Francisco de Borja Callejas1, Asunción Martínez-Antón1, Isam Alobid2, Mireya Fuentes1, Julio Cortijo3, César Picado4, Jordi Roca-Ferrer1, Joaquim Mullol5. 1. Clinical and Experimental Respiratory Immunoallergy, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain; CIBER of Respiratory Diseases (CIBERES), Barcelona, Spain. 2. Clinical and Experimental Respiratory Immunoallergy, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain; Rhinology Unit & Smell Clinic, ENT Department, Hospital Clínic, Universitat de Barcelona, Barcelona, Catalonia, Spain. 3. CIBER of Respiratory Diseases (CIBERES), Barcelona, Spain. 4. Clinical and Experimental Respiratory Immunoallergy, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain; CIBER of Respiratory Diseases (CIBERES), Barcelona, Spain; Pneumology & Respiratory Allergy Department, Hospital Clínic, Universitat de Barcelona, Barcelona, Catalonia, Spain. 5. Clinical and Experimental Respiratory Immunoallergy, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain; CIBER of Respiratory Diseases (CIBERES), Barcelona, Spain; Rhinology Unit & Smell Clinic, ENT Department, Hospital Clínic, Universitat de Barcelona, Barcelona, Catalonia, Spain.
Abstract
BACKGROUND: Primary human airway epithelial cells cultured in an air-liquid interface (ALI) develop a well-differentiated epithelium. However, neither characterization of mucociliar differentiation overtime nor the inflammatory function of reconstituted nasal polyp (NP) epithelia have been described. OBJECTIVES: 1st) To develop and characterize the mucociliar differentiation overtime of human epithelial cells of chronic rhinosinusitis with nasal polyps (CRSwNP) in ALI culture system; 2nd) To corroborate that 3D in vitro model of NP reconstituted epithelium maintains, compared to control nasal mucosa (NM), an inflammatory function. METHODS: Epithelial cells were obtained from 9 NP and 7 control NM, and differentiated in ALI culture for 28 days. Mucociliary differentiation was characterized at different times (0, 7, 14, 21, and 28 days) using ultrastructure analysis by electron microscopy; ΔNp63 (basal stem/progenitor cell), β-tubulin IV (cilia), and MUC5AC (goblet cell) expression by immunocytochemistry; and mucous (MUC5AC, MUC5B) and serous (Lactoferrin) secretion by ELISA. Inflammatory function of ALI cultures (at days 0, 14, and 28) through cytokine (IL-8, IL-1β, IL-6, IL-10, TNF-α, and IL-12p70) and chemokine (RANTES, MIG, MCP-1, IP-10, eotaxin-1, and GM-CSF) production was analysed by CBA (Cytometric Bead Array). RESULTS: In both NP and control NM ALI cultures, pseudostratified epithelium with ciliated, mucus-secreting, and basal cells were observed by electron microscopy at days 14 and 28. Displaying epithelial cell re-differentation, β-tubulin IV and MUC5AC positive cells increased, while ΔNp63 positive cells decreased overtime. No significant differences were found overtime in MUC5AC, MUC5B, and lactoferrin secretions between both ALI cultures. IL-8 and GM-CSF were significantly increased in NP compared to control NM regenerated epithelia. CONCLUSION: Reconstituted epithelia from human NP epithelial cells cultured in ALI system provides a 3D in vitro model that could be useful both for studying the role of epithelium in CRSwNP while developing new therapeutic strategies, including cell therapy, for CRSwNP.
BACKGROUND: Primary human airway epithelial cells cultured in an air-liquid interface (ALI) develop a well-differentiated epithelium. However, neither characterization of mucociliar differentiation overtime nor the inflammatory function of reconstituted nasal polyp (NP) epithelia have been described. OBJECTIVES: 1st) To develop and characterize the mucociliar differentiation overtime of human epithelial cells of chronic rhinosinusitis with nasal polyps (CRSwNP) in ALI culture system; 2nd) To corroborate that 3D in vitro model of NP reconstituted epithelium maintains, compared to control nasal mucosa (NM), an inflammatory function. METHODS: Epithelial cells were obtained from 9 NP and 7 control NM, and differentiated in ALI culture for 28 days. Mucociliary differentiation was characterized at different times (0, 7, 14, 21, and 28 days) using ultrastructure analysis by electron microscopy; ΔNp63 (basal stem/progenitor cell), β-tubulin IV (cilia), and MUC5AC (goblet cell) expression by immunocytochemistry; and mucous (MUC5AC, MUC5B) and serous (Lactoferrin) secretion by ELISA. Inflammatory function of ALI cultures (at days 0, 14, and 28) through cytokine (IL-8, IL-1β, IL-6, IL-10, TNF-α, and IL-12p70) and chemokine (RANTES, MIG, MCP-1, IP-10, eotaxin-1, and GM-CSF) production was analysed by CBA (Cytometric Bead Array). RESULTS: In both NP and control NM ALI cultures, pseudostratified epithelium with ciliated, mucus-secreting, and basal cells were observed by electron microscopy at days 14 and 28. Displaying epithelial cell re-differentation, β-tubulin IV and MUC5AC positive cells increased, while ΔNp63 positive cells decreased overtime. No significant differences were found overtime in MUC5AC, MUC5B, and lactoferrin secretions between both ALI cultures. IL-8 and GM-CSF were significantly increased in NP compared to control NM regenerated epithelia. CONCLUSION: Reconstituted epithelia from human NP epithelial cells cultured in ALI system provides a 3D in vitro model that could be useful both for studying the role of epithelium in CRSwNP while developing new therapeutic strategies, including cell therapy, for CRSwNP.
Authors: J Mullol; F B Callejas; E Méndez-Arancibia; M Fuentes; I Alobid; A Martínez-Antón; A Valero; C Picado; J Roca-Ferrer Journal: J Biol Regul Homeost Agents Date: 2010 Oct-Dec Impact factor: 1.711
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