IDENTIFICATION OF EOTAXIN-3 REGULATED GENES AND PROTEINS IN ALVEOLAR TYPE II CELL

Florida Agricultural And Mechanical Univ, Tallahassee FL

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Abstract

DESCRIPTION (provided by applicant): Asthma is a lung inflammatory disease characterized by infiltration of the airways by inflammatory cells especially the eosinophils. The presence of these cells in the airway is due to their attraction by CC (cystein-cystein) chemokines including CCL11 (eotaxin), CCL24 (eotaxin-2) and CCL26 (eotaxin-3). Indeed, high levels of eotaxins have been detected in patients with asthma. We recently reported that alveolar type II cells exposed to TH2 cytokines, interleukin (IL) 4/13, release massive quantities of CCL26. The reason for this burst of CCL26 release is unknown but could be due to its ability to regulate other proteins and genes. Type II alveolar and bronchial epithelial cells provide good models for studying the regulatory mechanisms of this chemokine because they constitutively express CCR3, the major CC-chemokine receptor of the eotaxins. Our specific hypothesis is that CCL26 induced by cytokines (IL-3 and IL-4) during inflammation may bind to its receptor on the same cell and regulate the expression of proinflammatory/anti-inflammatory cytokines/chemokines as well as cell proliferation and differentiation proteins. Our long term goal is to identify the proteins and genes regulated by CCL26 and establish their importance in progression of inflammation, cell differentiation and proliferation. The specific aims are: (1) Identify various proteins and genes expressed by alveolar type II cells by detecting the intracellular proteins and genes using RayBio human cytokine antibody arrays, Clonetech antibody microarrays and cDNA microarrays;(2) Assess the effect of CCL26 antisense and recombinant protein on the expression of these proteins using similar methods and confirming the results by western blot, immunocytochemistry and real time PCR in alveolar type II and BEAS-2B cells (3) Determine the role of CCL26 in alveolar type II cell differentiation and proliferation by determining the changes in type II and type I cell specific markers using western blot and real time PCR and assessing cell multiplication using the Biovision Quick Cell proliferation assay. Successful completion of these experiments will lead to discovery of new genes and proteins which could be targeted for formulation of asthma therapeutic agents. It will also provide knowledge on functional interaction of eotaxin 3 with other proteins. PUBLIC HEALTH RELEVANCE: Asthma being a complex disease, global interconnection, of different proteins and genes in enhancing its progression has not been investigated. Identifying proteins and genes regulated by eotaxin 3 which is a key chemokine involved in asthma etiology will improve understanding as well as reveal new targets that could be used for designing asthma and other lung inflammatory disease therapeutic agents.