Altered Expression of Substance P and NK1R in CCR3+ and CD123+HLA-DR− Basophils Under Airway Allergic Conditions

- ¹Allergy and Clinical Immunology Research Centre, The First Affiliated Hospital of Jinzhou Medical University, Jinzhou, China.
- ²Department of the PLA Center of Respiratory and Allergic Disease Diagnosing Management, General Hospital of Northern Theater Command, Shenyang, China.
- ³Translational Medicine Research Centre, Shenyang Medical College, Shenyang, China.
- ⁴Department of Otolaryngology Head and Neck Surgery, The First Affiliated Hospital of Jinzhou Medical University, Jinzhou, China.
- ⁵Department of Respiration, The First Affiliated Hospital of Jinzhou Medical University, Jinzhou, China.
Correspondence to Huiyun Zhang, MD, PhD. Allergy and Clinical Immunology Research Centre, the First Affiliated Hospital of Jinzhou Medical University, No. 2, Section 5, Renmin Street, Guta District, Jinzhou 121001, China. Tel: +86-416-4605081; Fax: +86-416-4605082; Email: zhanghuiyun2003@126.com

Correspondence to Shaoheng He, MD, PhD. Translational Medicine Research Centre, Shenyang Medical College, No. 146, Huanghe North Street, Shenyang 110034, China. Tel: +86-24-62214090; Fax: +86-24-62214089; Email: shoahenghe@126.com

Received February 24, 2022; Revised August 11, 2022; Accepted August 16, 2022.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Purpose

To explore expression of SP and NK1R in basophils of allergic asthma (AA), allergic rhinitis (AR) and AR combined with AA (ARA), and influence of allergens and immunoglobulin E (IgE) mediated mechanisms on SP and NK1R expression.

Methods

Expression of SP and NK1R was detected by flow cytometry, NK1R mRNA expression was detected by real time quantitative polymerase chain reaction (qPCR), and mouse AR and AA models were employed for in vivo study.

Results

SP⁺ and NK1R⁺ cells increased in CCR3⁺ and CD123⁺HLA-DR⁻ granulocytes of AA. PPE elevated proportions of SP⁺ cells in CCR3⁺ and CD123⁺HLA-DR⁻ granulocytes, whereas ASWE and HDME augmented SP⁺ cells in CD123⁺HLA-DR⁻ granulocytes of AR and ARA patients. ASWE, HDME and PPE increased proportions of NK1R⁺ cells in CCR3⁺ PBMC and CD123⁺HLA-DR⁻ granulocytes of AR patients. OVA, Der p1, IL-33, IL-37, IgE and SP enhanced NK1R expression on KU812 cells. NK1R expressing basophils were increased in blood of OVA sensitized and challenged AR and AA mice. FcεRI-KO AA mice seemed to have less NK1R⁺ basophils than WT AA mice in their blood.

Conclusion

CCR3⁺ and CD123⁺HLA-DR⁻ cells are likely involved in AA and AR via SP and NK1R. IgE-related mechanism may participate in upregulation of NK1R expression.

Keywords

Substance P; neurokinin 1 receptor; allergy; asthma; rhinitis; IgE; allergen

INTRODUCTION

Allergic asthma (AA) is a chronic, inflammatory condition of the lower airways characterized by largely reversible airflow obstruction, airway hyperresponsiveness, and wheezing.1 Allergic rhinitis (AR) is a disorder of the upper airways resulting from immunoglobulin E (IgE)-mediated inflammation of the nose upon contact of the nasal mucosa with allergens.2 Often, asthma and AR are comorbid conditions,2 with AR being a major risk factor for the occurrence of asthma.3 However, the pathophysiological mechanisms of these diseases remain obscure.

