Annexin A1 in plasma from patients with bronchial asthma: its association with lung function

Fifty asthmatic patients were recruited and followed for 6.6 ± 3.6 years, and plasma ANXA1 levels were determined during the stable and exacerbated states (Table 1). Experiments involving patients were approved by Soonchunhyang University’s institutional review board (IRB). Eight BALB/c mice were exposed to saline (control) or ovalbumin (OVA). Detailed analyses included evaluation of lung ANXA1 phospho Tyr21 levels and lung histology and FPR2 levels in bronchoalveolar lavage fluid (BALF) and lung tissue. Cells were treated with titanium dioxide nanoparticles (TiO₂) and dexamethasone (DEX). The animal studies were approved by Soonchunhyang University’s institutional animal care and use committee.

All patients were recruited from Soonchunhyang University, Bucheon Hospital. Asthma diagnoses were based on the Global Initiative for Asthma (GINA) guidelines [21]. This study used the same sample examined by Moon et al. [22] although several different measures and analyses are presented. The biospecimens and data used in this study were provided by the biobank of Soonchunhyang University Bucheon Hospital, a member of the Korean Biobank Network.

All subjects had a clinical diagnosis of asthma supported by one or more of the following criteria: 1) variation in the maximum diurnal peak expiratory flow >20% over the course of 14 days, 2) an increase in forced expiratory volume in 1 s (FEV₁) of >15% after inhalation of 200–400 μg albuterol, or 3) a 20% reduction in FEV₁ in response to a stimulating concentration of inhaled methacholine (PC20 methacholine) < 10 mg/ml. All subjects underwent standardized assessments, which included analyses of induced sputum specimens, complete differential blood cell counts, immunoglobulin E (IgE) measurements, chest posteroanterior radiography, allergy skin prick tests, and spirometry. All data were collected at the time of diagnosis, before administration of the asthma medication.

Among asthmatic patients from the hospital, asthmatics matched to normal controls in terms of age, sex, and BMI were selected for the study. Normal control subjects were recruited from among the spouses of the patients or members of the general population who answered negatively to a screening questionnaire regarding respiratory symptoms and other allergic diseases, had FEV₁ values >80% predicted, PC20 methacholine level > 10 mg/ml, and normal findings on simple chest radiographs. Of the subjects who completed a follow-up period of at least 2 years, 50 were diagnosed using the GINA guidelines [21].

Asthma exacerbation was analyzed in subjects who had completed regular follow-up for at least 2 years. Asthma exacerbation was defined by the GINA guidelines as episodes of progressively increasing shortness of breath, cough, wheezing, chest tightness, or some combination of these symptoms, accompanied by decreased expiratory airflow and use of systemic corticosteroids (tablets, suspensions, or injections) or an increase in dose from the stable maintenance dose for at least 3 days and a hospitalization or emergency department visit due to asthma, requiring systemic corticosteroids.

ANXA1 levels in the blood of asthmatic patients was measured by Enzyme-linked immunosorbent assay (ELISA; R&D System, Minneapolis, MN, USA). To compare results from different plates, the optical densities (ODs) of the test samples were adjusted relative to the positive and negative control samples supplied in each kit. The mean OD of duplicate wells was calculated. The index value of each test sample was defined by the following formula: index = (OD of test sample – OD of negative control)/(OD of positive control – OD of negative control) × 100. Low detection limits were set at 0.119 ng/mL for ANXA1 according to the manufacturer’s recommendation.

Female 6-week-old BALB/c mice (6 weeks of age, weighing 20–24 g) were purchased from Charles River Korea (Orient Bio Inc., Seongnam, Korea). All mice were sensitized by intraperitoneal injection on days 0 and 14 with 50 μg grade V chicken egg OVA (Sigma-Aldrich, St Louis, MO, USA) emulsified with 10 mg hydroxyl aluminum in 100 μl Dulbecco’s phosphate buffered saline (D-PBS). On days 21–23, all mice received intranasal challenges with 150 μg grade III OVA (Sigma-Aldrich) in 50 μl D-PBS. Control mice were sensitized and challenged with saline. On day 24, airway hyper responsiveness (AHR) was measured, BALF was collected, and lung tissue was processed for protein, RNA, and hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC).

