2X7R and P2Y2R because of their known affinities for ATP and because airway epithelial cells express these receptors (Fig. 6B). siRNA-mediated knockdown of P2X7R in NHBE cells led to a 93% decrease in P2X7R mRNA and inhibited the Alternaria-induced [Ca2+]i response by 80% (p < 0.01) (Fig. 6C). Similarly, siRNA-mediated knockdown of P2Y2R in NHBE cells led to a 95% decrease in P2Y2R mRNA and inhibited the Alternaria-induced [Ca2+]i response by 75% (p < 0.01). siRNA targeting P2X7R or P2Y2R did not affect basal [Ca2+]i. Thus, autocrine release of ATP and activation of P2 purinergic receptors appear to be involved in sensing exposure to aeroallergens and the subsequent increase in [Ca2+]i in airway epithelial cells.
ATP-mediated increases in [Ca2+]i induce IL-33 release
To examine the roles of ATP-induced [Ca2+]i increases in IL-33 release, NHBE cells were incubated with Alternaria extract in the presence of the Ca2+ chelators EGTA or BAPTA-AM. By immunohistochemistry, EGTA treatment blocked the Alternaria-induced transport of IL-33 from nucleus to cytosol (Fig. 7A). EGTA and BAPTA-AM inhibited the Alternaria-induced IL-33 release into the extracellular media (p < 0.01 and p < 0.05, respectively, n = 5), whereas basal IL-33 release with medium was unaffected (Fig. 7B). Conversely, to examine whether increases in [Ca2+]i are sufficient to induce IL-33 release, NHBE cells were incubated with a calcium ionophore, ionomycin, or with a Ca2+-ATPase inhibitor, thapsigargin, which depletes Ca2+ from reticular stores, leading to the activation of Ca2+ channels and Ca2+ entry (59). Ionomycin treatment localized IL-33 to cytoplasmic organelles with an apparent vesicular structure (Fig. 7C); in contrast, ionomycin did not affect the localization of nuclear HMGB1. Thapsigargin treatment showed IL-33 localization in the cytoplasm similar to ionomycin and did not affect localization of HMGB1. Furthermore, IL-33 was detected in the supernatants of NHBE cells treated with ionomycin or thapsigargin (p < 0.05) (Fig. 7D). Thus, increased [Ca2+]i is likely necessary and sufficient to release IL-33 from NHBE cells. Importantly, treatment of NHBE cells with wide-spectrum inhibitors for P2 purinergic receptors (i.e., oATP and suramin) (60) or enzymatic removal of ATP from the incubation medium by exogenous ATPase (i.e., apyrase) abrogated Alternaria-induced extracellular release of IL-33, suggesting critical roles for ATP and P2 purinergic receptors in IL-33 release (Fig. 7E). Indeed, depletion of specific transcripts for P2X7R or P2Y2R by siRNA inhibited IL-33 release from NHBE cells in response to Alternaria (p < 0.05, n = 5) (Fig. 7F).
ATP mediates IL-33 release and Th2-type innate immune responses in the airways of mice exposed to Alternaria
To test the importance of ATP as an innate sensor for allergen exposure in vivo, we used an airway model that exposed nonsensitized naive mice to Alternaria. As shown in Fig. 1 above, intranasally administered Alternaria extract rapidly increased BAL IL-33 within 1 h, followed by increased levels of lung IL-5 and IL-13 from innate sources by 6 h. We now used this model to examine the effects of blocking of P2 purinergic receptors in vivo with the same inhibitors as in the in vitro studies. Intranasally administered oATP plus Alternaria inhibited the Alternaria-induced increases in BAL IL-33 levels (p < 0.05) (Fig. 8A) and reduced both IL-5 and IL-13 production in the lungs (p < 0.05) (Fig. 8B). Similarly, mice exposed intranasally to Alternaria plus suramin showed minimal IL-33 release and IL-5 and IL-13 production.
