PURPOSE: Neutrophils are considered key effector cells in the pathogenic mechanisms of airway inflammation in asthma. This study assessed the activation status of neutrophils in adult asthmatics, and the therapeutic potential of FTY720, a synthetic sphingosine-1-phosphate analog, on activated neutrophils using an in vitro stimulation model. METHODS: We isolated peripheral blood neutrophils (PBNs) from 59 asthmatic patients (including 20 aspirin-exacerbated respiratory disease [AERD] and 39 aspirin-tolerant asthma [ATA] groups). PBNs were stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) or lipopolysaccharide (LPS) and their activation status was determined based on reactive oxygen species (ROS) production, cell surface expression of CD11b, interleukin (IL)-8 and matrix metallopeptidase (MMP)-9 release. PBNs were primed with FTY720 to evaluate its anti-inflammatory action. RESULTS: In vitro PBN stimulation with fMLP or LPS induced a significant increase in ROS/CD11b/IL-8/MMP-9 levels (P < 0.05 for all). In asthmatics, fMLP-induced ROS level was significantly correlated with values of forced expiratory volume in 1 second/forced vital capacity (r = −0.278; P = 0.036), maximal mid-expiratory flow (r = −0.309; P = 0.019) and PC20 methacholine (r = −0.302; P = 0.029). In addition, ROS levels were significantly higher in patients with AERD and in those with severe asthma than in those with ATA or non-severe asthma (P < 0.05 for all). FTY720 treatment could suppress ROS/CD11b levels, and LPS-induced IL-8 and MMP-9 levels (P < 0.05 for all). Responders to FTY720 treatment had significantly higher neutrophil counts in sputum (P = 0.004). CONCLUSIONS: Our findings suggest a useful in vitro PBN stimulation model for evaluating the neutrophil functional status and the therapeutic potentials of neutrophil-targeting candidates in asthmatics.
Adult ; Asthma ; Fingolimod Hydrochloride ; Forced Expiratory Volume ; Humans ; In Vitro Techniques ; Inflammation ; Interleukin-8 ; Interleukins ; Methacholine Chloride ; N-Formylmethionine Leucyl-Phenylalanine ; Neutrophil Activation ; Neutrophils ; Phenotype ; Reactive Oxygen Species ; Sputum ; Vital Capacity