Thirty-nine nonsmoking adults (men and women) were included into the study: 22 patients with persistent mild to moderate asthma defined according the GINA criteria and 17 healthy subjects who comprised the control group. The patients were recruited from the Department of Pulmonology and Immunology, Hospital of the Lithuanian University of Health Sciences, Kaunas. The study protocol was approved by the Regional Biomedical Research Ethics Committee of the Lithuanian University of Health Sciences (BE-2-13). Each participant was informed about ongoing investigation and gave his/her written consent.

Patients with asthma had a clinical history of the disease for ≥1 year, current symptoms, airway hyperresponsiveness and positive skin prick test (≥3 mm) in response to house dust mites Dermatophagoides pteronyssinus (D. pteronyssinus) or Dermatophagoides farinae (D. farinae). All patients were not using inhaled, nasal or oral steroids at least 1 month before visits, short-acting β₂ agonists at least 12 h and long-acting β₂ agonists at least 48 h prior the lung function test, antihistamines and antileukotrienes—7 days before skin prick test and prior the lung function test. None of the patients had a history of smoking. Baseline forced expiratory volume in 1 s (FEV1) was more than 70% of the predicted value in all patients. All healthy subjects were nonsmokers, without symptoms of rhinitis or asthma, with normal findings of spirometry and methacholine challenge, and all showed negative results of skin prick test. Demographic and clinical data are presented in Table 1.

Pulmonary function was tested using a pneumotachometric spirometer “CustovitM” (Custo Med, Germany). Baseline forced expiratory volume in 1 s (FEV1), forced vital capacity (FVC), and FEV1/FVC ratio were recorded as the highest of three reproducible measurements. The results were compared with the predicted values matched for age, body height, and sex according to the standard methodology.

Airway responsiveness was assessed as changes in airway function after challenge with inhaled methacholine using a reservoir method. Methacholine was nebulized into a 10-L reservoir with a pressure nebulizer (Pari Provocation I; Pari, Stanberg, Germany). Aerolized methacholine was inhaled through a one-way valve at 5-min intervals starting with 15-μg methacholine dose and doubling it until a 20% decrease in FEV1 from the baseline or the total cumulative dose of 3.87 mg was achieved. The bronchoconstricting effect of each dose of methacholine was expressed as a percentage of decrease in FEV1 from the baseline value. The provocative dose of methacholine causing a ≥20% fall in FEV1 (PD20) was calculated from the log dose-response curve by linear interpolation of two adjacent data points.

All patients were screened for allergy by the skin prick test using standardized allergen extracts (Stallergenes S.A., France) for the following allergens: D. pteronyssinus, D. farinae, cat and dog dandruff, 5 mixed grass pollen, birch pollen, mugwort, Alternaria, Aspergillus, and Cladosporium. Temoin was used for a negative control, and histamine hydrochloride (10 mg/mL) was used for a positive control. Skin testing was read 15 min after application. The results of skin prick test were considered positive if the mean wheal diameter was ≥3 mm.

After baseline FEV1 and FVC measurements, subjects inhaled 10 mL of sterile hypertonic saline solution (3, 4, or 5% NaCl, Ivex Pharmaceuticals, USA) at room temperature. Nebulised solution was given for three periods of 5 min at most by an ultrasonic nebuliser ULTRA-NEB™ (DeVilbiss, USA). The subjects were instructed to cough sputum into containers. If any symptoms occurred, nebulisation was discontinued. It was stopped after expectoration of an adequate amount of sputum. In order to detect a possible decrease in FEV1, spirometry was performed after each inhalation. Sputum was poured into a Petri dish and separated from saliva. A 4-fold volume of freshly prepared 0.1% dithiothreitol (DTT; Sigma-Aldrich) was added. The mixture was vortexed and placed on ice and rocked on an orbital shaker OS-10 (BIOSAN, Latvia) for 15 min. Next, an equal volume of phosphate-buffered saline solution (PBS; Sigma-Aldrich) was added to DTT. The cell pellet was separated using a 40-μm cell stainer (Becton Dickinson, USA). The mixture was centrifuged for 10 min at 4°C; the supernatant was aspirated and stored at −70°C for later analysis. The total cell counts, percentage of epithelial cells, and cell viability were investigated using a Neubauer hemocytometer (Heinz-Herenz, Germany) under a microscope (BX43, OLYMPUS, USA) by employing the Trypan blue exclusion method. The cytospin samples of induced sputum were prepared using a cytofuge instrument (Shandon Southern Instruments, USA). Supernatant with and without DTT was stored in 2 mL microtubes at −70°C for further researches. The prepared sputum cytospins were stained by the May-Grünwald-Giemsa method for differential cell counts. Cell differentiation was determined by counting approximately 400 cells in random fields of view under a light microscope, excluding squamous epithelial cells. The cells were identified by standard morphological criteria, nuclear morphology, and cytoplasmic granulation.