To determine whether deficiency in one of the P2 purinergic receptors is sufficient to modulate the innate Th2-type response to Alternaria, we administered Alternaria extract into the airways of P2X7−/− and P2Y2−/− mice (Fig. 8C). Production of both IL-5 and IL-13 was not affected in P2X7−/− mice. In contrast, production of IL-5 in the lungs was reduced in P2Y2−/− mice by 90% as compared with wild-type mice (p < 0.01). Similarly, IL-13 levels decreased by 70% in P2Y2−/− mice (p < 0.05). Thus, disrupting the ATP signaling pathway at the receptor level impairs allergen-induced IL-33 release and subsequent Th2-type immune responses in the airway mucosa.
IL-1 family cytokines (e.g., IL-1β, IL-33) may be sorted into two groups, depending on their production, regulation, extracellular release, and their cellular sources (11). Pro–IL-1β is synthesized when monocytes or macrophages are exposed to microbial or tissue injury products, and it is released extracellularly after proteolytic processing to mature IL-1β by caspase-1. In contrast, IL-33 is constitutively expressed and stored in the nucleus of epithelial and endothelial cells. IL33 and IL1RL1 (i.e., ST2) genes have been implicated in asthma in a recent large-scale genome-wide association scan study (61). Increased expression of IL-33 by epithelial cells is also observed in patients with asthma (62). However, the molecular mechanisms involved in IL-33 production and release remain enigmatic. The major finding in this report is that when airway epithelial cells are exposed to environmental allergens, they actively and rapidly release IL-33. Sustained increases in [Ca2+]i are sufficient and necessary to translocate and release IL-33. Furthermore, extracellular accumulation of ATP appears pivotal in regulating the Ca2+ response and subsequent IL-33 release. In mice exposed to an environmental allergen, in vivo blockade of the P2 purinergic receptor pathways inhibited IL-33 release and Th2 cytokine production, suggesting biological significance of the ATP-mediated IL-33 release. Our findings are consistent with reports that showed biological activity for unprocessed full-length pro–IL-33 and showed that the processing and release of IL-33 probably does not involve caspase-1 (35). Thus, airway epithelial cells may use ATP to sense noncytolytic stress in the air we breathe and to initiate Th2-type immune responses by releasing preformed, full-length IL-33.
Previous models proposed IL-33 as an alarmin, which is released during necrotic cell death associated with tissue damage or infection, similarly to a prototypic alarmin, HMGB1 (36). In theory, potential cytotoxic effects of allergen extracts might release IL-33 from airway epithelial cells. However, direct toxic effects appear unlikely because cell membrane integrity was not compromised after cells were exposed to Alternaria extract and after they released IL-33. Pharmacological inhibitors of P2 purinergic receptors and Ca2+-chelating agents also abolished IL-33 release in vitro and in vivo.
Extracellular ATP serves as a danger signal to alert the immune system of tissue stress or damage (63). Increased levels of ATP are observed in BAL fluids of patients with asthma or sensitized mice challenged with an Ag (64). Neutralization of ATP inhibits Th2 sensitization to inhaled Ags and ongoing Th2-type airway inflammation (64). We also found that exposure to aeroallergens induces rapid extracellular release of ATP (Fig. 5), and neutralization of ATP or blocking of the P2 purinergic pathway inhibits IL-33 release and early innate Th2 type immune responses to an inhaled allergen (Figs. 7, 8). Taken together, these findings suggest that extracellular ATP plays pivotal roles in various stages of Th2-type immune responses and inflammation in the airways.
