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Allergens stimulate store-operated calcium entry and cytokine production in airway epithelial cells

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ABSTRACT

Aberrant immune responses to environmental allergens including insect allergens from house dust mites and cockroaches contribute to allergic inflammatory diseases such as asthma in susceptible individuals. Airway epithelial cells (AECs) play a critical role in this process by sensing the proteolytic activity of allergens via protease-activated receptors (PAR2) to initiate inflammatory and immune responses in the airway. Elevation of cytosolic Ca2+ is an important signaling event in this process, yet the fundamental mechanism by which allergens induce Ca2+ elevations in AECs remains poorly understood. Here we find that extracts from dust mite and cockroach induce sustained Ca2+ elevations in AECs through the activation of Ca2+ release-activated Ca2+ (CRAC) channels encoded by Orai1 and STIM1. CRAC channel activation occurs, at least in part, through allergen mediated stimulation of PAR2 receptors. The ensuing Ca2+ entry then activates NFAT/calcineurin signaling to induce transcriptional production of the proinflammatory cytokines IL-6 and IL-8. These findings highlight a key role for CRAC channels as regulators of allergen induced inflammatory responses in the airway.

No MeSH data available.


Cockroach allergen extracts activate store-operated CRAC channels in BEAS-2B cells.(A–C) [Ca2+i] imaging showing responses of individual BEAS-2B cells to cockroach allergen extracts (10 μg/mL) administered in (A) 2 mM Ca2+ ringer’s solution, (B) Ca2+ free ringer’s solution or (C) 2 mM Ca2+ ringer’s solution in the presence of the CRAC channel inhibitor BTP2 (500 nM). (D) Average [Ca2+i] response of the individual cells shown in (A–C). (E–F) Summary of the average rise in [Ca2+i] 600 seconds after addition of cockroach extract (E) and the integral of the [Ca2+]i signal during application of the allergen. (F). (G) Ca2+ imaging trace showing SOCE in BAES-2B cells. SOCE was induced by depleting ER Ca2+ stores with 1 µM thapsigargin in a Ca2+-free Ringer’s solution and readding 2 mM Ca2+ following store depletion. Pre-treating cells with BTP2 (500 nM) strongly inhibits SOCE. (H) Summary of average rise in cytosolic Ca2+ levels 200 seconds after re-addition of 2 mM Ca2+ ringer’s following store-depletion. Data are mean ± SEM of 34-47 cells. Representative of 5 independent experiments. **P < 0.01, ***P < 0.001, Ck. Ext, cockroach extract.
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f1: Cockroach allergen extracts activate store-operated CRAC channels in BEAS-2B cells.(A–C) [Ca2+i] imaging showing responses of individual BEAS-2B cells to cockroach allergen extracts (10 μg/mL) administered in (A) 2 mM Ca2+ ringer’s solution, (B) Ca2+ free ringer’s solution or (C) 2 mM Ca2+ ringer’s solution in the presence of the CRAC channel inhibitor BTP2 (500 nM). (D) Average [Ca2+i] response of the individual cells shown in (A–C). (E–F) Summary of the average rise in [Ca2+i] 600 seconds after addition of cockroach extract (E) and the integral of the [Ca2+]i signal during application of the allergen. (F). (G) Ca2+ imaging trace showing SOCE in BAES-2B cells. SOCE was induced by depleting ER Ca2+ stores with 1 µM thapsigargin in a Ca2+-free Ringer’s solution and readding 2 mM Ca2+ following store depletion. Pre-treating cells with BTP2 (500 nM) strongly inhibits SOCE. (H) Summary of average rise in cytosolic Ca2+ levels 200 seconds after re-addition of 2 mM Ca2+ ringer’s following store-depletion. Data are mean ± SEM of 34-47 cells. Representative of 5 independent experiments. **P < 0.01, ***P < 0.001, Ck. Ext, cockroach extract.

