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

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.

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Dust mite allergen extracts activate CRAC channels in BEAS-2B cells.(A) HDM extract (nDerP, 18 μg/mL) induces a cytosolic Ca2+ signal that is blocked by BTP2. (B,C) Summary of the average rise in [Ca2+i] 200 seconds after addition of HDM extract (B) and the integral Ca2+ signal (D–F) Knockdown of STIM1 or Orai1 inhibits nDerP induced Ca2+ signals. (D) Average trace showing the effects of siRNA mediated knockdown of STIM1 and ORAI1 on HDM allergen-induced Ca2+ signals. Summary of (E) average cytosolic Ca2+ levels 540 seconds after addition of dust mite allergen extract and (F) integrated Ca2+ signal. Data are mean ± SEM of 32–57 cells. Representative of 3–5 independent experiments. **P < 0.01, ***P < 0.001, nDerP, dust mite extract.
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f3: Dust mite allergen extracts activate CRAC channels in BEAS-2B cells.(A) HDM extract (nDerP, 18 μg/mL) induces a cytosolic Ca2+ signal that is blocked by BTP2. (B,C) Summary of the average rise in [Ca2+i] 200 seconds after addition of HDM extract (B) and the integral Ca2+ signal (D–F) Knockdown of STIM1 or Orai1 inhibits nDerP induced Ca2+ signals. (D) Average trace showing the effects of siRNA mediated knockdown of STIM1 and ORAI1 on HDM allergen-induced Ca2+ signals. Summary of (E) average cytosolic Ca2+ levels 540 seconds after addition of dust mite allergen extract and (F) integrated Ca2+ signal. Data are mean ± SEM of 32–57 cells. Representative of 3–5 independent experiments. **P < 0.01, ***P < 0.001, nDerP, dust mite extract.

Mentions: Previous studies have shown that exposure to HDM can trigger allergic inflammation in asthmatic patients12829. Components of HDM, including Der p1, Der p3 and Der p9 exhibit proteolytic activity that leads to activation of PAR2 receptors, which in turn plays a critical role in mediating the inflammatory effects of HDM830. HDM has also been shown to activate Ca2+ signals in airway epithelial cells in both primary epithelial cells and cell lines, and this is believed to occur through both PAR2-dependent and -independent mechanisms83031. However, whether HDM can activate CRAC channels has not been studied. When administered in a Ca2+-free Ringer’s solution, HDM induced only a transient Ca2+ signal indicating that the extract causes Ca2+ release from internal stores (Supplementary Fig. S2). In the presence of extracellular Ca2+, however, HDM extracts activated a sustained Ca2+ signal in BEAS-2B cells that was inhibited by the CRAC channel inhibitor BTP2 (Fig. 3A–C, Supplementary Fig. S2). Further, knockdown of the CRAC channel proteins STIM1 and Orai1 significantly abrogated the average sustained Ca2+ signals seen in response to HDM (Fig. 3D–F). These results indicate that HDM allergens mobilize cellular Ca2+ elevations in bronchial epithelial cells by depleting ER Ca2+ stores and activating CRAC channels encoded by STIM1 and Orai1.


Allergens stimulate store-operated calcium entry and cytokine production in airway epithelial cells
Dust mite allergen extracts activate CRAC channels in BEAS-2B cells.(A) HDM extract (nDerP, 18 μg/mL) induces a cytosolic Ca2+ signal that is blocked by BTP2. (B,C) Summary of the average rise in [Ca2+i] 200 seconds after addition of HDM extract (B) and the integral Ca2+ signal (D–F) Knockdown of STIM1 or Orai1 inhibits nDerP induced Ca2+ signals. (D) Average trace showing the effects of siRNA mediated knockdown of STIM1 and ORAI1 on HDM allergen-induced Ca2+ signals. Summary of (E) average cytosolic Ca2+ levels 540 seconds after addition of dust mite allergen extract and (F) integrated Ca2+ signal. Data are mean ± SEM of 32–57 cells. Representative of 3–5 independent experiments. **P < 0.01, ***P < 0.001, nDerP, dust mite extract.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5015156&req=5

f3: Dust mite allergen extracts activate CRAC channels in BEAS-2B cells.(A) HDM extract (nDerP, 18 μg/mL) induces a cytosolic Ca2+ signal that is blocked by BTP2. (B,C) Summary of the average rise in [Ca2+i] 200 seconds after addition of HDM extract (B) and the integral Ca2+ signal (D–F) Knockdown of STIM1 or Orai1 inhibits nDerP induced Ca2+ signals. (D) Average trace showing the effects of siRNA mediated knockdown of STIM1 and ORAI1 on HDM allergen-induced Ca2+ signals. Summary of (E) average cytosolic Ca2+ levels 540 seconds after addition of dust mite allergen extract and (F) integrated Ca2+ signal. Data are mean ± SEM of 32–57 cells. Representative of 3–5 independent experiments. **P < 0.01, ***P < 0.001, nDerP, dust mite extract.
Mentions: Previous studies have shown that exposure to HDM can trigger allergic inflammation in asthmatic patients12829. Components of HDM, including Der p1, Der p3 and Der p9 exhibit proteolytic activity that leads to activation of PAR2 receptors, which in turn plays a critical role in mediating the inflammatory effects of HDM830. HDM has also been shown to activate Ca2+ signals in airway epithelial cells in both primary epithelial cells and cell lines, and this is believed to occur through both PAR2-dependent and -independent mechanisms83031. However, whether HDM can activate CRAC channels has not been studied. When administered in a Ca2+-free Ringer’s solution, HDM induced only a transient Ca2+ signal indicating that the extract causes Ca2+ release from internal stores (Supplementary Fig. S2). In the presence of extracellular Ca2+, however, HDM extracts activated a sustained Ca2+ signal in BEAS-2B cells that was inhibited by the CRAC channel inhibitor BTP2 (Fig. 3A–C, Supplementary Fig. S2). Further, knockdown of the CRAC channel proteins STIM1 and Orai1 significantly abrogated the average sustained Ca2+ signals seen in response to HDM (Fig. 3D–F). These results indicate that HDM allergens mobilize cellular Ca2+ elevations in bronchial epithelial cells by depleting ER Ca2+ stores and activating CRAC channels encoded by STIM1 and Orai1.

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.


Related in: MedlinePlus