By immunohistochemistry, both HMGB1 and IL-33 were localized in the nuclei of NHBE cells (Fig. 3). However, HMGB1 did not translocate to the cytoplasm when epithelial cells were exposed to Alternaria extract or to ionomycin, suggesting that the Ca2+ signal is unlikely to regulate HMGB1 release. Activated macrophages secrete HMGB1 after hyperacetylation of lysine residues (65), and necrotic cells release HMGB1 during cellular damage (45). Thus, distinct intracellular mechanisms may regulate the extracellular release of alarmin-like proteins, and different classes of molecules may be released depending on the cells’ conditions or cell types. Generally, in vivo cell necrosis triggers an acute inflammatory response by releasing DAMPs (66). Our freeze/thaw airway epithelial cell lysates were highly immunogenic and induced production of Th1 (IFN-γ) and Th2 cytokines (IL-5, IL-13) in the DC and CD4+ T cell coculture system. Thus, because aeroallergens induced ATP release, but no apparent cellular injury, they may trigger IL-33 release without DAMP release, resulting in a Th2-type immune response. Conversely, massive cellular damage to airway epithelial cells by microbial infection or toxic chemicals may release DAMPs as well as IL-33, resulting in Th1 and Th2 responses or a Th1-dominant response. The selective release of alarmin-like molecules from the airway epithelium appears to modulate the magnitude and nature of inflammation.
IL-33 release by airway epithelial cells may involve different innate immunological stimuli other than conventional TLRs. In this study, while TLR agonists induced IL-6 production by airway epithelial cells, TLR agonists in vitro or LPS in vivo did not induce IL-33 release. Furthermore, in mice exposed to Alternaria, IL-33 release was induced even in MyD88-deficient mice. Proinflammatory stimuli, such as IL-1β and TNF-α, inhibit transcription of IL-33 in endothelial cells (16). Thus, IL-33 production and release are independent of, or rather negatively regulated by, the TLRs and the NF-κB signaling pathway. Instead, in airway epithelial cells, the ionomycin- or thapsigargin-induced increases in [Ca2+]i were sufficient to translocate IL-33 from nucleus to cytoplasm and to the extracellular milieu. In human embryonic kidney (HEK-293) cells transfected with IL-1β genes, sustained increases in [Ca2+]i produced rapid secretion of mature IL-1β and unprocessed pro–IL-1β (67). In monocytes and macrophages, Ca2+ was involved in P2X7R-mediated secretion of mature IL-1β, while K+ efflux was involved in enzymatic processing of pro–IL-1β to mature IL-1β (67). Thus, the Ca2+-dependent secretory pathway/apparatus for IL-1 family cytokine release may be ubiquitously expressed regardless of the cell type, and increases in [Ca2+]i may be critical in secretion, but not processing, of IL-1 family molecules. Because IL-33 requires no proteolytic processing by caspase-1, the increases in [Ca2+]i may be sufficient to mobilize and release IL-33 from airway epithelial cells.
As potential therapeutic targets, the molecules playing major roles in Ca2+ signal regulation and IL-33 release are of interest. We found that ATP is released within 60 s when airway epithelial cells are exposed to aeroallergens. Generally, ATP is stored at millimolar concentrations within cells, and disrupting plasma membrane integrity leads to ATP extracellular release (68). In both animals and plants, ATP is also released in noncytolytic conditions, such as membrane deformation, mechanical stress, osmotic stress, and exposure to proteases (69–71). Thus, ATP can be used to communicate tissue distress or injury to the immune system, promoting appropriate responses, for example, inflammation, restoration of homeostasis, and adaptation to the altered cellular conditions or environment. Activation of P2X7R by ATP facilitates the rapid influx of extracellular Na+ and Ca2+ and efflux of intracellular K+ (72). In optic nerve glia, low ATP concentrations mediate the increase in [Ca2+]i through P2Y2R and, at high concentrations, P2X7R is also activated, resulting in robust and sustained increases in [Ca2+]i (73). In our study of human airway epithelial cells, both P2X7R and P2Y2R were involved in the Ca2+ response and IL-33 release in vitro. Broad-spectrum inhibitors of P2 purinergic receptors and mice deficient in the P2Y2R gene showed a marked inhibition of innate Th2 cytokine production in response to Alternaria in vivo. Thus, the P2Y2R and perhaps the P2X7R play major roles in detecting the danger signal, extracellular ATP, and in triggering IL-33 release and the subsequent Th2-type responses. Some caution may be necessary to interpret the roles of specific receptors, however. Whereas the involvement of P2Y2R was observed in both human epithelial and mouse in vivo models, there appears to be discrepancy regarding the involvement of P2X7R. Gene-deficient mice might have unknown immunological abnormalities that we may not be aware of, or they might compensate for the deficiency in one gene (e.g., P2X7R) by upregulating other genes. The coupling of receptors to their effector molecules may be different between mice and humans. Furthermore, the mouse in vivo system may involve other cell types more than airway epithelial cells, which may overcome the deficiency in one cell type. Any of these mechanisms may produce the different observations between the mouse and human models.