In recent years, CCR3⁺ and CD123⁺HLA-DR⁻ cells4 in peripheral blood peripheral blood mononuclear cells (PBMCs) have been recognized as basophils, which are primary effector cells of allergy, including AA5 and AR.6 Prior studies have shown that AA patients exhibit phenotypically distinct basophil populations in the peripheral blood, some of which respond robustly to IgE-mediated activation.5 Basophils have been identified in the nasal washes of patients with AR and are thought to be the dominant source of histamine in late-phase responses to allergen challenge in patients.6 Studies have identified that basophils are highly enriched in the post-mortem lung tissue of patients who have died of asthma as well as in bronchial biopsies of patients with asthma.7

The involvement of substance P (SP) in basophil degranulation and accumulation has been highlighted.8 SP proved to be a potent chemoattractant for human basophils in vitro acting via its specific receptor neurokinin-1 receptor (NK1R) in basophils.9 Increased amounts of SP are found in sputum and BAL fluid of asthmatics10 ^, 11 as well as in nasal secretions of AR patients.12 SP causes many of the typical changes observed in asthmatic airways, including bronchoconstriction,13 increased mucus secretion, and plasma leakage.14 Some studies have shown upregulated NK1R expression in asthmatic lung compared to normal controls.15 These findings suggest a role of SP and NK1R in allergic inflammatory processes. However, little is known about the expression of SP and NK1R in basophils in patients with AR and AA.

It has been reported that the house dust mite (HDM) protease Dermatophagoide pteronyssinus (Der p) 1 can promote the production of interleukin (IL)-4, IL-5 and IL-13 from human basophils.16 In a murine model of HDM-induced airway inflammation, basophils were found to play a direct role in promoting optimal Th2 cytokine responses.17 However, the influence of allergens on the expression of SP and NK1R in basophils has not been investigated.

Therefore, the aim of the study is to investigate the expression of SP and NK1R in basophils of allergic airway diseases, and the influence of allergens and IgE-mediated mechanisms on SP and NK1R expression in basophils. We found that IgE-mediated mechanisms can affect NK1R expression.

MATERIALS AND METHODS

Reagent

The following reagents were purchased from Biolegend (San Diego, CA, USA): human Fc receptor blocking solution, red blood cell lysis buffer, Zombie Aqua^TM Fixable Viability kit, BV421-conjugated mouse anti-human CCR3 antibody, APC-conjugated anti-human CCR3 antibody, PE-conjugated mouse anti-human CD123 antibody, FITC-conjugated anti-human CD123 antibody, PerCP-conjugated mouse anti-human HLA-DR antibody, APC/Cy7-conjugated anti-human HLA-DRα antibody, APC-conjugated rabbit anti-mouse CD49b antibody, and PE/Cy7-conjugated rabbit anti-mouse FcεRIα antibody. APC-conjugated mouse anti-human NK1R antibody and its isotype control antibody APC-conjugated mouse IgG3 were obtained from R&D Systems (Minneapolis, MN, USA). FITC-conjugated rabbit anti-human SP antibody and its isotype control antibody FITC-conjugated rabbit IgG were obtained from LifeSpan BioSciences (Seattle, WA, USA). AF488-conjugated rabbit anti-mouse NK1R antibody and its isotype control antibody AF488-conjugated rabbit IgG, PE-conjugated rabbit anti-mouse NK1R antibody and its isotype control antibody PE-conjugated rabbit IgG were obtained from Novus (Centennial, CO, USA). Cytofix/Cytoperm™Fixation/Permeabilization kit was obtained from BD Biosciences Pharmingen (Bedford, MA, USA). Fetal bovine serum (FBS, HyClone, Logan, UT, USA) and RPMI 1640 were from Gibco BRL (Grand Island, NY, USA). Ovalbumin (OVA, grade V), DNase I and Trypan blue dye were purchased from Sigma-Aldrich (St Louis, MO, USA). Artemisia sieversiana wild allergen extract (ASWE), HDM extract (HDME) or Platanus pollen allergen extract (PPE) were purchased from Macro Union Pharmaceutical Co. Ltd. (Beijing, China). Human IL-3, human IL-31, human IL-33, and human IL-37 were obtained from Peprotech (London, UK). Human IgE, IgA, IgD, IgM, IgG and anti-IgE antibody were obtained from Abcam (London, UK). HDM allergen Der p 1 was synthesized by Beijing Protein Innovation Co., Ltd. (Beijing, China). Oligonucleotide primers for real time quantitative polymerase chain reaction (qPCR) were synthesized by Invitrogen Biotechnology Co. (Shanghai, China). Trizol reagent was obtained from Invitrogen (Carlsbad, CA, USA). cDNA was synthesized by using a PrimeScript RT reagent kit. The resultant cDNA was subjected to qPCR that was performed with a LightCycler using a SuperScript III Platinum SYBR Green 2-step qPCR kit. RNA was extracted by using a TaKaRa Mini BEST Universal RNA Extraction kit. Quantification of mRNA expression level was performed by applying TaKaRa SYBR Premix EX Tag kit (TaKaRa, Beijing, China). Most of the general chemicals, such as salts and buffer components were of analytical grade.