Mentions: There is a strong correlation between sensitization and allergy to inhaled cockroach extracts and the incidence of acute asthmatic attacks2425. In vitro and in vivo studies have shown that extracts from cockroach have proteinase activity and stimulate PAR2 receptors to mediate their inflammatory effects910. Induction of cytosolic Ca2+ fluxes in response to cockroach extracts has been shown in alveolar A549 cells26, cultured human keratinocytes27 and KNRK cells, a rat kidney cell line10. However, the pathways mediating these Ca2+ fluxes are unknown. We found that administration of cockroach extract to BEAS-2B cells in a 2 mM Ca2+ Ringer’s solution produced a biphasic rise in cytoplasmic Ca2+: a rapid initial spike followed by sustained Ca2+ signals that lasted more than 10 minutes (Fig. 1A). In most cells, the sustained component of the Ca2+ response consisted of an elevated baseline with an oscillating component superposed on the baseline. The sustained signals elicited by cockroach extract were almost completely abolished in a Ca2+ free Ringer’s solution, suggesting that Ca2+ influx across the plasma membrane was needed for this Ca2+ signal (Fig. 1B). Moreover, the CRAC channel inhibitor, BTP2, significantly inhibited both the plateau Ca2+ signals as well as the oscillating component (Fig. 1C), indicating that the sustained Ca2+ signals arise from the opening of CRAC channels. Fig. 1A–C show traces from individual cells, whereas the average [Ca2+]i changes and the integrated area under the curve during the time period of allergen treatment are summarized in Fig. 1D–F. To confirm the inhibitory effects of BTP2 on CRAC channel activation in BEAS-2B cells more directly, we activated SOCE using thapsigargin, a SERCA pump inhibitor that irreversibly depletes ER Ca2+ stores. SOCE was significantly inhibited by BTP2 at the same concentration that was used to inhibit allergen induced Ca2+ signal (Fig. 1G,H). Based on these results, we conclude that cockroach extracts induce long lasting Ca2+ signals in bronchial epithelial cells by activating SOCE through CRAC channels.