Peripheral blood samples for eosinophil isolation were collected into sterile Vacutainer tubes with EDTA. Polymorphonuclear leukocytes (PMN) were isolated by high-density gradient centrifugation. The whole blood was layered on Ficoll-Paque PLUS (GE Healthcare, Finland) and centrifuged at 1000 g force for 30 min at room temperature, and then PMN were separated by hypotonic lysis of erythrocytes. Eosinophils were separated using a magnetic eosinophil isolation kit (Miltenyi Biotec, USA) according to the procedure previously described (Januskevicius et al., 2016). Manufacturer confirms that eosinophils separation kit do not influence eosinophils viability and separation efficiency are more than 97%. Eosinophils were counted using an ADAM automatic cell counter (Witec AG, Switzerland)—eosinophils viability must to be found at least 98%. To check eosinophil purity after magnetic separation, a diluted eosinophil suspension was fixed with methanol on a glass plate, stained with May–Grunwald–Giemsa dyes, and inspected by light microscopy to determine whether there was any other cell in the suspension.

Healthy human ASM cells, immortalized by stable expression of hTERT, as described previously (Gosens et al., 2006), were used for experiments. For all experiments, the same hTERT-ASM cell line was used at Passage 5 for all 39 investigating subjects, thus avoiding changes in ASM activity and viability that could result from repeated thawing and passage. Cells were cultivated in 75 cm² Falcon culture flasks under standard culture conditions of 5% CO₂ in air at 37°C with medium renewal every 2–3 days. For all experiments, passaged cells were grown on plastic dishes in Dulbecco's modified eagle medium (DMEM) (GIBCO®; Life Technologies, Paisley, UK) supplemented with streptomycin/penicillin (2% v/v; GIBCO®; Life Technologies), amphotericin B (1% v/v; GIBCO®; Life Technologies), and fetal bovine serum (10% v/v; GIBCO®; Life Technologies). After reaching sufficient confluency, cells were passaged by trypsinization. Cells were serum deprived in DMEM supplemented with antibiotics and insulin, transferrin, and selenium reagent (GIBCO®; Life Technologies) before each experiment to stop cell proliferation and avoid possible errors in proliferation and gene expression measurements due to the effects of mediators of bovine serum in growth medium.

Whole eosinophils were separated into 3 groups depended by peptides were incubated with: control eosinophils—without incubation with peptides; eosinophils incubated with integrins blocking peptide RGDS; eosinophils incubated with integrins non-blocking peptide (negative control) GRADSP. Respectively amount of eosinophils suspension in serum-free growth medium was taken and peptide solution was added to the final concentration of 0.125 mg/ml. Eosinophils with peptides were incubated for 1 h at 37°C. After incubation eosinophils were centrifuged, growth medium was removed and fresh serum-free growth medium was added.

Cell cultivation dishes with approximately 2 × 10⁵ ASM cells were prepared and co-cultures was made by adding eosinophil suspension with 0.5 × 10⁵ viable eosinophils to the ASM cells.

To observe and visualize cell growth, an inverted microscope (CETI Inverso TC100; Medline Scientific, Oxford, UK) was used which had a 10 × /22 mm wide-field eyepiece, a phase objective of 10 × /0.25, and an installed CETI Si-5C camera (Si-Cap software, ver. 2.1, 2012).

Airway smooth muscle (ASM) cells were seeded in six-well plates (2 × 10⁴ per well) and grown in DMEM//10% FBS for 3 days. Medium was removed and wells were washed twice with warm PBS. In serum-free DMEM (supplemented with 1% insulin-transferrin-selenium reagent) cells were incubated for 1 day, after which a co-culture with eosinophils was prepared.