We were surprised by the rapid (by 12 h after allergen exposure) production of IL-5 and IL-13 in the lungs of naive mice; this cytokine response was independent of adaptive immunity. Endogenous IL-33 and its receptor, ST2, mediated this “innate” Th2 cytokine response. Robust IL-6 production was also observed within 12 h of Alternaria exposure (Fig. 2), suggesting that this cytokine may be involved in Th2-type immune responses of the airways to inhaled allergens (74). A major question remains regarding the cellular source of innate IL-5 and IL-13. Recently, exogenous IL-25 and IL-33 or infection with helminths induced proliferation of novel non-T/non-B cells that produced abundant IL-5 and IL-13 (12, 30, 31, 37, 75, 76). These innate IL-13–expressing cells are likely dispersed throughout the animals’ bodies (32). Indeed, we found comparable lineage−CD25+CD44+ cells in lungs of naive mice. These lineage−CD25+CD44+ cells expressed Sca-1, IL-7Rα, ST2, ICOS, and MHC class II, presented lymphocytic morphology, and produced a large quantity of IL-5 and IL-13 when cultured with IL-33 in vitro (data not shown). These novel non-T/non-B lymphocytes cells may mediate early innate Th2-type immune responses after airway exposure to airborne allergens. Further studies are necessary to verify this speculation. Our studies provide a mechanistic explanation for the initiation phase of such innate Th2-type immunity, namely the ATP- and Ca2+-dependent release of stored IL-33 from structural cells. In recent years, considerable progress has been made to understand better the innate immune responses to infection and tissue injury (6, 66). Our observations add to this knowledge. The airway’s innate Th2-type responses may be triggered by the epithelial cells’ ability to sense noncytotoxic cellular stress by means of ATP. Thus, some chronic inflammatory conditions in humans, such as asthma, may be better understood in this broader context of inflammation rather than the classic models of infection and injury. Manipulating tissue stress responses by intervening in the ATP/P2 purinergic receptor pathways may prevent development and exacerbation of such diseases.
The authors have no financial conflicts of interest.
We thank Dr. Larry R. Pease and Dr. Andrew N. McKenzie for providing MyD88−/− and ST2−/− mice, respectively. We thank Cheryl R. Adolphson for editorial assistance, LuRaye S. Eischens for secretarial help, and Gail M. Kephart and Peter J. Maniak for technical assistance.
This work was supported in part by National Institutes of Health Grants AI34486 and AI49235 (to H.K.) and DK074010 and HL095811 (to S.M.O.), as well as by the Mayo Foundation.
Abbreviations used in this article:
- bronchoalveolar lavage
- bronchial epithelial cell growth medium
- intracellular calcium concentration
- calcein AM
- calcein acetoxymethyl
- damage-associated molecular pattern
- dendritic cell
- ethidium homodimer-1
- high mobility group box-1
- lactate dehydrogenase
- normal human bronchial airway epithelial
- periodate oxidized ATP
- polyinosinic-polycytidylic acid
- pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid
- small interfering RNA.
- Received September 8, 2010.
- Accepted January 19, 2011.
- Copyright © 2011 by The American Association of Immunologists, Inc.