Mice were anesthetized with 2.5 mg/kg tiletamine and xylazine (Zoletil and lumpum; Bayer Korea Co, Seoul, Korea), and AHR was assessed following challenges with 0, 5, 20, or 100 mg/ml methacholine (Sigma-Aldrich). Measurements of airway hyperresponsiveness were conducted using an animal pulmonary instrument (OCP-3000) 1 min after each dose with 3 min between doses. The following day, BALF was obtained, centrifuged, and the supernatant stored (−20 °C). The cell pellet was resuspended for cell counting, and cytospin slides were prepared for stained with modified Diff-Quick stain. Differential cell counting was performed on at least 500 cells in each slide using standard morphological criteria under a light microscope. A portion of the lung was fixed in 4% phosphate-buffered paraformaldehyde, embedded in paraffin, sectioned (4 μm), and stained (H&E and IHC staining).

Mouse lung sections were deparaffinized and rehydrated in an ethanol gradient series. The sections were incubated with 1.4% hydrogen peroxide in methanol for 30 min to block endogenous peroxidases, with 1.5% horse serum to block non-specific binding, and then with the anti-rabbit ANXA1 primary antibody (1200, Thermo Fisher, Rockford, IL, USA). The next day, sections were treated with the ABC kit (Vector Laboratories, Burlingame, CA, USA). The color reaction was developed by staining with liquid DAB+ substrate (Golden Bridge International Inc., Mukilteo, WA, USA). After immunohistochemical staining, the slides were counterstained with Harris’s hematoxylin for 1 min. Images were analyzed with the ImageJ program (National Institutes of Health, Bethesda, Md).

The extracted lung tissues were homogenized in a protein lysis solution containing 50 mM Tris-HCl (pH 7.4), 50 mM NaCl, 0.1% SDS, 1% Triton X-100, 0.5 mM EDTA, and 100 mM PMSF in distilled water. After centrifuged at 14,000 rpm for 30 min at 4 °C, the soluble materials were collected. Mouse lung lysates and bronchoalveolar lavage fluid (BALF) proteins were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked in TBS with 5% skim milk and 0.1% Tween 20 for 1 h at room temperature before incubating with rabbit anti-ANXA1 (1:1000, Thermo Fisher) or rabbit anti-FPRL1/FPR2 (Novus Biologicals, Littleton, USA) or rabbit anti-ANXA1 (phospho Tyr21) (1:500, Genetex, San Antonio, TX, USA) (overnight, 4 °C). The membranes were then incubated for 1 h at room temperature with an HRP-conjugated secondary antibody (1:5000, Santa Cruz Biotechnology, Dallas, USA). Detection was performed using an enhanced chemiluminescence (ECL) plus Western Blot Detection System (ATTO, Tokyo, Japan) on X-ray film. The relative protein levels were determined by quantitative densitometry and were normalized to anti-β-actin monoclonal antibody (1:5000, Sigma-Aldrich) levels.

Normal human bronchial primary epithelial cells (NHBE) were purchased from Lonza (Lonza, Basel, Switzerland, cat#. CC-2540). NHBE cells were plated at 3000 cells/cm² in culture 75 cm² flasks in bronchial epithelial cell growth medium supplemented with the BEGM BulletKit™ (Bronchial Epithelial Cell Growth Medium) (Lonza, cat#: CC-3170) and cultured at 37 °C in a 5% CO₂ incubator. The medium was changed every 48 h, and the cells were grown to 80–90% confluence for 5 to 6 days. NHBE cells (second-passage) with density 1.5 × 10⁶ cells/ml were seeded on in 6-well plate with BEGM medium. The 24 h prior to the experiment, the medium was changed to BEBM basal medium. The cells were exposed to 100 μM TiO₂ and 10 μM DEX (Sigma-Aldrich, Melbourne, VIC, Australia). Additionally, control cell lines are not exposed to DEX and TiO₂. Cells were treated with different doses time (4, 8, 24 h).

