1. Introduction
Bronchial asthma is the most common chronic airway disease with over 250 million patients worldwide [
1]. It is characterized by chronic airway inflammation and remodeling, which result in a variety of respiratory symptoms such as wheezing, heavy cough, and shortness of breath [
2]. The severity of asthma varies from patient to patient depending on genetic as well as environmental factors [
2]. In most cases, asthma originates from an exposure to an environmental allergen followed by IgE-dependent sensitization [
3]. Although asthma can be divided into several different subphenotypes, T helper type 2 (Th2) response-driven allergic asthma is the most prominent [
4].
Asthma can only be controlled rather than cured. However, since the pathogenesis of asthma, especially allergic asthma, has been well researched during the last few decades, an increasing number of advanced treatments are being developed. According to the international guidelines of the Global Initiative for Asthma, the basic treatment of asthma involves administration of inhaled corticosteroids (ICS) [
5]. If the symptoms remain uncontrolled after the ICS treatment, administration of a long-acting β
2-agonist needs to be added to the treatment [
6]. If such treatments cannot completely control the asthmatic symptoms, additional treatments are induced to control the severe asthma. One such additional method includes the administration of macrolide antibiotics that have immunomodulatory and anti-inflammatory effects in addition to their antimicrobial activity [
7]. Recently, monoclonal antibody (mAb) drugs have been widely used in the treatment of various diseases, because they can aim for specific targets in a defined pathway. Several types of mAbs have been developed for the treatment of asthma. Anti-IgE mAb (omalizumab) was the first successful mAb that helps patients with asthma through prevention of mast cell and basophil degranulation by blocking IgE binding to FcεRI [
8,
9]. However, because of the complex pathology of asthma, they cannot be effective in every patient, which makes it necessary to develop precise treatments for specific phenotypes of asthma. For example, anti-IL-5 mAb (Reslizumab) and anti-IL-5Rα mAb (Benralizumab) have been developed to treat eosinophilic asthma because IL-5 is an important cytokine for the maturation and activation of eosinophils [
10]. Although anti-IL-5 or anti-IL-5Rα mAbs have therapeutic effects on patients with allergic asthma and eosinophilia, no therapeutic effects were observed in patients with moderate or neutrophilic asthma [
11].
In most tissues, leukocytes are generally recruited through a cascade of four steps: rolling, adhesion, crawling, and transmigration [
12]. In allergic asthma, inflammatory mediators are released after allergen exposure, which induce the expression of E- and P-selectins on the endothelial surface at the site of inflammation. These two selectins have similar functions in mediating leukocyte infiltration. The sialyl Lewis X (sLe
x) glycans on P-selectin glycoprotein ligand 1 (PSGL-1), which is expressed on the surface of leukocytes, bind to P- and E-selectins and capture the leukocytes in the blood flow, allowing them to roll along the direction of the blood flow [
13]. It has been proven that without the binding of PSGL-1 to E- or P-selectin, leukocytes cannot start rolling on the endothelial surface and the subsequent cascade of events cannot happen [
14,
15], leading to suppressed leukocyte infiltration to the sites of inflammation [
16]. Therefore, the leukocyte rolling step is a reasonable target for the treatment of asthma.
Based on the abovementioned mechanisms for leukocyte infiltration, we hypothesized that targeting sLe
x, the ligand for P- and E-selectin, with specific anti-glycan antibodies would be a potential therapy for asthma. In our previous study, we developed an anti-sLe
x mAb, termed F2, that specifically reacts with sLe
x glycans expressed in both humans and mice [
17]. The glycan array analysis showed that the mAb F2 specifically bond to sLe
x, but not to other glycans. Administration of F2 into mice significantly blocked lymphocyte homing to peripheral lymph nodes and suppressed leukocyte infiltration in a murine contact hypersensitivity model. Based on these findings, we hypothesized that mAb F2 should have therapeutic effects against asthma. In this study, to explore whether sLe
x could be a target for the treatment of allergic asthma, we investigated the expression of sLe
x in mouse eosinophils using the mAb F2 and examined its therapeutic effects in a murine model of allergen-induced asthma.
