Female BALB/c mice (6 weeks old; weight, 20 ± 2 g) were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). The animals were housed under specific pathogen-free conditions and were maintained on a 12-hour light/dark cycle with food and water provided ad libitum. All animal experiments conducted in this study were approved by the Institutional Animal Care and Use Committee of Ajou University (IACUC 2017-0068).
Eosinophilic asthma mouse model and tirofiban treatment
BALB/c mice were intraperitoneally sensitized with 10 µg/mg ovalbumin (OVA) and aluminum hydroxide solution on days 0 and 14, followed by 3 nebulized OVA challenges on days 28-30 using an ultrasonic nebulizer (NE-SM1; Ktmed Inc., Seoul, South Korea) . On each challenge day, 5 mg/kg tirofiban was administered intraperitoneally 30 minutes before the challenge. The mice were assayed for the next experiments 48 hours after the last challenge.
Airway resistance measurement and sample collection
The FlexiVent system (Scireq, Montreal, QC, Canada) was employed to measure airway resistance. On the day indicated, the mice were anesthetized with pentobarbital sodium and intubated with a cannula. After connecting them to a computer-controlled small-animal ventilator, the mice were ventilated with a tidal volume of 10 mL/kg at a frequency of 150 breaths/min. The baseline airway resistance (RL) of each mouse was recorded. Subsequently, a dilution series of acetyl-β-methylcholine chloride (MCh) from 3.12 to 50 mg/mL were gradually introduced to the mice, and the RL values at each concentration were recorded.
Harvest of bronchoalveolar lavage (BAL) fluid and lung histology analysis
After measuring airway resistance, BAL fluid was harvested with a wash of 1 mL of PBS plus 2% bovine serum albumin (Sigma Aldrich, St. Louis, MO, USA). After the BAL fluid was centrifuged at 1,500 rpm for 5 minutes at 4°C, leukocytes were quantified with a hemocytometer, and differential cell counts were performed by counting at least 200 cells on cytospin slides stained with Wright–Giemsa stain. The supernatant was collected and stored at −70°C until further analysis. Tissue sections were evaluated using ImageJ (National Institutes of Health, Bethesda, MD, USA). To detect inflammatory cells, sections were stained with haematoxylin and eosin, and mucus-containing cells were stained with periodic acid-Schiff (PAS). The number of inflammatory cells per μm2 of perivascular and peribronchial areas and the number of mucus-containing cells per μm2 of basement membrane were determined.
Measurement of cytokine levels
The levels of IL-4, IL-5, IL-13 (eBioscience, San Diego, CA, USA), PF-4 (Abcam, Cambridge, UK), and eosinophilic cationic protein (ECP) (MyBiosource, Inc., San Diego, CA, USA) in the BAL fluid were measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions.
Identification of PEA in whole blood
Flow cytometry of PEA in whole blood was performed as previously described . Mouse whole blood was harvested by cardiac puncture into tubes containing 3.8% sodium citrate to prevent coagulation. The whole blood was incubated with phycoerythrin (PE)-conjugated anti-mouse Siglec-F and fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD41 for 30 minutes at room temperature (RT) in the dark. Next, red blood cells (RBCs) were lysed with the RBC Lysis/Fixation solution (Biolegend, San Diego, CA, USA) for 10 minutes and washed once with 1× PBS. The cells were analyzed immediately by flow cytometry with the BD FACSCanto II (BD Bioscience, San Diego, CA, USA) as previously described . A leukocyte gate was set in terms of size and granularity. Within the leukocyte gating, eosinophils were labeled by PE-conjugated anti-mouse Siglec-F. PEA was identified as CD41+ eosinophils (Siglec-F+/CD41+), and at least 1,000 events were recorded for each sample. A pooled sample from 3 mice was used to obtain a sufficient number of eosinophils for the analysis.
IF was performed using the immunofluorescence technique on 5mm -thick paraffin sections. After deparaffinization, tissue sections were sequentially incubated with blocking buffer (0.05% PBS-Tween 20 containing 5% bovine serum albumin and 10% normal donkey serum) for 1 hour at RT and then incubated with rabbit anti-EPX antibody (Bioss Antibodies), rat anti-PSGL-1 antibody (Ray Biotech) overnight at 4°C. For IF labeling, the sections were incubated with appropriate secondary antibodies. The Alexa Fluor 594 donkey anti-rat antibody (Life Technologies) and Alexa Fluor 488 donkey anti-rabbit antibody (Life Technologies) were applied for 50 minutes at 37°C. Finally, after nuclear staining with DAPI (0.5 mg/mL) for 5 minutes, biomeda mounting solution (Biomeda) was dropped on the glass slide, and the coverslips were inverted and placed onto glass microscope slides.
Detection MAC-1 of by western blotting
Thirty-five micrograms of protein was isolated from each tissue homogenate with RIPA buffer. Proteins was separated 12% SDS-PAGE gel and transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). Blocking in 5% skim milk (Sigma Aldrich) in Tris buffered saline containing 0.01% Tween 20 (TBST-T) for 1h at room temperature. The membranes were probed with primary antibodies against MAC-1 (bs-1014R, Bioss antibodies) and β-actin antibody (sc-47778, Santa Cruz Biotechnology) was used as an internal control. After extensive washing in Tween-TBS, The membranes were incubated with biotinylated secondary antibody for 1h at room temperature. Antibody binding was visualized using an ECL detection kit (GE Healthcare, Little Chalfont, UK), and images were acquired using a gel doc system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Antibodies and reagents
The antibodies used against the mouse target proteins were anti-PSGL-1 antibody (Q14242; Ray Biotech, USA), anti-EPX antibody (bs-3881R; Bioss Antibodis), Alexa Fluor 488-conjugated donkey anti-rabbit IgG (A21206), and Alexa Fluor 594-conjugated donkey anti-rat IgG (A21209) (ThermoFisher Scientific, Waltham, MA, USA). PerCP-conjugated anti-Ly6G (127654, Biolegend), APC-conjugated CD11c (117309, Biolegend), PE-conjugated anti-Siglec-F (552126, BD Bioscience), FITC-conjugated anti-CD41 (133904, Biolegend), and PE/Cy7-conjugated anti-CD62P (148310, Biolegend) antibodies were used for flow cytometry and cell sorting. All drugs used for treatment were obtained from Sigma-Aldrich unless indicated otherwise.
Data are presented as mean ± standard error. The differences between treatment groups were assessed using one-way analysis of variance and Tukey’s post hoc test unless indicated otherwise. The differences between the stimulated and control cells in the in vitro assay were assessed using the Wilcoxon signed-rank test. All statistical analyses were performed by using SPSS software version 23.0 (SPSS Inc., Chicago, IL, USA), and a P-value of <0.05 was considered significant.