Osteopontin expression and relation to disease severity in human asthma

Study population

A total of 54 persistently asthmatic patients (35 with MMA and 19 with SA) and 17 non-atopic healthy controls participated in the present study and were followed for ≥1 yr. From this population, 29 patients with steady-state asthma (12 MMA and 17 SA) and nine healthy controls consented to undergo research fibreoptic bronchoscopy with endobronchial biopsy and BALF collection when possible. Serum samples were collected from all participants in the steady state and the 17 patients that exhibited an exacerbation during the study. In addition, a subpopulation of 17 patients with MMA and SA agreed to receive 2 weeks of placebo-controlled treatment with oral corticosteroids (11 patients received 40 mg prednisolone and six received placebo; 2:1 ratio) in order to evaluate the effect of steroids on serum OPN expression. Informed and written consent was obtained from all participating subjects, and the corresponding protocol was approved by the Hospital Research Ethics Committee of Athens Chest Hospital, as well as the Greek National Organization for Medicines (both Athens, Greece). Subject characteristics are summarised in table 1, and patient groups and procedures are presented in a flow diagram (fig. 1).

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Figure 1–

Flow diagram showing patient groups and procedures included in the present study. MMA: mild-to-moderate asthma; SA: severe asthma; BALF: bronchoalveolar lavage fluid.

Asthma was defined as a clear clinical history with current symptoms plus 15% reversibility in forced expiratory volume in 1 s (FEV₁) after two puffs of β₂-agonist and/or positive methacholine challenge. All patients had a physician-confirmed diagnosis of persistent asthma and were receiving therapy depending on their asthma severity, according to Global Initiative for Asthma guidelines 16. Asthma exacerbations were defined as events of severe deterioration of symptoms and rescue medication use that required the use of systemic corticosteroids, or an increase from a stable maintenance dose, for ≥3 days 17. All asthmatic patients had been attending the asthma outpatient clinic of Athens Chest Hospital for ≥2 yrs prior to the study. None of the patients were treated with theophylline, antileukotrienes or anti-immunoglobulin (Ig)E during the study. In addition, none of the participants had shown upper respiratory tract infection for 2 months prior to bronchoscopy or serum sample collection. Healthy individuals had no history of asthma or any other chronic disease, were not receiving any medication and gave normal spirometric results. All subjects participating in the study were nonsmokers. Atopy was defined according to the recently revised nomenclature of the World Allergy Organization 18, and was assessed by means of skin-prick tests for 18 common aeroallergens (HAL Allergy Benelux, Leiden, the Netherlands).

Pulmonary function testing

Pulmonary function tests were performed on the same day prior to bronchoscopy or on the day that serum samples were collected, as previously described 19.

Fibreoptic bronchoscopy

Bronchoscopy was performed on an outpatient basis at the Athens Chest Hospital according to published recommendations 20. All bronchoscopic procedures were performed by the same experienced bronchoscopist using a flexible bronchoscope (WM-N60 mobile workstation; Olympus, Tokyo, Japan). After inspection of the bronchial tree, a mini-lavage was performed according to the most recent European Respiratory Society Task Force guidelines 21. Briefly, 100 mL pre-warmed 0.9% saline were instilled into the right middle lobe in 20-mL aliquots and then gently aspirated. The first aliquot collected was discarded. Only BALF samples with a recovery rate of ≥60% were kept for further analysis, which was the case in six healthy controls and 17 patients with asthma. Endobronchial biopsy specimens were obtained following BALF collection, from various sites of the subsegmental carinae of the right lower lobes or right middle lobe. A minimum of three bronchial biopsy specimens were taken from each subject.

Sample collection and storage

Blood samples were centrifuged for 15 min at 1,000×g at room temperature. BALF samples were centrifuged for 15 min at 300×g at 4°C. Serum and BALF supernatant samples were aliquoted and stored at -80°C within 2 h after collection. BALF cell pellets were resuspended in 1 mL PBS and used for total and differential cell counts (table 2). Peripheral blood cell counts were also performed (table 3).