3. Discussion
In our study, asthma was first induced in WT mice and FucT-IV/VII DKO mice lacking sLe
x glycans. Previous research using the DKO mice showed that leukocytes from these mutant mice express no E- and P-selectin ligand activity and completely lack selectin-dependent cell adhesion [
18]. We therefore hypothesized that the lack of glycan-dependent leukocyte recruitment in the DKO mice should result in the attenuation of leukocyte infiltration in asthma. As expected, eosinophils from the DKO mice almost completely disappeared from the BALF after repeated OVA administration, indicating the importance of sLe
x in the infiltration of eosinophils in the asthma model. These results are consistent with the prior research using PSGL-1-deficient mice [
24] in which eosinophil recruitment was reduced in the asthma model, suggesting that the inhibition of glycan-selectin interaction would be a reasonable approach for the treatment of asthma [
25].
Inhibition of glycan-selectin interaction can be achieved by either blocking the selectins or blocking the ligands for selectins. Efforts have been made to develop selectin inhibitors that block selectin-glycan dependent leukocyte rolling. An oligosaccharide analog of sLe
x, called CY-1503, showed protective effects in a pig model of acute lung injury by interrupting the binding of sLe
x to P- and E-selectin [
26]. However, it was proved to have poor efficacy in clinical trials of the reperfusion injury, which might result from the poor metabolic stability in vivo [
27]. Non-oligosaccharide synthetic inhibitor TBC1269, also known as bimosiamose, is a pan-selectin antagonist against all the three types of selectins [
28]. It showed therapeutic effects in a sheep model of allergic asthma [
29]. However, the results of clinical trials using TBC1269 had diversity via the approach of drug administration. It reduced the late asthmatic reactions by 50% in asthma patients via the administration by inhalation [
30], but showed no effects via intravenous administration [
31]. Although the clinical trials of TBC1269 provided some promise as potential therapeutics for asthma, the overall strategy against selectins has been still challenging.
Although glycosylation of sLe
x on PSGL-1 plays an important role for the glycan-selectin interaction, PSGL-1 does not always function as a main ligand for E-selectin [
25], which makes targeting sLe
x glycans more reasonable for drug development. In addition, P-selectin is temporally expressed on endothelial cells upon stimulation, which makes it hard to define the right time window to block P-selectin itself. After expression, P-selectin is internalized within a short time period by endocytosis and newly synthesized P-selectin is stored in Weibel–Palade bodies in endothelial cells [
32]. In contrast, the sLe
x glycans are always expressed on the surfaces of effector cells such as eosinophils. Therefore, it is likely that blocking sLe
x glycans, the ligand for selectins, can be a more effective way for the treatment of asthma. Surprisingly, no such ligand inhibitors have been tested in clinical trials.
Protein-based selectin ligand inhibitors are expected to have better metabolic stability and thus better protective effects, although such trials are still under development [
25,
27]. We hypothesized that our mAb F2 can be one of such drug discovery candidates against asthma. It binds to sLe
x with high specificity and showed strong suppressive effects in blocking eosinophil infiltration in our asthma model. The number of total leukocytes and eosinophils in the BALF was strongly suppressed in the F2-administered group. In addition, the results of the histological analysis suggested a relationship between eosinophil infiltration and allergic responses, including overproduction of mucin in the airway and fibrosis of the respiratory tracts. Blocking eosinophil infiltration also suppressed the allergic immune responses in the F2-administered mice. The levels of IgE in the serum along with the Th2 cytokine, eosinophil-related chemokines, and granule protein were suppressed, indicating that the allergic immune response caused by the intranasal challenge was relieved by F2 administration. The reduced production of the Th2 cytokine IL-5 after F2 administration was also confirmed at the protein level using ELISA. These findings indicate that targeting sLe
x using the mAb F2 in a murine asthma model resulted in its therapeutic effects.
