Interactions between alveolar epithelial cells and neutrophils under pro-inflammatory conditions

BACKGROUND Intercellular communication is essential for defense and survival of the organism. The aim of the study was to find out whether there is an active crosstalk between airway cells constituting the first line of defense, alveolar epithelial cells (A549) and neutrophils, following activation with pro-inflammatory stimuli in vitro and to explore whether this communication is altered in chronic obstructive pulmonary disease (COPD), a condition characterized by chronic airway and lung inflammation. METHODS Blood neutrophils from healthy subjects and COPD patients were co-cultured with A549 cells in pure medium and in medium containing lipopolysaccharide (LPS), peptidoglycan (PGN), or tumor necrosis factor. The expression of Toll-like receptor 2 (TLR2), Toll-like receptor 4 (TLR4), and CD14 on the cell surface of neutrophils was assessed by flow cytometry, and release of CXCL8 (IL-8) and the soluble CD14 (sCD14) was measured in the supernatant with enzyme-linked immunosorbent assay (ELISA). RESULTS On neutrophils, the surface expression of TLR2 was diminished following activation with all three pro-inflammatory stimuli, and membrane bound (mCD14) and TLR4 expression were significantly increased in co-cultures compared to single cell cultures, irrespective of pro-inflammatory stimulation. There was a correlation between CXCL8 and sCD14 in LPS-stimulated co-cultured cells (r=0.82; p<0.01). CONCLUSION An active crosstalk between A549 cells and blood neutrophils was clearly demonstrated, both in unstimulated cells and following activation with pro-inflammatory stimuli, in vitro. Co-culturing implied synergy and correlation between LPS-induced release of sCD14 and CXCL8, which indicates that sCD14 may be donated by neutrophils to epithelial cells facilitating TLR4-signaling. Furthermore, TLR2 on neutrophils was found to be down-regulated by pro-inflammatory stimuli.