Allergens stimulate store-operated calcium entry and cytokine production in airway epithelial cells
Cockroach allergen extracts activate store-operated CRAC channels in BEAS-2B cells.(A–C) [Ca2+i] imaging showing responses of individual BEAS-2B cells to cockroach allergen extracts (10 μg/mL) administered in (A) 2 mM Ca2+ ringer’s solution, (B) Ca2+ free ringer’s solution or (C) 2 mM Ca2+ ringer’s solution in the presence of the CRAC channel inhibitor BTP2 (500 nM). (D) Average [Ca2+i] response of the individual cells shown in (A–C). (E–F) Summary of the average rise in [Ca2+i] 600 seconds after addition of cockroach extract (E) and the integral of the [Ca2+]i signal during application of the allergen. (F). (G) Ca2+ imaging trace showing SOCE in BAES-2B cells. SOCE was induced by depleting ER Ca2+ stores with 1 µM thapsigargin in a Ca2+-free Ringer’s solution and readding 2 mM Ca2+ following store depletion. Pre-treating cells with BTP2 (500 nM) strongly inhibits SOCE. (H) Summary of average rise in cytosolic Ca2+ levels 200 seconds after re-addition of 2 mM Ca2+ ringer’s following store-depletion. Data are mean ± SEM of 34-47 cells. Representative of 5 independent experiments. **P < 0.01, ***P < 0.001, Ck. Ext, cockroach extract.
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f1: Cockroach allergen extracts activate store-operated CRAC channels in BEAS-2B cells.(A–C) [Ca2+i] imaging showing responses of individual BEAS-2B cells to cockroach allergen extracts (10 μg/mL) administered in (A) 2 mM Ca2+ ringer’s solution, (B) Ca2+ free ringer’s solution or (C) 2 mM Ca2+ ringer’s solution in the presence of the CRAC channel inhibitor BTP2 (500 nM). (D) Average [Ca2+i] response of the individual cells shown in (A–C). (E–F) Summary of the average rise in [Ca2+i] 600 seconds after addition of cockroach extract (E) and the integral of the [Ca2+]i signal during application of the allergen. (F). (G) Ca2+ imaging trace showing SOCE in BAES-2B cells. SOCE was induced by depleting ER Ca2+ stores with 1 µM thapsigargin in a Ca2+-free Ringer’s solution and readding 2 mM Ca2+ following store depletion. Pre-treating cells with BTP2 (500 nM) strongly inhibits SOCE. (H) Summary of average rise in cytosolic Ca2+ levels 200 seconds after re-addition of 2 mM Ca2+ ringer’s following store-depletion. Data are mean ± SEM of 34-47 cells. Representative of 5 independent experiments. **P < 0.01, ***P < 0.001, Ck. Ext, cockroach extract.
Mentions: There is a strong correlation between sensitization and allergy to inhaled cockroach extracts and the incidence of acute asthmatic attacks2425. In vitro and in vivo studies have shown that extracts from cockroach have proteinase activity and stimulate PAR2 receptors to mediate their inflammatory effects910. Induction of cytosolic Ca2+ fluxes in response to cockroach extracts has been shown in alveolar A549 cells26, cultured human keratinocytes27 and KNRK cells, a rat kidney cell line10. However, the pathways mediating these Ca2+ fluxes are unknown. We found that administration of cockroach extract to BEAS-2B cells in a 2 mM Ca2+ Ringer’s solution produced a biphasic rise in cytoplasmic Ca2+: a rapid initial spike followed by sustained Ca2+ signals that lasted more than 10 minutes (Fig. 1A). In most cells, the sustained component of the Ca2+ response consisted of an elevated baseline with an oscillating component superposed on the baseline. The sustained signals elicited by cockroach extract were almost completely abolished in a Ca2+ free Ringer’s solution, suggesting that Ca2+ influx across the plasma membrane was needed for this Ca2+ signal (Fig. 1B). Moreover, the CRAC channel inhibitor, BTP2, significantly inhibited both the plateau Ca2+ signals as well as the oscillating component (Fig. 1C), indicating that the sustained Ca2+ signals arise from the opening of CRAC channels. Fig. 1A–C show traces from individual cells, whereas the average [Ca2+]i changes and the integrated area under the curve during the time period of allergen treatment are summarized in Fig. 1D–F. To confirm the inhibitory effects of BTP2 on CRAC channel activation in BEAS-2B cells more directly, we activated SOCE using thapsigargin, a SERCA pump inhibitor that irreversibly depletes ER Ca2+ stores. SOCE was significantly inhibited by BTP2 at the same concentration that was used to inhibit allergen induced Ca2+ signal (Fig. 1G,H). Based on these results, we conclude that cockroach extracts induce long lasting Ca2+ signals in bronchial epithelial cells by activating SOCE through CRAC channels.

View Article: PubMed Central - PubMed

ABSTRACT

Aberrant immune responses to environmental allergens including insect allergens from house dust mites and cockroaches contribute to allergic inflammatory diseases such as asthma in susceptible individuals. Airway epithelial cells (AECs) play a critical role in this process by sensing the proteolytic activity of allergens via protease-activated receptors (PAR2) to initiate inflammatory and immune responses in the airway. Elevation of cytosolic Ca2+ is an important signaling event in this process, yet the fundamental mechanism by which allergens induce Ca2+ elevations in AECs remains poorly understood. Here we find that extracts from dust mite and cockroach induce sustained Ca2+ elevations in AECs through the activation of Ca2+ release-activated Ca2+ (CRAC) channels encoded by Orai1 and STIM1. CRAC channel activation occurs, at least in part, through allergen mediated stimulation of PAR2 receptors. The ensuing Ca2+ entry then activates NFAT/calcineurin signaling to induce transcriptional production of the proinflammatory cytokines IL-6 and IL-8. These findings highlight a key role for CRAC channels as regulators of allergen induced inflammatory responses in the airway.

No MeSH data available.