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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.

No MeSH data available.


STIM1 and Orai1 mediate Ca2+ entry evoked by cockroach allergen extracts.(A) Western blot showing STIM1 and Orai1 expression in BEAS-2B cells and the effects of siRNA knockdown of STIM1 or Orai1. (B) Ca2+ traces showing the effects of siRNA knockdown of STIM1 and Orai1 on cockroach allergen-induced Ca2+ signals. A scrambled siRNA sequence was used as control. (C,D) Summary of average cytosolic Ca2+ levels 798 seconds after addition of cockroach allergen extract (8 μg/mL) (C) and the integral [Ca2+] signal following addition of cockroach extract (D). (E–G) Ca2+ imaging traces of individual cells treated with either a scrambled control siRNA (E) or siRNA against Stim1 (F) or Orai1 (G) showing effects of the knockdown on cockroach allergen-induced Ca2+ signals. Data are mean ± SEM of 29–38 cells. Representative of 3 independent experiments. **P < 0.01.
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f2: STIM1 and Orai1 mediate Ca2+ entry evoked by cockroach allergen extracts.(A) Western blot showing STIM1 and Orai1 expression in BEAS-2B cells and the effects of siRNA knockdown of STIM1 or Orai1. (B) Ca2+ traces showing the effects of siRNA knockdown of STIM1 and Orai1 on cockroach allergen-induced Ca2+ signals. A scrambled siRNA sequence was used as control. (C,D) Summary of average cytosolic Ca2+ levels 798 seconds after addition of cockroach allergen extract (8 μg/mL) (C) and the integral [Ca2+] signal following addition of cockroach extract (D). (E–G) Ca2+ imaging traces of individual cells treated with either a scrambled control siRNA (E) or siRNA against Stim1 (F) or Orai1 (G) showing effects of the knockdown on cockroach allergen-induced Ca2+ signals. Data are mean ± SEM of 29–38 cells. Representative of 3 independent experiments. **P < 0.01.

Mentions: We have previously shown in AECs that SOCE is mediated by the CRAC channel proteins, STIM1 and Orai115. Knockdown of STIM1 and Orai1 using siRNA reduced expression of these proteins (Fig. 2A and Supplementary Fig. S1) and significantly reduced the amplitude of Ca2+ elevation induced by cockroach extract (Fig. 2B). Both the average amplitude of Ca2+ signal and the integrated Ca2+ signal over time was significantly attenuated in siSTIM1 and siOrai1 treated cells (Fig. 2C,D). Knockdown of STIM1 by siStim1 showed good specificity and did not have any effect on STIM2 expression (Supplementary Fig. S1). Interestingly, analysis of single cell Ca2+ responses revealed that a proportion of cells in both siSTIM1 and siOrai1 treated samples showed Ca2+ oscillations (Fig. 2F,G). It is likely that, given the incomplete knockdown of STIM1 and Orai1 by siRNA (Fig. 2A), these oscillatory signals are mediated by the residual CRAC channel machinery. In contrast, cells treated with the siRNA control showed a more heterogeneous response with individual cells showing a sustained increase in Ca2+ signal with oscillatory Ca2+ signals riding on top of the elevated baseline, which accounted for the higher average Ca2+ response (Fig. 2E). We also note that the average Ca2+ elevation in response to cockroach extracts seen in siControl treated cells was lower than in untransfected control cells (Fig. 1D) likely due to the cell stress induced by cell transfection with lipofectamine. Taken together, these results demonstrate that STIM1 and Orai1 make essential contributions to the Ca2+ elevations in AECs following exposure of the cells to cockroach extracts.


Allergens stimulate store-operated calcium entry and cytokine production in airway epithelial cells
STIM1 and Orai1 mediate Ca2+ entry evoked by cockroach allergen extracts.(A) Western blot showing STIM1 and Orai1 expression in BEAS-2B cells and the effects of siRNA knockdown of STIM1 or Orai1. (B) Ca2+ traces showing the effects of siRNA knockdown of STIM1 and Orai1 on cockroach allergen-induced Ca2+ signals. A scrambled siRNA sequence was used as control. (C,D) Summary of average cytosolic Ca2+ levels 798 seconds after addition of cockroach allergen extract (8 μg/mL) (C) and the integral [Ca2+] signal following addition of cockroach extract (D). (E–G) Ca2+ imaging traces of individual cells treated with either a scrambled control siRNA (E) or siRNA against Stim1 (F) or Orai1 (G) showing effects of the knockdown on cockroach allergen-induced Ca2+ signals. Data are mean ± SEM of 29–38 cells. Representative of 3 independent experiments. **P < 0.01.
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Related In: Results  -  Collection

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f2: STIM1 and Orai1 mediate Ca2+ entry evoked by cockroach allergen extracts.(A) Western blot showing STIM1 and Orai1 expression in BEAS-2B cells and the effects of siRNA knockdown of STIM1 or Orai1. (B) Ca2+ traces showing the effects of siRNA knockdown of STIM1 and Orai1 on cockroach allergen-induced Ca2+ signals. A scrambled siRNA sequence was used as control. (C,D) Summary of average cytosolic Ca2+ levels 798 seconds after addition of cockroach allergen extract (8 μg/mL) (C) and the integral [Ca2+] signal following addition of cockroach extract (D). (E–G) Ca2+ imaging traces of individual cells treated with either a scrambled control siRNA (E) or siRNA against Stim1 (F) or Orai1 (G) showing effects of the knockdown on cockroach allergen-induced Ca2+ signals. Data are mean ± SEM of 29–38 cells. Representative of 3 independent experiments. **P < 0.01.
Mentions: We have previously shown in AECs that SOCE is mediated by the CRAC channel proteins, STIM1 and Orai115. Knockdown of STIM1 and Orai1 using siRNA reduced expression of these proteins (Fig. 2A and Supplementary Fig. S1) and significantly reduced the amplitude of Ca2+ elevation induced by cockroach extract (Fig. 2B). Both the average amplitude of Ca2+ signal and the integrated Ca2+ signal over time was significantly attenuated in siSTIM1 and siOrai1 treated cells (Fig. 2C,D). Knockdown of STIM1 by siStim1 showed good specificity and did not have any effect on STIM2 expression (Supplementary Fig. S1). Interestingly, analysis of single cell Ca2+ responses revealed that a proportion of cells in both siSTIM1 and siOrai1 treated samples showed Ca2+ oscillations (Fig. 2F,G). It is likely that, given the incomplete knockdown of STIM1 and Orai1 by siRNA (Fig. 2A), these oscillatory signals are mediated by the residual CRAC channel machinery. In contrast, cells treated with the siRNA control showed a more heterogeneous response with individual cells showing a sustained increase in Ca2+ signal with oscillatory Ca2+ signals riding on top of the elevated baseline, which accounted for the higher average Ca2+ response (Fig. 2E). We also note that the average Ca2+ elevation in response to cockroach extracts seen in siControl treated cells was lower than in untransfected control cells (Fig. 1D) likely due to the cell stress induced by cell transfection with lipofectamine. Taken together, these results demonstrate that STIM1 and Orai1 make essential contributions to the Ca2+ elevations in AECs following exposure of the cells to cockroach extracts.

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.