Receiver operating characteristic (ROC) curve, the area under the curve (AUC) was calculated using significant predictors (as determined via multivariate regression) to derive best suitable cut-off values and to assess model discrimination and predictive accuracy. Determination of optimal cutoff points were conducted with SPSS statistical software package (ver. 20.0; SPSS Inc.; Chicago, IL, USA).

The data were double-entered into the SPSS statistical software package (20).

Data with a normal distribution were described by means and standard deviations and compared using a two-sample t-test; data with non-parametric distributions were described by the median and interquartile ranges (IQR) and compared using Mann-Whitney U-test (intergroup analysis) or Pearson’s χ² test for normally distributed, skewed, and categorical data, respectively. Differences between the patient populations were analyzed by χ² test with Fisher’s exact test when low expected cell counts were encountered. Different correlation coefficients were calculated the Spearman’s correlation analysis. p < 0.05 was deemed to indicate statistical significance.

Plasma ANXA1 levels in asthmatic patients (0.570 ± 0.043 ng/ml) were higher than those in healthy control subjects (0.312 ± 0.078 ng/ml; Interquartile range, IQR: 0.0–0.611), (p < 0.01) (Fig. 1a). Plasma ANXA1 levels were significantly lower in exacerbated patients (0.497 ± 0.058 ng/ml; IQR: 0.038–0.850) compared with stable patients (0.645 ± 0.061 ng/ml; IQR: 0.447–0.871), (p < 0.05) (Fig. 1a). Therefore, this study calculated the best cut-off values of the Annexin-A1 ELISA in patients. By receiver operating characteristic curve (ROC), the best cut-off value of Annexin-A1 ELISA was 0.5023 ng/mL in patients (sensitivity and specificity were 68% and 99.68%, resp.), and the area under curve (AUC) was 0.680 (95% CI; 0.596–0.829), (p < 0.001) (Fig. 1b).

The population characteristics are shown in Table 1. Plasma ANXA1 levels in controlled asthmatic patients were correlated with forced expiratory volume in 1 s (FEV₁%) pred. (r    = −0.191, p = 0.033) (Fig. 2a) and FEV₁/forced vital capacity (FVC), (r = −0.202, p = 0.024) (Fig. 2b). Smoking asthmatics tended to have lower levels of ANXA1 compared with non-smoking asthmatics (4.14 ± 0.31 vs. 4.56 ± 0.99 ng/ml; p > 0.05). There was no relationship between ANXA1 and age, smoking amount, body mass index, PC20, white blood cell count, or total IgE levels.

Airway hyperresponsiveness (AHR) was increased in the OVA sensitized and OVA challenged mice (OVA-OVA mice group) compared with saline sensitized and air challenged mice (Control mice group) (Fig. 3a). Inflammatory cells, including eosinophils, macrophages, lymphocytes and neutrophils were increased in the BAL fluid in OVA-OVA mice compared with control mice group (Fig. 3b). The results demonstrated that induced the infiltration of inflammatory cells into the BAL fluid of the OVA-OVA challenged mice.

ANXA1 protein levels were significantly increased in lung tissue (Fig. 4) and bronchoalveolar lavage fluid (BALF) (Fig. 5) of mice sensitized and challenged with OVA (OVA-OVA mice) compared with mice sensitized with saline and challenged with air (control mice). FPR2 (Fig. 6) protein levels were significantly increased in the lung tissue of OVA-OVA mice compared with control mice. ANXA1-phospho Tyr21 protein levels were significantly increased in the lung tissue (Fig. 7) of OVA-OVA mice compared with control mice. ANXA1 protein levels were increased (Fig. 8) in dexamethasone (DEX) and titanium dioxide (TiO₂) treated NHBE cells at 4, 8, 24 h (p < 0.05, controls vs. DEX or DEX + TiO₂ or TiO₂).