ELISA

OPN concentration in serum and BALF was determined in duplicate using an ELISA kit (DY1433; R&D Systems, Wiesbaden, Germany) according to the manufacturer’s guidelines. Serum samples were diluted 50-fold before measurement. OPN levels in all samples were higher than the calculated detection limit of the assay (0.045 ng·mL⁻¹). The intra- and inter-assay variability were determined to be 7.8 and 9.8%, respectively. The stability of the frozen serum and BALF samples was tested in 16 randomly selected samples from both patients (n = 10) and controls (n = 6), as previously described 19. The evaluation of the stability of OPN did not show significant differences among the four measurements performed in the serum (1st week: 39.93 (24.99–54.72) ng·mL⁻¹, after 4 weeks: 41.61 (25.58–50.61) ng·mL⁻¹, after 8 weeks: 40.59 (23.48–52.35) ng·mL⁻¹, and after 12 weeks: 38.89 (23.35–49.75) ng·mL⁻¹; p = 0.1) and BALF samples (1st week: 1.036 (0.939–1.329) ng·mL⁻¹, after 4 weeks: 0.962 (0.785–1.312) ng·mL⁻¹, after 8 weeks: 1.037 (0.698–1.357) ng·mL⁻¹, and after 12 weeks: 0.9915 (0.755–1.279) ng·mL⁻¹; p = 0.29).

Immunohistochemistry for OPN

Immunohistochemical staining for OPN was performed as previously described 14. Bronchial biopsy specimens were fixed in 10% neutral buffered formalin overnight and embedded in paraffin. Sections 3–4 μm thick were deparaffinised in xylol and rehydrated in a graded alcohol series. Endogenous peroxidase activity was inhibited using 0.3% H₂O₂ in methanol for 15 min. Heat-induced epitope retrieval was performed in pre-heated 10 mM citrate buffer (pH 6.0) using a microwave oven (600 W) for 15 min. Blocking of unspecific binding sites was performed by incubating slides with 10% goat serum for 30 min. Mouse anti-human monoclonal OPN antibody (diluted in PBS containing 1% bovine serum albumin BSA and 0.1% Triton X-100; MAB1433; R&D Systems; 1 μg·mL⁻¹) was applied overnight at 4°C, followed by a 45-min incubation at room temperature with a biotinylated secondary goat anti-mouse antibody (BA-9200; Vector Laboratories, Burlingame, CA, USA). Slides were developed with the streptavidin–biotin complex–peroxidase reagent and the peroxidase substrate diaminobenzidine (DAB substrate kit (SK1400); Vector Laboratories) according to the manufacturer’s instructions. Negative controls were performed by substitution of the primary antibody with the same concentration of the corresponding isotypic IgG control (R&D Systems). All sections were counterstained with haematoxylin. All stained sections were coded and examined in a blinded manner at the end of the study. All available sections from each patient were evaluated. Biopsy specimens were considered suitable for examination when there was ≥1.0 mm of basement membrane length with intact epithelium and ≥0.1 mm² of subepithelial area. At least three suitable biopsy sections were examined per subject.

Quantitative measurements of OPN-positive cells in the bronchial tissue were performed as previously described 14. Briefly, OPN-positive and -negative cells were counted in each biopsy specimen separately in the intact epithelium and the submucosa. Bronchial epithelial cells positive for the OPN monoclonal antibody were expressed as a percentage of total epithelial cells, whereas positive subepithelial inflammatory cells were expressed as a percentage of total cells. The intra-observer error was assessed by performing three separate counts of the same section on different occasions and was calculated to be <8%.

RBM thickness and goblet cell number measurements

Analysis of RBM thickness and subepithelial infiltration was performed on sections stained with haematoxylin–eosin as previously described 22. Briefly, basement membrane thickness was measured in micrometers by performing measurements at 20-μm intervals along the whole length of the membrane, taking only the adequately preserved and oriented area into account. A semi-quantitative scoring system, ranging 0–3, was used to grade the number of inflammatory cells infiltrating the subepithelial layer (table 2). Goblet cells were examined on lung sections stained with periodic acid–Schiff (395B-1KT; Sigma-Aldrich, Steinheim, Germany). The reaction of periodic acid selectively oxidises glucose residues and creates aldehydes that react with the Schiff reagent, staining carbohydrates in goblet cells a purple-magenta colour. Goblet cells were quantified as the number of positively staining cells per millimetre of basement membrane, as previously described 23. Sections were observed using an Axioskop40 microscopy system (Carl Zeiss Jena, Jena, Germany) coupled with an AxioCam MRc digital camera, and basement membrane thickness measurements were performed using AxioVision v4.5.0.0 image analysis software.