Eosinophil adhesion was measured for up to 240 min thereafter, removing non-adherent cells by aspiration, then washing cultures twice with warm PBS. Adherent eosinophil abundance in the cultures was determined by measuring residual eosinophil peroxidase (EPO) activity as previously described (White et al., 1991). Because intercellular EPO levels may be intrinsically lower in eosinophils from asthmatic individuals owing to degranulation (Krug et al., 1999), increased EPO protein levels detected by eosinophil adhesion assay in the co-culture models can be assigned as a higher number of eosinophils stably adhering to the ASM cell surface.

To assay EPO activity, 462 μL DMEM without phenol red and 462 μL EPO substrate (1 mM H₂O₂, 1 mM o-phenylenediamine, 0.1% Triton X-100 in Tris buffer; pH 8.0) were added to each well. After 30 min at 37°C, 231 μL of 4 M H₂SO₄ was added to each well to stop the reaction and absorbance was measured at 490 nm using a microplate reader. Results are expressed relative to substrate oxidation levels in control cells (ASM cells without incubation with eosinophils). Light absorbance in control cell wells is presented as 100% and eosinophil adhesion is expressed as the difference from the control value.

Cells for proliferation measurements were grown in six-well cluster plates in conditions described previously. Healthy ASM cells were co-cultured with their respective eosinophil group isolated from asthma patients and healthy individuals for 72 h. Following co-culture, cells were washed once with warm PBS, incubated for 4 min with warm PBS supplemented with 1 mM EDTA to detach eosinophils from ASM cells, and washed again with warm PBS to remove any remaining EDTA. Proliferation was evaluated by incubating wells with Hank's balanced salt solution containing alamarBlue® (10% v/v; Invitrogen™; Life Technologies, Paisley, UK). Conversion of alamarBlue® to the reduced form was dependent on the metabolic activity of ASM cells and was assayed by dual-wavelength spectrophotometry at wavelengths of 570 nm and 600 nm. As indicated by the manufacturer, the degree of alamarBlue® conversion is proportional to the number of viable cells. Data are expressed as the percentage increase or decrease in alamarBlue® conversion by ASM cells compared with control cells (ASM cells cultured without eosinophils), which did not proliferate during the co-culturing period because of serum-free growth medium.

TGF-β₁ and ECP protein levels in co-culture growth medium were measured using ELISA according to the manufacturer's instructions. Twenty-four hours before experiments, growth medium with fetal bovine serum was changed to fresh serum-free medium at the same volume for all wells. After a 24-h co-culture, growth medium was collected in new 10 mL tubes, centrifuged at 400 × g for 10 min at 25°C to separate non-attached cells, and 2 mL cryogenic tubes containing 1 mL supernatant were frozen at −80°C for protein level analysis. The results are expressed as protein level per 1 mL growth medium. LDL for TGF-β₁ is 23.44 pg/mL, for ECP–1.9 pg/ml.

Total RNA was extracted using a miRNeasy mini kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Real-time PCR was performed using a Power SYBR® Green RNA-to-C_T™ 1-Step kit (Applied Biosystems, Foster City, CA) in a 7500 Fast Real-Time PCR system as follows: reverse transcription at 48°C for 30 min; activation of AmpliTaq Gold® DNA polymerase, UP (Ultra-Pure) at 95°C for 10 min; 40 cycles of denaturation at 95°C for 15 s; and annealing and extension at 60°C for 1 min. Real-time PCR data were analyzed using the comparative cycle threshold method (the amount of target gene was normalized to the endogenous reference gene 18S ribosomal RNA). A difference of 1 C_t (ΔC_t = 1, where C_t is the amplification cycle number) after normalization to the reference gene indicates a 2-fold higher expression level of the investigated gene. Relative differences were determined by normalization of test sample ΔC_q values to control sample ΔC_q values with the equation ΔC_q(control)−ΔC_q(test), and the differences in fold were expressed by 2^ΔΔCt value. Primers used to analyze gene expression are shown in Table 2.

Statistical analysis was performed with GraphPad Prism 6 for Windows (ver. 6.05, 2014; GraphPad Software Inc., San Diego, CA). Normally distributed data are mean and standard error of the mean (SEM). Significant differences between two independent groups were determined using the Mann–Whitney U-test. The Wilcoxon matched-pairs signed rank test was used for dependent groups. A p < 0.05 was considered statistically significant.