One might argue that preventing eosinophil infiltration into the sites of inflammation would lead to eosinophilia in the blood and affect the function of other tissues. However, we observed that the deficiency or blockage of sLe
x resulted in a decrease in the percentage of eosinophils in the peripheral blood in the asthma model. As eosinophils are differentiated and proliferated in the bone marrow [
33], we further investigated the eosinophils in the bone marrow in the allergic asthma model. As expected, the differentiation of eosinophils showed a decrease in the bone marrow in the DKO mice, and the WT mice treated with the mAb F2 after induction of asthma. Notably, IL-5 is responsible for the proliferation of eosinophils in the bone marrow and their translocation from the bone marrow into the blood [
34]. In addition, previous research has shown that circulating IL-5, but not local IL-5, is required for the development of an allergic asthma model [
35]. The immune cells, which can release IL-5, mainly include Th2 cells and innate lymphoid cells (ILC) [
36]. It has been shown that mature eosinophils can promote Th2 cytokine production from Th2 cells in allergic situations [
37]. Therefore, the blockage of eosinophil infiltration into the lungs in our study could have suppressed the release of IL-5 from Th2 cells. These results suggest that the deficiency or blockage of sLe
x locally can have a suppressive effect on eosinophil development in the bone marrow in asthma.
Based on the results of this study, we hypothesized that a positive feedback loop is involved in the progression of allergic asthma (
Figure 7). The initial part of the positive feedback loop might start from the inhalation of allergens, resulting in the activation of pathogenic Th2 cells in the lungs [
38]. The activated Th2 cells would then release Th2 cytokines including IL-5, IL-4, and IL-13. IL-5 is released into the peripheral blood to induce the maturation and proliferation of the eosinophil precursors in the bone marrow [
34]. IL-4 and IL-13 facilitate the class switching of B cells to release IgE [
39], which binds to mast cells. After repeated antigen exposure, histamine is released by the mast cells to damage the epithelial cells and activate the alveolar macrophages to release the CCL11 [
40], which attracts the eosinophils to the tissue. Eosinophils would then be recruited to the asthmatic lung through interaction with its sLe
x glycans and endothelial P-selectin to damage the airway epithelial cells by releasing the granule proteins such as MBP [
41] and to promote the Th2 cells to release more IL-4, IL-5, and IL-13, which results in the amplification of the whole inflammatory cascade [
42]. This form of positive feedback loop would result in the recruitment of more eosinophils to the lungs and worsen the inflammation. Our rolling assay proved that sLe
x is critical for the rolling of eosinophils on P-selectin expressing cells, so that the subsequent infiltration cascade [
43] can be stopped by blocking the function of sLe
x. Therefore, the results of this study collectively support the idea that the mAb F2 not only inhibits selectin-mediated infiltration of eosinophils into the asthmatic lung, but also breaks the cycle of the positive feedback loop in our OVA-induced asthma model.
Limitations of this research have to be mentioned. As previously reported, F2 also binds to a sulfated structure of sLe
x, called 6-sulfo sLe
x, expressed on the high endothelial venules (HEVs) in lymph nodes [
17]. 6-sulfo sLe
x serves as a ligand for L-selectin, which is responsible for the lymphocytes homing to lymph nodes [
44]. However, the effects of blocking L-selectin-ligand interactions could not be assessed in our present study, because F2 serves as a pan-selectin inhibitor which binds to a common glycan structure found in the ligands for L-, P- and E-selectins. Prior research based on L-selectin-deficient mice showed partially decreased airway hyperresponsiveness in a murine model of asthma, but the airway inflammation and leukocyte recruitment were unchanged in the KO mice compared with those in WT mice [
45], indicating that L-selectin was not as important as P- and E-selectin in a murine model of allergic asthma. Although in a sheep model of asthma, a monoclonal antibody called MECA-79 reactive against sulfated sLe
x showed suppressive effects in both the airway hyperresponsiveness and L-selectin-mediated leukocyte recruitment through ectopically induced HEV-like blood vessels [
46], this difference of L-selectin-dependency in the asthma models might be possibly due to the species difference. Considering the above as well as the results of our present study, we think it likely that the suppressive effects of F2 should be mainly mediated by blocking P- and E-selectin ligands, although the involvement of 6-sulfo-sLe
x, a major ligand for L-selectin, in the asthma model needs further investigation.
