A 3-D Well-Differentiated Model of Pediatric Bronchial Epithelium Demonstrates Unstimulated Morphological Differences Between Asthmatic and Nonasthmatic Cells

Abstract

There is a need for reproducible and effective models of pediatric bronchial epithelium to study disease states such as asthma. We aimed to develop, characterize, and differentiate an effective, an efficient, and a reliable three-dimensional model of pediatric bronchial epithelium to test the hypothesis that children with asthma differ in their epithelial morphologic phenotype when compared with nonasthmatic children. Primary cell cultures from both asthmatic and nonasthmatic children were grown and differentiated at the air-liquid interface for 28 d. Tight junction formation, MUC5AC secretion, IL-8, IL-6, prostaglandin E2 production, and the percentage of goblet and ciliated cells in culture were assessed. Well-differentiated, multilayered, columnar epithelium containing both ciliated and goblet cells from asthmatic and nonasthmatic subjects were generated. All cultures demonstrated tight junction formation at the apical surface and exhibited mucus production and secretion. Asthmatic and nonasthmatic cultures secreted similar quantities of IL-8, IL-6, and prostaglandin E2. Cultures developed from asthmatic children contained considerably more goblet cells and fewer ciliated cells compared with those from nonasthmatic children. A well-differentiated model of pediatric epithelium has been developed that will be useful for more in vivo like study of the mechanisms at play during asthma. (Pediatr Res 67: 17-22, 2010)

Secretory Phospholipases A 2 Are Secreted From Ciliated Cells and Increase Mucin and Eicosanoid Secretion From Goblet Cells

“…Although the HBE cells used in this study were from subjects without lung disease, it has been reported that bronchial epithelial cell cultures from subjects with asthma contain many more goblet cells and fewer ciliated cells compared with those from healthy subjects. 28 Conclusions Th e fi ndings in the current study suggest that sPLA 2 are released from ciliated airway epithelial cells and act on goblet cells in a paracrine manner to stimulate LTB 4 , LTC 4 , and MUC5AC secretion. We reported previously that IL-33 acts directly on goblet-enriched cells, but not ciliated-enriched cells, to stimulate IL-8 production, suggesting that beyond secreting mucus, goblet cells are infl ammatory eff ector cells.…”

“…27,28 For the apical surface view, cultured HBE cell membranes were cut from the insert using a scalpel and were fi xed in 3% formaldehyde. For the z-stack view, the cell membrane was embedded in Tissue-Tek OCT Compound (Sakura FineTechnical Co Ltd), frozen in liquid nitrogen, and stored at -80°C.…”

In vitro modeling of respiratory syncytial virus infection of pediatric bronchial epithelium, the primary target of infection in vivo

“…PBECs were obtained from healthy children undergoing elective surgery (n = 5) (42). The generation and characterization of WD-PBEC cultures were described previously and are outlined in SI Materials and Methods (21,43). Briefly, primary cells were expanded in collagen-coated flasks until almost confluent and then were transferred onto collagen-coated semipermeable (0.4-μm pore size, 12-mm diameter) membrane supports.…”

Effects of IL-13 on Mucociliary Differentiation of Pediatric Asthmatic Bronchial Epithelial Cells

“…Under basal unstimulated conditions, we have previously demonstrated that cultures from asthmatic PBEC, PBEC(A), children contained significantly more goblet cells and fewer ciliated cells compared with those from normal PBEC, PBEC(N), children (16).…”

“…We have established a 3D well-differentiated model of PBECs, which, we believe, mimics the bronchial epithelium in vivo (16). Under basal unstimulated conditions, we have previously demonstrated that cultures from asthmatic PBEC, PBEC(A), children contained significantly more goblet cells and fewer ciliated cells compared with those from normal PBEC, PBEC(N), children (16).…”