The main characteristics of the study participants are shown in Table 1. A total of 39 non-smoking adults participated in the study (22 steroid-free patients with intermittent or mild-to-moderate persistent asthma, and 17 healthy participants who constituted the control group). There were no significant differences in age, gender balance or FEV₁ existed between groups, however, the asthma group was unique in exhibiting airway hyperresponsiveness (PD20) as well as skin prick positivity. Notably, all asthma patients had significantly higher numbers of peripheral blood eosinophils, and increased eosinophil number in induced sputum (Table 1).

We observed that asthmatic eosinophils had 3.7 ± 1.0-fold of α4, 4.6 ± 1.4-fold of αM, 4.1 ± 1.1-fold of β₁ and 16.8 ± 4.9-fold of β₂ more mRNA, compared to eosinophils from healthy individuals (Figure 1A). We also determined the effects of eosinophil adhesion to cultured human ASM cells on the abundance of α4, αM, β₁, and β₂ mRNA (Figure 1B). We collected adherent asthmatic and healthy eosinophils after 24 h of co-culture with ASM cells, and determined that asthmatic eosinophils exhibited a 6.0 ± 1.8-fold increase in α4 mRNA compared to eosinophil that were not incubated with ASM cells (p < 0.05). In striking contrast, eosinophils from healthy donors exhibited no change in α4, β1, and β2 mRNA after 24 co-culture with human ASM cells (Figure 1B). We measured no change in other integrins subunits mRNA abundance after co-culture with ASM cells in eosinophils from either healthy or asthmatic donors.

From the calibration curve, eosinophil number significantly correlated with light absorbance value (Figure 2A). One hour was the optimal period of incubation with RGDS peptide (0.125 mg/mL) for sufficient formation of the electrostatic junction between the eosinophil integrin active center and the amino acid peptides. Moreover, in pre-experiments, we determined the most effective RGDS concentration that would have a significant effect on suppressing eosinophil integrin and determined that negative control GRADSP had no integrins blocking properties as EPO substrate oxidation by non-treated eosinophils and eosinophils incubated with GRADSP was equal (Figure 2B).

For all eosinophil adhesion measurements, the same eosinophil preparation protocol was used. This was a 1-h incubation with 0.125 mg/mL RGDS peptide or its negative control, GRADSP.

Eosinophils isolated from the asthma group showed significantly increased adhesion to ASM cells at all time points, with an average value of 58.83 ± 4.50%. Integrin-blocking peptide had a significant effect on reducing eosinophil adhesion in both the control and asthma groups, with similar percentage values. In the control group, adhesion decreased by an average of 9.54 ± 0.71% (Figure 2C), while blocking of eosinophil integrin in the asthma group reduced adhesion by an average of 13.53 ± 2.70% (Figure 2D).

We investigated differences in eosinophil TGF-β₁ gene expression between the asthma group and the control group. We found that the gene was expressed to a greater extent in the asthma group (4.32 ± 0.68–fold) compared with the control group (Figure 3A). Eosinophils from both groups were observed to induce TGF-β₁ gene expression in ASM cells, but the effect in the asthma group was significantly higher compared with the control group (asthma group gene overexpression: 3.50 ± 0.44–fold vs. control group gene overexpression: 2.26 ± 0.32–fold; p < 0.05) (Figure 3B).

Considering that direct contact with ASM cells is necessary to induce the effects of eosinophils, we investigated changes in TGF-β₁ gene expression after blocking eosinophil integrins. Incubation with RGDS reduced the influence of eosinophils from the control group from 2.26 ± 0.32–fold to 1.14 ± 0.34–fold, and from 3.50 ± 0.44–fold to 2.08 ± 0.46–fold for the asthma group (p < 0.05) compared with eosinophils incubated with GRADSP, the negative control (Figure 3B).