Patients and samples

A total of 38 AR, 30 AA, 16 AR combined with AA (ARA) and 21 healthy control (HC) subjects were recruited in the study. The diagnosing criterion of AA was conformed to the Global Initiative for Asthma,18 and diagnosis for AR in this experiment are in line with the 2015 AR Clinical Practice Guidelines issued by the American Academy of Otorhinolaryngology Head and Neck Surgery,19 ARA was diagnosed based on the AR and its impact on asthma,20 Samples were obtained at the First Affiliated Hospital of Jinzhou Medical University, China. The informed consent from each volunteer according to the declaration of Helsinki and agreement with the ethical committee of the First Affiliated Hospital of Jinzhou Medical University (approved number: KY201405) was obtained. The general characteristics of the patients and control subjects are summarized in Table 1. The blood from each patient with AR, AA, ARA and HC subject was taken in outpatient clinics. From each individual, 10 mL of peripheral blood was taken into an EDTA containing tube before centrifugation at 450 g for 10 minutes. The cells were used for flow cytometric analysis, and plasma was collected and frozen at −80°C until use.

Table 1
General characteristics of the patients with AR, AA, ARA and HC subjects

Click for larger image
Click for full table
Download as Excel file

Animals

BALB/c mice (6–8 weeks) were obtained from Vital River Laboratory Animal Technology Co. Ltd (Beijing, China). They were housed in the Animal Experimental Center of Jinzhou Medical University in a specific pathogen-free environment with free access to standard rodent chow and water at a constant temperature of 23°C–28°C and relative humidity of 60%–75%. The animal experiment procedures were approved by the Animal Care Committee at the First Affiliated Hospital of Jinzhou Medical University.

C57-wild type (WT) mice and C57-FcεRI knock out (KO) mice (6–8 weeks) were obtained from Gem Pharmatech Co, Ltd (Jiangsu, China). FcεR1α-KO mouse model had been established by targeted disruption of the gene encoding the FcεRI-α chain, exton 3 and exton 4 (also available at http://www.informatics.jax.org/allele/summary?markerId=MGI:95494&alleleType= Targeted).21 In our study, the exon 1-5 of FcεRIα gene was knocked out and validated by PCR of genomic DNA.

Mouse AR model sensitized by OVA was mainly adopted from a previous study.22 Briefly, OVA sensitized and OVA challenged (OVA-OVA) mice, and OVA-sensitized and normal saline (NS)-challenged (OVA-NS) mice were sensitized on days 0, 7, 14 and 21 with an intranasal injection of 50 μg of OVA and 1.5 mg of Al(OH)₃ suspended in NS to a total volume of 0.5 mL. Control mice (NS-sensitized and NS-challenged, NS-NS) received only the equal volume (0.5 mL) of NS on the same days. On day 28, OVA-OVA mice were challenged with nasal drops of either 20 μL of 1% OVA for 7 days, and OVA-NS mice and NS-NS mice were challenged with 20 μL of NS for 7 days, 10 μL on each side of the nasal cavity. On the last day, 3 hours after nasal drip being completed, each animal was sacrificed and its blood was collected for analysis.

