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Notes: Background Prominent infiltration of eosinophils in airway mucosa is the pathognomonic sign of asthma. The role of airway epithelial cells in eosinophil infiltration, however, has not been fully elucidated.Objective The aim of this study is to develop a new in vitro transmigration system composed of airway epithelial cells and extracellular matrix, and to investigate the role of airway epithelial cells in eosinophil infiltration.Methods A layer of type I collagen gel was formed in Netwell™, and BEAS-2B bronchial epithelial cells were cultured on the gel. Then the wells covered with epithelial monolayer were filled with medium, inverted, and new upper chambers were constructed on the gel side by applying a ring cap. After further incubation with or without exogenous cytokines for 48 h, eosinophils or neutrophils were loaded in upper chambers (the gel side) and cells transmigrated to lower chambers (the epithelial cell side) were counted. Immunohistochemical analyses were also performed.Results While a simple collagen gel hardly promoted eosinophil migration even in the presence of eotaxin or RANTES, significant numbers of eosinophils migrated to lower chambers in the presence of the epithelial cells. Replacement of medium in the lower chamber (the epithelial cell side) with fresh medium, addition of exogenous eotaxin or RANTES in the upper chamber (the gel side), or pre-treatment of eosinophils with anti-CCR3 all inhibited transmigration. We found that the epithelial cells produced and deposited extracellular matrix proteins such as type IV collagen onto the type I collagen gel. Separately, we found that type IV collagen itself was capable of enhancing eotaxin-induced eosinophil migration in a standard chemotaxis assay. Neutrophils also efficiently migrated in the present transmigration system. Pre-treatment of epithelial cells with TNF-α and IL-4 enhanced eosinophil transmigration, while that of neutrophils was enhanced by TNF-α but suppressed by IL-4.Conclusion By utilizing a new in vitro transmigration system mimicking the airway mucosa, we have demonstrated that airway epithelial cells play an essential role in transmigration of eosinophils and that multiple factors such as chemokines, extracellular matrix proteins and exogenous inflammatory cytokines are involved in efficient transmigration.