Administration of the mAb F2 achieved great therapeutic effects in the mouse model of asthma. Since F2 is a mouse mAb, it cannot be directly applied to asthma patients unless it is humanized. However, this study strongly supports the idea that drug development based on the control of sLex-dependent eosinophil infiltration could be applied as a novel treatment for allergic asthma.
4. Materials and Methods
4.1. Animals
Female C57BL/6J mice, aged 8–10 weeks, were purchased from Charles River Laboratories Japan and housed in the animal room of Chiba University. Mice doubly deficient in fucosyltransferase-IV (FucT-IV) and FucT-VII were generated and housed as previously described [
18]. All mice were treated in accordance with the guidelines of the Chiba University Animal Care and Use Committee.
4.2. OVA-Induced Mouse Model of Asthma
For sensitization, the mice received intraperitoneal injection of OVA (100 μg per mouse dissolved in 200 μL sterile PBS, Grade V, Sigma-Aldrich, St. Louis, MO, USA) emulsified in 100 μL ImjectAlum (Thermo Fisher Scientific, Waltham, MA, USA) on days 0 and 7. The mice were anesthetized using 3% isoflurane and then challenged by intranasal administration of 100 μg OVA in sterile PBS, for 5 consecutive days starting on day 14. In the antibody administration experiments, 200 μg mAb F2, which was purified from ascites and examined as previously described [
17], was administered intraperitoneally to each mouse on days 14, 16, and 18 before the immunization. The control mice received an intraperitoneal injection of 200 μL sterile-PBS.
4.3. Harvesting of Bronchoalveolar Lavage Fluid (BALF) and Lung Tissues
Twenty-four hours after the final intranasal challenge, the mice were anesthetized with sodium pentobarbital. The trachea was exposed and a 22G indwelling needle was inserted into it. The airway and lungs were washed with 1.5 mL ice-cold PBS supplemented with 2% FBS. The lung was filled with a 0.5 mL O.C.T. compound (Sakura, Tissue-Tek, Torrance, CA, USA) and stored at −80 °C until use. In some experiments, the lung was filled with 0.5 mL 10% formalin neutral buffer solution and then excised and fixed in 10 mL formalin for histological analysis. In other experiments, the lung was submerged in TRIzolTM Reagent (Thermo Fisher Scientific, Waltham, MA, USA) and stored at −80 °C for total RNA extraction.
4.4. Flow Cytometric Analysis of BALF, Blood and Bone Marrow
The BALF was filtered through a 70 μm nylon mesh and centrifuged at 440× g for 5 min at 4 °C. The supernatant was collected and stored at −80 °C for protein measurement. For the collection of bone marrow cells, the hind femur was dissected, and the cells were washed with 2 mL RPMI-1640 (Thermo Fisher Scientific, Waltham, MA, USA) using a 26 G needle. The red blood cells in the BALF and bone marrow were lysed using ammonium-chloride-potassium buffer (150 mM NH4Cl, 10 mM KHCO3, and 1 mM EDTA-2Na, pH 7.2). The remaining cells (leukocytes) were preincubated with anti-mouse CD16/32 antibody (Tonbo, San Diego, CA, USA, clone: 2.4G2) for 10 min at 4 °C to block the Fc receptors, and then with the following antibodies for 20 min at 4 °C: PE-Cy7-conjugated anti-mouse CD45 (BioLegend, San Diego, CA, USA, clone: 30-F11), APC-conjugated anti-mouse CD11c (BioLegend, San Diego, CA, USA, clone: N418), PE-conjugated anti-mouse Siglec-F (BD Biosciences, San Jose, CA, USA, Clone: E50-2440), FITC-conjugated anti-mouse CD4 (eBioscience, San Diego, CA, USA, Clone: RM4.5), PE-Cy7-conjugated anti-mouse CCR3 (BioLegend, San Diego, CA, USA, Clone: J073E5), and biotin-conjugated mAb F2. The samples were washed with PBS containing 0.1% BSA and centrifuged at 440× g for 5 min at 4 °C. The cells were incubated with BV421-conjugated streptavidin (BioLegend, San Diego, CA, USA, #405225) for 20 min at 4 °C, centrifuged at 440× g for 5 min at 4 °C, resuspended in 300 μL filtered-PBS and subjected to FACS analysis (Beckman Coulter, CytoFlex, Brea, CA, USA). The data were processed and analyzed using the FlowJo software version 10.6.1. (BD Biosciences, San Jose, CA, USA).