Double immunofluorescence

Double immunofluorescence staining was performed in order to identify the cellular source of OPN. Antigen retrieval was performed (S2368 and S2369; Dako, Glostrup, Denmark) and then sections were blocked for 30 min in 10% goat serum, followed by incubation with primary antibodies (diluted in Tris-buffered saline containing 1% BSA and 0.1% Triton X-100) for 1 h at room temperature. The primary antibodies that were used in appropriate combinations were as follows: mouse anti-human OPN (MAB1433; R&D Systems; 2 μg·mL⁻¹), rabbit anti-human OPN (O7264; Sigma-Aldrich; 5 μg·mL⁻¹), smooth muscle α-actin (α-SMA) (Ab5694; Abcam, Cambridge, UK; 1:100), T-lymphocytes (CD3) (M7254; Dako; 1:5), B-lymphocytes (CD19) (M7296; Dako; 1:5), mast cells (mast cell tryptase) (clone AA1 (M7052); Dako; 1:50), neutrophils (neutrophil elastase) (clone NP57 (M0752); Dako; 1:100) and eosinophils (EG2 (No. 10-9196-01); Kabi Pharmacia Diagnostics, Uppsala, Sweden; 1:50). The secondary antibodies used were the Alexa Fluor (AF) 555-labelled goat F(ab’)₂ anti-mouse and the AF488-labelled goat F(ab')₂ fragment fluorescent anti-rabbit secondary monoclonal antibodies (A11070 and A21425, respectively; Molecular Probes; 5 μg·mL⁻¹; incubation for 40 min at room temperature). Antigen retrieval was not performed for the OPN/neutrophil elastase and OPN/EG2 combinations, as it was found destructive of the epitopes. Finally, tissue sections were mounted with ProLong Gold Antifade Reagent (P36931; Molecular Probes, Eugene, OR, USA), which also contained 4′-6-diamidino-2-phenylindole (DAPI) as a nuclear counterstain, and were examined using an Axioplan2 microscope (Carl Zeiss Jena), with filters set for AF488 and AF555.

Confocal laser microscopy

Specimens that showed double staining on fluorescence microscopy were further examined using a confocal laser microscope. Fluorescence images of the tissue sections were acquired, using an Axiovert 200/LSM 510 META confocal microscopy system (Carl Zeiss Jena). DAPI was excited by the 405-nm laser diode and the fluorescence was collected using a long-pass (LP) 420 emission filter; the green (AF488) antibody was excited by the 488-nm argon ion laser line and the fluorescence was collected using a band-pass (BP) 505–530 emission filter, and the red (AF555) antibody was excited by the 543-nm green helium–neon laser line and the fluorescence was collected using an LP560 emission filter. Tissue sections were visualised using the confocal microscope at 1,024×1,024-pixel resolution through a Plan-Neofluar 40×/1.3 oil differential interference contrast objective with eight-times averaging in sequential scanning (multi-track) mode with the pinhole set to obtain an optical section of ∼1 μm in all channels.

Statistical analysis

Data are expressed as median with interquartile range (first and third quartiles) unless specified otherwise. Normality was assessed using Kolmogorov–Smirnov and D’Agostino–Pearson omnibus normality tests. Two-group comparisons were performed using the nonparametric Mann–Whitney U-test, whereas three-group comparisons were performed using the nonparametric Kruskal–Wallis one-way ANOVA accompanied by Dunn’s post hoc correction. Stability data were evaluated using the nonparametric Friedman test followed by Dunn’s post hoc analysis. The nonparametric Wilcoxon matched-pair test was applied to evaluate OPN expression in the stable state and during exacerbation. Correlation coefficients were calculated using the nonparametric Spearman’s rank method. A statistical software package was used for all data analysis and graph preparation (Prism v5; GraphPad, San Diego, CA, USA). A p-value of <0.05 was considered significant.