For mouse AA model sensitized by OVA, which was largely adopted from previous study.23 OVA-OVA mice and OVA-NS mice were actively sensitized on days 0 and 7 with intra-peritoneal injection of 10 μg of OVA and 1 mg of Al(OH)₃ suspended in NS to a total volume of 0.5 mL. NS-NS mice received only the equal volume (0.5 mL) of NS on the same days. On days 14 to 21, OVA-OVA mice were exposed daily to aerosolized 10 mg/mL of OVA over a 30-minute period, OVA-NS mice and NS-NS mice were exposed daily to NS over a 30-minute period. On the last day, 24 hours after aerosol inhalation being completed, animal was sacrificed and its blood was collected for analysis.23

Flow cytometry analysis of the expressions of SP and NK1R in CCR3⁺ and CD123⁺HLA-DR⁻ blood granulocytes and PBMCs

The procedure was mainly adopted from previously published work.24 Briefly, blood cells were challenged with or without allergens ASWE, HDME or PPE (all at concentrations of 0.1 and 1.0 μg/mL) for 60 minutes at 37°C, and 2 μg/mL of brefeldin A was also added into the tube. Cells were then incubated with human Fc receptor blocking solution for 15 minutes, and each labelled monoclonal antibody including BV421-conjugated mouse anti-human CCR3 antibody, PE-conjugated mouse anti-human CD123 antibody and PerCP-conjugated mouse anti-human HLA-DR antibody were added into the tube at room temperature for 15 minutes in the dark. Following ligation of red blood cells, white blood cells were fixed and permeabilized by using the Cytofix/Cytoperm^TM Fixation/Permeabilization Kit according to the manufacturer’s instructions. APC-conjugated mouse anti-human NK1R antibody and FITC-conjugated rabbit anti-human SP antibody were then added to the testing tube, and APC-conjugated mouse IgG3 and FITC-conjugated rabbit IgG were used as isotype controls. Cells were incubated at 4°C for 30 minutes in the dark. Finally, cells were resuspended in fluorescence activated cell sorting (FACS)-flow solution and analyzed with FACS Verse flow cytometer (BD Biosciences, San Jose, CA, USA). A total of 10,000 events in live cell gate were analyzed for each sample. Data were analyzed with FlowJo software version 7.0 (Treestar, Ashland, OR, USA). Dead cells and doublets were excluded from analysis by live/dead cell dyes. Granulocytes and PBMCs in human blood leukocytes were defined according to high side scatter (SSC)-A (SSC-Ahigh forward scatter [FSC]-A) and low SSC-A (SSC-Alow FSC-A) by flow cytometry, respectively.25

Flow cytometric cell sorting

Fresh peripheral blood samples were first incubated with human Fc receptor-blocking solution for 10 minutes and then stained with FITC-conjugated anti-human CD123, APC-conjugated anti-human CCR3 and APC/Cy7-conjugated anti-human HLA-DRα Abs. Target cells in granulocytes and PBMC were sorted into individual tubes containing RPMI 1640 medium supplemented with 3% FBS on a 3-laser SH800 sorter (Sony Biotechnology, San Jose, CA, USA) in purity mode.

Preparation of cytospin slides and cell count

Cytocentrifuge slides were prepared with Shanton cytospin 4 (Thermo Fisher Scientific, Waltham, MA, USA) and stained with Wright Giemsa stain according to the manufacturer’s instructions. Based on the morphology of leukocyte, differential cell counts under the microscope were performed with a minimum count of 100 cells. The results were expressed as percentage of each cell type out of total cells counted per preparation.

Flow cytometry analysis of the expressions of NK1R on mouse blood basophils

To detect expression of NK1R in mouse blood basophils, cells were incubated with TruStain fcX^TM (anti-mouse CD16/32) and a live/dead cell dye (Zombie NIR^TM Fixable Viability Kit) for 15 minutes, and each labelled monoclonal antibody including BV510-conjugated rabbit anti-mouse CD49b and PE/Cy7-conjugated rabbit anti-mouse FcεRIα was added into the tubes. This was followed by adding PE-conjugated rabbit anti-mouse NK1R antibody into the tube and incubated at room temperature for 15 minutes in the dark. Cells were resuspended in FACS-flow solution and analyzed with a FACS Verse flow cytometer for mouse blood cells.

