Survanta®, Curosurf®, and the major surfactant phospholipid dipalmitoylphosphatidylcholine (DPPC) on interleukin-8 (IL-8) gene and protein expression in human A549 lung epithelial cells using immunoassay and PCR

This article is available online at http://www.jlr.org crucial sites for innate immune responses. In addition to providing a physico-chemical barrier, the respiratory epithelium can respond to inhaled micro-organisms and irritants by releasing a variety of infl ammatory mediators ( 1–4 ). Inappropriate or excessive innate immune responses of respiratory epithelia could likely contribute to the development of respiratory diseases, as activation of these cells is documented in a variety of human diseases, such as asthma, chronic obstructive pulmonary disease, cystic fi brosis, and respiratory infections ( 2, 5–7 ). Stimulation of respiratory epithelial cells with infl ammatory mediators and infectious stimuli causes the increased expression and secretion of a number of cytokines with chemoattractant and pro-infl ammatory functions ( 1, 3, 4, 8, 9 ). Interleukin-8 (IL-8), a principal chemokine released by lung epithelial cells, plays a pivotal role in the recruitment of infl ammatory cells into the lung ( 10, 11 ). In addition, this cytokine upregulates the expression of adhesion molecules on neutrophils ( 12 ), promotes their transendothelial migration ( 13 ), and stimulates the oxidative burst and the release of lysosomal enzymes ( 14 ). Thus, IL-8 plays a pivotal role in the recruitment of neutrophils into the lung and their activation to clear infection. However, several studies have suggested that overexpression of IL-8 is central to pathophysiologic changes in the airways and to the severity of airfl ow obstruction in chronic infl ammatory lung diseases, such as chronic obstructive pulmonary disease, cystic fi brosis, and bronchiectasis ( 9, 15–17 ). There is also accumulating evidence that Gram-negative bacterial infections play a role in the pathology and exacerbation of the infl ammatory responses Abstract In addition to providing mechanical stability, growing evidence suggests that surfactant lipid components can modulate infl ammatory responses in the lung. However, little is known of the molecular mechanisms involved in the immunomodulatory action of surfactant lipids. This study investigates the effect of the lipid-rich surfactant preparations Survanta®, Curosurf®, and the major surfactant phospholipid dipalmitoylphosphatidylcholine (DPPC) on interleukin-8 (IL-8) gene and protein expression in human A549 lung epithelial cells using immunoassay and PCR techniques. To examine potential mechanisms of the surfactant lipid effects, Toll-like receptor 4 (TLR4) expression was analyzed by fl ow cytometry, and membrane lipid raft domains were separated by density gradient ultracentrifugation and analyzed by immunoblotting with anti-TLR4 antibody. The lipid-rich surfactant preparations Survanta®, Curosurf®, and DPPC, at physiological concentrations, signifi cantly downregulated lipopolysaccharide (LPS)-induced IL-8 expression in A549 cells both at the mRNA and protein levels. The surfactant preparations did not affect the cell surface expression of TLR4 or the binding of LPS to the cells. However, LPS treatment induced translocation of TLR4 into membrane lipid raft microdomains, and this translocation was inhibited by incubation of the cells with the surfactant lipid. This study provides important mechanistic details of the immune-modulating action of pulmonary surfactant lipids. —Abate, W., A. A. Alghaithy, J. Parton, K. P. Jones, and S. K. Jackson . Surfactant lipids regulate LPSinduced interleukin-8 production in A549 lung epithelial cells by inhibiting translocation of TLR4 into lipid raft domains. J. Lipid Res . 2010. 51: 334–344.