Accumulation of intraepithelial mast cells with a unique protease phenotype in T(H)2-high asthma

Abstract

Background: Previously, we found that mast cell tryptases and carboxypeptidase A3 (CPA3) are differentially expressed in the airway epithelium in asthmatic subjects. We also found that asthmatic subjects can be divided into 2 subgroups ("T(H)2 high" and "T(H)2 low" asthma) based on epithelial cell gene signatures for the activity of T(H)2 cytokines.

Objectives: We sought to characterize intraepithelial mast cells (IEMCs) in asthma.

Methods: We performed gene expression profiling in epithelial brushings and stereology-based quantification of mast cell numbers in endobronchial biopsy specimens from healthy control and asthmatic subjects before and after treatment with inhaled corticosteroids (ICSs). We also performed gene expression and protein quantification studies in cultured airway epithelial cells and mast cells.

Results: By means of unsupervised clustering, mast cell gene expression in the airway epithelium related closely to the expression of IL-13 signature genes. The levels of expression of mast cell genes correlate positively with lung function improvements with ICSs. IEMC density was 2-fold higher than normal in subjects with T(H)2-high asthma compared with that seen in subjects with T(H)2-low asthma or healthy control subjects (P = .015 for both comparisons), and these cells were characterized by expression of tryptases and CPA3 but not chymase. IL-13 induced expression of stem cell factor in cultured airway epithelial cells, and mast cells exposed to conditioned media from IL-13-activated epithelial cells showed downregulation of chymase but no change in tryptase or CPA3 expression.

Conclusion: IEMC numbers are increased in subjects with T(H)2-high asthma, have an unusual protease phenotype (tryptase and CPA3 high and chymase low), and predict responsiveness to ICSs. IL-13-stimulated production of stem cell factor by epithelial cells potentially explains mast cell accumulation in T(H)2-high asthmatic epithelium.

FIG 1

Epithelial gene expression of mast cell proteases, as determined by means of gene array (top row) and qPCR (bottom row). Lines represent median values. A, Asthmatic; H, healthy. *P < .0001.

FIG 2

Heat map showing genes in cluster number 24 from unsupervised hierarchical clustering of gene expression data from epithelial brushings of asthmatic subjects (T_H2 high, n = 22; T_H2 low, n = 20) and healthy control subjects (n = 28). The color bar along the top indicates subject status: green, healthy; blue, T_H2-low asthma; red, T_H2-high asthma.

FIG 3

Baseline mast cell protease gene expression is positively correlated with improvement in lung function from ICSs. Baseline (pretreatment) tryptase (A) and CPA3 (B) mRNA expression levels (as measured by means of qPCR) in subjects subsequently randomized to inhaled fluticasone correlated with treatment-related improvements in lung function at 4 weeks.

FIG 4

Density of mast cells within airway epithelium among subjects stratified by disease status (A) and T_H2 subgroup (B). Lines represent median values, and boxes represent interquartile ranges. A, Asthmatic; H, healthy. *P = .011, Kruskal-Wallis test.

FIG 5

Immunohistochemistry in endobronchial biopsy specimens for mast cells and basophils. A and B, Serial sections show tryptase-positive cells within the epithelium (arrows; Fig 5, A) and absence of basophils (Fig 5, B). C and D, Serial sections show tryptase-positive cells within the epithelium (arrow; Fig 5, C) and chymase-positive cells in the submucosa (arrow; Fig 5, D). E, Anti-CPA3 shows CPA3-positive cells (arrows). Additional immunostaining is shown in Fig E5.

FIG 6

IL-13 induces epithelial cell production of SCF. qPCR (A) and protein (B) levels in basal media after 4 days of treatment with control (Grey media), IL-13, TNF-α, and IL-1β. Results are shown as the fold induction over control averaged across 4 different donor cell lines. *P = .02 and **P = .004 versus control.

FIG 7

CM from IL-13–treated epithelial cells suppresses mast cell chymase but not tryptase or CPA3. Cord blood–derived mast cells were grown in CM from human epithelial cells treated with control media or IL-13 (10 ng/mL; CM/IL-13). A and B, qPCR data (means ± SDs). C, Immunostaining of mast cells for tryptase, CPA3, and chymase. *P = .002.