Changes in gene expression had an influence on circulating TGF-β₁ protein levels in growth medium of co-cultures. With ELISA of co-culture supernatant, we found that the highest protein level was in medium from co-cultures of ASM cells and eosinophils that were not subject to integrin-blocking from the asthma group (270.9 ± 18.5 pg/mL). For the TGF-β₁ level from ASM cells cultivated alone under the same conditions, there was an average difference of 162.7 pg per mL in the asthma group compared with the control group. In the control group, TGF-β₁ levels were lower because there was less expression of this growth factor gene in eosinophils and ASM cells after exposure (the concentration in ASM cells cultivated alone was 78.1 ± 14.3 pg/mL, while in co-cultures of control eosinophils and ASM cells, the protein level increased to 143.9 ± 29.1 pg/mL). Blocking eosinophil integrins with RGDS peptide significantly reduced the TGF-β₁ level in growth medium of co-cultures of eosinophils from the asthma group and ASM cells (reduced by 31 pg. per mL; p < 0.05). In the control group, a reduction was also seen, but because of too much variation in data, the p-value was over the significance limit (Figure 3C).

As studies have reported that ECP stimulated TGF-β₁ production in lung fibroblasts (Zagai et al., 2007), we tested the hypothesis that ECP released from eosinophils can induce overexpression of TGF-β₁ in ASM cells. To this end, we collected serum-free growth medium from co-cultures of eosinophils and ASM cells and used ELISA to estimate ECP protein levels.

The highest ECP protein level was observed in medium from co-cultures of control eosinophils without integrin blocking and ASM cells (1118.0 ± 132.4 pg/mL in eosinophils without contact to exogenous proteins, and 1196.0 ± 140.7 in eosinophil incubated with integrin non-binding peptide GRADSP). ECP protein levels were significantly lower in medium containing eosinophils from the asthma group (827.7 ± 118.3 pg per 1 mL in co-cultures with control eosinophils, and 865.2 ± 123.5 pg per 1 mL of growth medium in co-cultures with ASM cells and eosinophils incubated with the negative control GRADSP). Integrin-blocking peptide had a similar effect on reducing ECP production in both the asthma and control groups. Blocking eosinophil integrin in the control group showed a decrease in ECP level of 271.6 pg per 1 mL, whereas the asthma group showed a decrease of 139.6 pg per mL compared with eosinophils incubated with the negative control GRADSP (Figure 4).

We found that exposure to eosinophils from both the asthma and control groups increased WNT-5a gene expression in ASM cells, but the asthma group showed a significantly higher effect (asthma group increase in gene expression of the ligand: 2.95 ± 0.46–fold vs. control group increase: 1.79 ± 0.16–fold; p < 0.05). Reducing the ability of eosinophils to adhere to the surface of ASM cells or ECM protein by integrin-blocking using the RGDS peptide had a significant inhibitory effect on WNT-5a gene expression. Decreased TGF-β₁ levels in co-cultures of ASM cells and asthma or control eosinophils using integrin-blocking reduced WNT-5a gene expression, and significant increases in expression were not observed (Figure 5A).

We found that eosinophils from the asthma and control groups showed an increase in fibronectin gene expression in ASM cells by 3.51 ± 0.46–fold and 3.82 ± 0.41–fold, respectively, without any significant differences between the groups (Figure 5B). Eosinophils from the asthma group had a significantly greater effect on type-1 collagen gene expression in ASM cells compared with eosinophils from the control group (eosinophils from the control group showed an increase in gene expression of 3.47 ± 0.37–fold vs. the asthma group increase of 4.67 ± 0.53–fold; p < 0.05) (Figure 5C).

Eosinophil integrin-blocking had similar significant effects in both groups. Fibronectin gene expression showed a reduction in the asthma group to 2.50 ± 0.57–fold compared with the control group which was 2.30 ± 0.24–fold. Importantly, suppression of eosinophil integrin reduced the increased effect of eosinophils on collagen gene expression (eosinophils from the control group with integrin-blocking showed an increase in collagen gene expression by 2.46 ± 0.31–fold vs. 2.81 ± 0.53–fold for the asthma group) (Figures 5A,B).

Eosinophils isolated from peripheral blood of asthma patients had a greater effect on ASM cell proliferation compared with eosinophils from healthy control participants because of increased adhesion ability. After eosinophils from the asthma group were incubated for 72 h, we found that ASM cells showed increased proliferation of 22.41 ± 3.78%, whereas eosinophils from the control group only increased by 6.90 ± 1.53%.

After ASM cells were incubated with eosinophils from the asthma group and integrins were blocked, ASM cell proliferation reduced to 11.52 ± 2.50%, while eosinophil integrin-blocking in the control group showed that the proliferative effect was reduced to 2.85 ± 0.82% (Figure 6).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.