Primary human airway epithelial cell-dependent inhibition of human lung mast cell degranulation

Abstract

Introduction: Chronic mast cell activation is a characteristic feature of asthma. BEAS-2B human airway epithelial cells (AEC) profoundly inhibit both constitutive and IgE-dependent human lung mast cell (HLMC) histamine release. The aim of this study was to examine the regulation of HLMC degranulation by primary AEC from healthy and asthmatic subjects, and investigate further the inhibitory mechanism.

Methods: HLMC were co-cultured with both BEAS-2B and primary AEC grown as monolayers or air-liquid interface (ALI) cultures.

Results: Both constitutive and IgE-dependent HLMC histamine release were attenuated by BEAS-2B, primary AEC monolayers and ALI cultures. This occurred in the absence of HLMC-AEC contact indicating the presence of a soluble factor. Unlike healthy ALI AEC, asthmatic ALI-AEC did not significantly reduce constitutive histamine release. AEC inhibitory activity was transferable in primary AEC monolayer supernatant, but less active than with Transwell co-culture, suggesting that the inhibitory factor was labile. The AEC inhibitory effects were attenuated by both AEC wounding and pertussis toxin, indicating the involvement of a G(0)/G(i) receptor coupled mechanism. Solid phase extraction of lipids (<10 kDa) removed the AEC inhibitory activity. The lipid derivatives resolving D1 and D2 and lipoxin A(4) attenuated HLMC histamine release in a dose-dependent fashion but were not detectable in co-culture supernatants.

Conclusions: Primary AEC suppress HLMC constitutive and IgE-dependent histamine secretion through the release of a soluble, labile lipid mediator(s) that signals through the G(0)/G(i) receptor coupled mechanism. Manipulation of this interaction may have a significant therapeutic role in asthma.

Figure 1. Constitutive (16 h) and IgE-dependent (30 min) HLMC histamine secretion in monoculture and in the presence of BEAS-2B cells, either in contact or on either side of a Transwell membrane (BEAS-2B on the underside of the membrane, HLMC on the upperside of the membrane).

Mean ± SEM ng/10⁶ cells (N = 3 HLMC donors).

Figure 2. Constitutive (16 h) and IgE-dependent (30 min) HLMC histamine secretion in monoculture or in the presence of BEAS-2B cells separated by a Transwell membrane (HLMC in the bottom chamber, BEAS-2B in the top chamber).

Retained cell histamine content prior to activation is also shown. n = 7 HLMC donors. Mean ± SEM ng/10⁶ cells.

Figure 3. The effects of HLMC co-culture with healthy and asthmatic ALI AEC on constitutive (16 h) and IgE-dependent (30 min) HLMC histamine secretion compared to HLMC cultured alone, n = 6 HLMC donors.

Mean ± SEM ng/10⁶ cells. (p<0.05*, p<0.01**, p<0.001***, ns = not significant).

Figure 4. HLMC histamine release in different culture media.

E – primary AEC monolayer supernatant; E-SPE – SPE-processed (lipid-deplete) AEC supernatant; BEGM-SPE – SPE-processed BEGM media; BEGM – unprocessed media. Mean ± SEM of 4 experiments using 2 AEC donors and 2 HLMC donors. HLMCs cultured in AEC supernatant showed suppressed histamine release (p<0.05) whereas there is no significant suppression in lipid-deplete supernatant.

Figure 5. The effects of AH6809, epithelial wounding and pertussis toxin on epithelial-dependent inhibition of HLMC histamine secretion n = 4 HLMC donors, mean ± SEM ng/10⁶ cells.

(p<0.05*, p<0.01**, P<0.001***, ns = not significant).

Figure 6. The effects of lipoxin A4, resolvin D1 and resolvin D2 on HLMC IgE-dependent degranulation.

Mean ± SEM from 7 HLMC donors. p<0.001 for all mediators (repeated measures ANOVA).

Figure 7. LC-MS calibration curve for triplicate injections of resolvin D1 standard with a linear regression best fit line.

Integrated peak area refers to the 375.2 → 141.2 ion pair.