4.5. Enzyme-Linked Immunosorbent Assay (ELISA) for the Measurement of Serum IgE and IL-5
To detect OVA-specific IgE in mouse serum, the wells of a 96-well ELISA plate (Costar Assay Plate, half area, CORNING, Corning, NY, USA) were coated overnight with 2.0 μg/mL anti-mouse IgE (BioLegend, San Diego, CA, USA, clone: RME-1). After coating, the wells were blocked with 3% BSA in PBS for 1 h at room temperature. Diluted serum samples were added to the wells and incubated for 2 h. After washing with PBS containing 0.05% Tween 20, the wells were incubated with HRP-conjugated anti-mouse IgE (1:2000 dilution, Southern Biotech, Birmingham, AL, USA) for 1 h. After washing, 25 μL 1-step Ultra ELISA substrate (Thermo Fisher Scientific, Waltham, MA, USA) was added to the wells, and the optical density was measured at 450 nm using a 96-well spectrometer (Spectra Rainbow Thermo, TECAN, Menendorf, Switzerland). To determine the concentration of Th2 cytokine IL-4 and IL-5, the ELISA MAX Deluxe Set Mouse IL-4 (BioLegend, San Diego, CA, USA) and ELISA MAX Deluxe Set Mouse IL-5 (BioLegend, San Diego, CA, USA) were used, and all the steps were performed according to the manufacturer’s instructions.
4.6. Real-Time Quantitative PCR
Total RNA was extracted from the upper lobe of the lung tissues using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthesized using ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan), and the concentration was determined using NanoDrop One (Thermo Fisher Scientific, Waltham, MA, USA). Reverse Transcription quantitative PCR (RT-qPCR) was performed using the THUNDERBIRD SYBR qPCR Mix (Toyobo, Osaka, Japan). The expression of each mRNA was normalized to the expression of β-actin using the ΔΔCt method according to the manufacturer’s instructions (Thermal Cycler Dice TP870, Takara Bio Inc, Shiga, Japan). The primer sets used were as follows: β-actin, 5′-CATCCGTAAAGACCTCTATGCCAAC-3′ and 5′-ATGGAGCCACCGATCCACA-3′; IL-4, 5′-TCTCGAATGTAC-CAGGAGCCATATC-3′ and 5′-AGCACCTTGGAAGCCCTACAGA-3′; CCL11, 5′-TCCACAGCGCTTCTATTCCTG-3′ and 5′-TAAAGCAGCAGGAAGTTGGGA-3′; IL-5, 5′-AGCACAGTGGTGAAAGAGACCTT-3′ and 5′-TCCAATGCATAGCTGGTGATTT-3′; IL-13, 5′-AGACCAGACTCCCCTGTGCA-3′ and 5′-TGGGTC-CTGTAGATGGCATTG-3′; MBP, 5′-TTTGCAAACTTGACAAGACCCA-3′ and 5′-TGCATCCCTAGGCAAGTTCTC-3′.
