Allergy Asthma Immunol Res.  2019 May;11(3):381-393. 10.4168/aair.2019.11.3.381.

Evaluation of Neutrophil Activation Status According to the Phenotypes of Adult Asthma

• Kim SH
• Uuganbayar U
• Trinh HK
• Pham D
• Kim N
• Kim M
• Sohn H
• Park HS

• KMID: 2442049
• DOI: http://doi.org/10.4168/aair.2019.11.3.381

Abstract

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.

Keyword

Neutrophils; asthma; reactive oxygen species; FTY720

MeSH Terms

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
Fingolimod Hydrochloride
Interleukin-8
Interleukins
Methacholine Chloride
N-Formylmethionine Leucyl-Phenylalanine
Reactive Oxygen Species

Figure

• 

  Fig. 1 In vitro PBN activation under fMLP or LPS stimulation. PBNs were isolated from asthmatics, stimulated under (A) fMLP stimulation or (B) LPS stimulation in 1 hour. The MFI of DCF fluorescence of PBNs and CD11b expression percentage were measured by flow cytometry; IL-8 and MMP-9 in the supernatants were measured by enzyme-linked immunosorbent assay. Data are presented as means ± standard deviation. P values were analyzed by paired t-test. fMLP, N-formyl-methionyl-leucyl-phenylalanine; LPS, lipopolysaccharide; PBN, peripheral blood neutrophil; IL, interleukin; MMP, matrix metallopeptidase; ROS, reactive oxygen species.

• 

  Fig. 2 Correlations of PBN-derived ROS production with the severity of airway obstruction and bronchial hyper-reactivity. PBNs from asthmatics were stimulated with fMLP and measured for the DFC fluorescence intensity. Correlations of ROS and (A) FEV1/FVC; (B) MMEF (%) predicted values; (C) PC20. P values were analyzed by Pearson correlation coefficient analysis. FEV1, forced expiratory volume in 1 second; FEV1/FVC ratio, a ratio of forced expiratory volume in 1 second to forced volume vital; MMEF, maximal mid-expiratory flow; PC20 methacholine, methacholine provocative concentration causing a 20% drop in FEV1; ROS, reactive oxygen species; MFI, mean fluorescence intensity.

• 

  Fig. 3 Inhibitory action of FTY720 on neutrophil activation in asthmatics. PBNs were isolated from asthmatics, treated with FTY720 and stimulated under (A) fMLP stimulation or (B) LPS stimulation in 1 hour. P values were analyzed by paired t test. fMLP, N-formyl-methionyl-leucyl-phenylalanine; LPS, lipopolysaccharide; PBN, peripheral blood neutrophil; IL, interleukin; MMP, matrix metallopeptidase; ROS, reactive oxygen species.

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