ISPUB.COM The Internet Journal of Asthma, Allergy and Immunology Volume 7 Number 2 1 of 14 Eosinophil Interaction With Matrix-Producing Lung Cells: An In Vitro Model System Employing AML14.3D10 Eosinophil-Like Cells To Interact With AH1F Fibroblasts And A549 Epithelial Cells J Meerschaert, K Nkhata, J Sedgwick, W Busse, D Mosher Citation J Meerschaert, K Nkhata, J Sedgwick, W Busse, D Mosher. Eosinophil Interaction With Matrix-Producing Lung Cells: An In Vitro Model System Employing AML14.3D10 Eosinophil-Like Cells To Interact With AH1F Fibroblasts And A549 Epithelial Cells. The Internet Journal of Asthma, Allergy and Immunology. 2008 Volume 7 Number 2. Abstract The extracellular matrix protein fibronectin is found in the airway space after bronchial provocation and can extend the life of eosinophils in vitro. Herein we describe a system to study the effect of co-culture of eosinophil-like cells with cell lines derived from tissues. We compared integrin engagement on human peripheral blood eosinophils to several eosinophil-like cell lines, including EoL-3, AML 14.3D10, and HL-60 clone 15 cells. Effects of co-incubation of AML 14.3D10 cells with adherent cell AH1F fibroblasts or A549 epithelial cells were examined for adhesion, metabolic activity (reduction of cytochrome C), and fibronectin production. AML 14.3D10 cells had the ability to bind to the adherent cells, however the interaction was not reduced by blocking integrins on the eosinophil-like cells. The amount of fibronectin found in association with the extracellular matrix of AH1F fibroblasts or A549 epithelial cells as measured by ELISA was not altered when AML 14.3D10 cells were included as a co-culture. The co-cultures were separated into three fractions to measure fibronectin that was secreted into the medium or fibronectin associated with non-adherent cells, and also fibronectin associated with adherent cell extracellular matrices. Fibronectin was detected all three fractions, indicating that secreted fibronectin was not limited to incorporation within the extracellular matrix of adherent cells. Thus both fibroblasts and epithelial cells are capable of producing soluble cellular fibronectin and either may be the source of soluble cellular fibronectin found in the lungs of asthmatics after bronchoprovocation. INTRODUCTION Chronic asthma is characterized by airway remodeling and eosinophil efflux into the airway space. We have demonstrated a relationship between the matrix protein fibronectin and survival of eosinophils in in vitro cultures [1]. This soluble form of cellular (also known as tissue) fibronectin is found in increased amounts in the airway space BAL fluid of atopic subjects upon stimulation with antigen [2]. It is clear that the soluble form of cellular fibronectin causes human eosinophils to live longer in culture due to an autocrine production of GM-CSF [1]. It is not known the cellular origin of soluble cellular fibronectin within the airway space. Possibilities include lung tissue fibroblasts or airway epithelial cells. Integrins are the cell surface receptors for matrix proteins and certain Ig-family adhesion molecules. Eosinophils express alpha 4-beta1 and alpha 4-beta 7 integrins that bind to fibronectin and VCAM [3]. Engagement of the beta 7 integrin with monoclonal antibodies or the soluble form of cellular fibronectin results in increased message for GM- CSF and enhanced in vitro survival [1]. However, engagement of alpha 4-beta 1 by monoclonal antibodies did not have the same effect and neither did the soluble form of VCAM indicating a specific interaction of fibronectin with beta 7 is needed. The message for fibronectin can be spliced into two major forms coding for either the plasma form of fibronectin found in the blood (produced as the primary splice variant in hepatocytes) or as the tissue (herein referred to as “cellular”) form produced by cells throughout the body such as fibroblasts. When secreted from cells in tissues, fibronectin can be integrated into the extracellular matrix and this process is highly controlled [4,5]. The fate of cellular fibronectin secreted by tissue cells that is not incorporated into matrix is not fully appreciated. At least