4.7. Immunofluorescence
The lung tissue blocks frozen with the O.C.T. compound (Sakura, Tissue-Tek, Torrance, CA, USA) were cut into 12-µm-thick sections using a cryostat (HM525, Thermo Fisher Scientific, Waltham, MA, USA). For immunohistochemistry, the sections were fixed with ice-cold acetone, and the non-specific binding sites were blocked with PBS containing 3% BSA (Sigma-Aldrich, St. Louis, MO, USA) for 1 h. After washing, the sections were incubated overnight with the primary antibodies. The primary antibodies used were as follows: Alexa Fluor 488-conjugated anti-mouse CD45 (BioLegend, San Diego, CA, USA, Clone: 30-F11), Alexa Fluor 647-conjugated anti-mouse CD31 (BioLegend, San Diego, CA, USA, Clone:MEC13.3), Alexa Fluor 488-conjugated anti-mouse CD11c (BioLegend, San Diego, CA, USA, Clone: N418), and BV421-conjugated anti-mouse Siglec-F mAb (BD Biosciences, San Jose, CA, USA, clone: E50-2440). Sections stained with Alexa Fluor 488-conjugated anti-mouse CD45 and Alexa Fluor 647-conjugated anti-mouse CD31 mAbs were further incubated with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) for 1 h. After incubation, the sections were mounted with Fluoromount (Diagnostic BioSystems, Pleasanton, CA, USA) and air-dried for over 30 min. For hematoxylin and eosin (H&E) staining, the sections were fixed with 10% buffered formalin for 5 min. After washing with PBS, the sections were stained with hematoxylin for 5 min and then with eosin for 5 min. The sections were dehydrated with gradient ethanol and xylene before mounting with Mount-Quick (Daido Sangyo, Tokyo, Japan). All images were obtained using a fluorescence microscope (BZ-9000; Keyence, Osaka, Japan).
4.8. Histological Analysis
The lungs of mice were filled with 10% formalin (0.5 mL) after sacrifice. The collected lungs were fixed in 10 mL 10% formalin at room temperature overnight. The fixed lungs were washed twice with PBS and dehydrated for 1 h in the following order: 70%, 80%, 90% and 100% ethanol, and 100% xylene. After dehydration, the lungs were embedded in paraffin. Sections (2 μm) were cut and dried in air for 16 h at room temperature, and then deparaffinized in the order of 100% xylene, 100%, 90%, 80% and 70% ethanol. For H&E staining, the sections were stained with Mayer’s hematoxylin for 7 min and then with eosin for 5 min after washing with deionized water for 10 min. For Masson’s trichrome staining, the sections were stained with Weigert’s iron hematoxylin for 5 min, followed by washing in deionized water for 10 min. The sections were then stained with 0.8% Orange G solution for 10 min, Masson solution (Muto Pure Chemicals Co., Tokyo, Japan) for 7 min, 5% phosphotungstic acid solution for 15 min, and 2% Lightgreen solution (Muto Pure Chemicals Co., Tokyo, Japan) for 5 min. Between each step, the sections were washed with 1% acetic acid solution for 5 s. For periodic acid-Schiff staining, the sections were stained with 0.5% periodic acid solution for 10 min, followed by washing in deionized water. The sections were then stained with Schiff solution (Muto Pure Chemicals Co., Tokyo, Japan) for 20 min and sulfurous acid solution (Wako Co., Osaka, Japan) for 3 min three times. The sections were then stained with Mayer’s hematoxylin for 3 min, followed by washing in running water for 10 min. After staining, the sections were dehydrated in the following order: 70%, 80%, 90% and 100% ethanol, and 100% xylene before mounting with Mount-Quick (Daido Sangyo, Tokyo, Japan). All images were obtained using a fluorescence microscope (BZ-9000; Keyence, Osaka, Japan). A histology score was determined by the severity of leukocyte infiltration as follows: 0, normal; 1, less than 10 infiltrated leukocytes around the airway in the field; 2, 10 to 100 infiltrated leukocytes around the airway in the field; 3, moderate infiltration with a thin ring-shaped leukocyte infiltration (containing 100 to 1000 cells) around the airway in the field; and 4, severe infiltration with a thick ring-shaped leukocyte infiltration (containing more than 1000 cells) around the airway in the field captured using the 10× objective lens. Four fields were counted for each section and the histology score was calculated.
4.9. Ex Vivo Induction of Eosinophils
Bone marrow from C57BL/6J wild-type (WT) mice or FucT-IV/VII-doubly deficient (DKO) mice were collected as described above. Mouse hematopoietic progenitor cells were isolated from the bone marrow using the MojosortTM hematopoietic progenitor cell isolation kit (BioLegend, San Diego, CA, USA) according to the manufacturer’s instructions. The isolated cells were diluted to 105 cells/mL with IMDM medium (Thermo Fisher Scientific, Waltham, MA, USA supplemented with 10% FBS (BioWest, Riverside, MO, USA), 10 mM HEPES (Sigma-Aldrich, St. Louis, MO, USA), and 100 U/mL Penicillin-100 μg/mL Streptomycin (Wako Co., Osaka, Japan) and incubated in 24-well plates. Next, 100 ng/mL mouse FLT3 ligand (BioLegend, San Diego, CA, USA, #579702) and 100 ng/mL mouse SCF (BioLegend, San Diego, CA, USA, #550704) were added to the culture medium on days 0 and 2. The medium was partly replaced with medium supplemented with 10 ng/mL mouse IL-5 (BioLegend, San Diego, CA, USA, #581502) on days 4, 7, and 10. Eosinophil induction was determined using flow cytometry.
4.10. Eosinophil Rolling Assay
Mouse P-selectin (CD62P) cDNA was prepared by RT-PCR using total RNA from the mouse bone marrow and cloned into pcDNA3.1(+) Neo (Invitrogen, Waltham, MA, USA) expression vector. CHO-K1 cells were transfected with the expression vector, mouse P-selectin/pcDNA3.1(+) Neo, using NeonTM Transfection System (Thermo fisher Scientific, Waltham, MA, USA), and cultured in the presence of 1.2 mg/mL G418 (Nacalai tesque, INC., Kyoto, Japan). After cloning by limiting dilution, cells expressing mouse P-selectin were selected by flow cytometry using anti-mouse CD62P antibody (BD PharmingenTM, Franklin Lake, NJ, USA) and APC-conjugated anti-rat IgG F(ab’)2 (Santa Cruz Biotechnology, Dallas, TX, USA). CHO-K1 cells stably expressing mouse P-selectin were then cultured as monolayers in 35-mm culture dishes (Corning, Corning, NY, USA). Bone marrow-derived eosinophils were fluorescently labeled with 1 μM CFSE (Thermo Fisher Scientific, Waltham, MA, USA, Molecular Probes®, Eugene, OR, USA) at 37 °C for 20 min and incubated with or without 10 μg/mL purified F2 for 10 min at room temperature. After incubation, the dishes were equipped with a parallel plate flow chamber (GlycoTech Co., Gaithersburg, USA), according to the manufacturer’s instructions. CFSE-labeled eosinophils from the WT and DKO mice were resuspended in the rolling assay buffer (20 mM HEPES-NaOH, 150 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, and pH 7.4) containing 0.1% BSA at a density of 4 × 106 cells/mL and introduced into the flow chamber at a wall shear stress of 3.0 dynes/cm2 using a syringe pump Model 11 Plus (Harvard Apparatus Co., Holliston, MA, USA). Images were captured using an OptiMOSTM Scientific CMOS camera (QImaging Co., Kent, UK) equipped with an inverted microscope (Zeiss Axiovert S100, White Plains, NY, USA) and analyzed using Fiji ImageJ.
4.11. Migration Assay
Bone marrow cells were prepared as described above and diluted to 106 cells/mL. The diluted cells were incubated with PBS or 10 μg/mL F2 in PBS on ice for 20 min. Five hundred μl RPMI-1640 (Thermo Fisher Scientific, Waltham, MA, USA) was added to each well of the 24-well plate. Recombinant murine CCL11 (Biolegend, San Diego, CA, USA) was added to the medium at the concentration of 50 ng/mL. The uncoated transwell was inserted into each well carefully. After applying the cell suspension (2 × 105 cells per well), the 24-well plate was incubated at 37 °C for 4 h. After incubation, the number of eosinophils in the lower chamber was analyzed by flow cytometry.
4.12. Statistical Analysis
The data are presented as the mean ± SEM, as indicated in the figure legends. The statistical significance of differences was assessed using the Student’s t-test. Comparison including three groups was analyzed with one-way analysis of variance (ANOVA) and post hoc Tukey’s HSD test. Statistical significance was set at p < 0.05. Statistical analyses were performed using GraphPad Prism 7.0.