Cell line and culture

The KU812 human basophil line was purchased from American Type Culture Collection. Cells were cultured in an RPMI 1640 medium supplemented with 10% heat-inactivated FBS and 100 units·mL⁻¹ penicillin/streptomycin in 75 cm²-tissue culture flasks (Falcon, Mexico City, Mexico) at 37°C in a 5% (v/v) CO₂, water-saturated atmosphere.

Flow cytometric analysis of NK1R expression on KU812 cells

The challenging procedure for KU812 cells was mainly adopted from a method previously described by Zhong et al.24 for P815 cells. Briefly, cultured KU812 cells at a density of 0.5 × 10⁶ cells/mL were incubated with various concentrations of OVA and Der p 1, IL-33, IL-37 and SP, and ASWE (3.0 μg/mL), HDME (3.0 μg/mL), IL-3 (3.0 ng/mL), IL-31 (10.0 ng/mL), thymic stromal lymphopoietin (TSLP, 10.0 ng/mL), regulated upon activation, normal T-cell expressed and secreted (RANTES, 0.1 μg/mL), tryptase (1.0 μg/mL), histamine (1.0 μg/mL), olopatadine hydrochloride (Olo, 10.0 μg/mL), prostaglandin D2 (PGD2, 0.1 μg/mL), BAY-u-3405 Ramatroban (BAY, 0.35 μg/mL), leukotriene C4 (LTC4, 300 nM), montelukast sodium (Mon, 900 nM), human IgE (1.0 μg/mL), anti-IgE antibody (1.0 μg/mL), human IgA (1.0 μg/mL), human IgD (1.0 μg/mL), human IgM (1.0 μg/mL), human IgG (1.0 μg/mL), calcium ionophore (CI, 1.0 μM) or phosphate buffered saline (PBS, pH 7.4) for 2 or 16 hours at 37°C. Cells were then harvested and centrifuged at 300 g for 8 minutes at 4°C before the culture supernatant being collected and frozen at −80°C. Cell pellets were resuspended for flow cytometry and quantitative real time (q)PCR analysis, respectively. The challenge tests were repeated 4 times. For those blocking compounds, they were preincubation with the corresponding reagents for 30 minutes at room temperature before adding to cells.

To examine NK1R expression, KU812 cells were incubated with human Fc receptor blocking solution and Zombie NIR dye 24 for 15 minutes and then stained with APC-conjugated mouse anti-human NK1R antibody. Following washing with PBS, cells were analyzed by flow cytometry as described above.

qPCR analysis of expression of NK1R mRNA in KU812 cells

cDNA generated from total RNA of KU812 cells was used as templates, and qPCR assay was performed as described by Zhang et al.26 by using specific primers of NK1R and β-actin as listed in Table 2. Briefly, qPCR was performed with the SYBR Premix Ex Taq II Kit on a Real-time Thermal Cycler (Thermo Fisher Scientific). Each reaction contains 10 μL of 2×SYBR green Master Mix, 300 nM oligonucleotide primers, and 10 μL of the cDNA. Untreated controls were chosen as the reference samples, and the ΔCt for all experimental samples were subtracted by the ΔCt for the control samples (ΔΔCt). The magnitude change of test gene mRNA was expressed as 2^−ΔΔCt. Each measurement of a sample was conducted in duplicate.

Table 2
Specific primers of NK1R

Click for larger image
Click for full table
Download as Excel file

Statistical analysis

Statistical analyses were performed by using SPSS version 13.0 software (SPSS, Inc., Chicago, IL, USA). Human peripheral blood basophil data are displayed as a scatter plot, where Kruskal-Walles analysis indicated significant differences between groups, for the pre-planned comparisons of interest, the paired Mann-Whitney U test was employed. Data from KU812 cell lines were presented as mean ± standard error of mean and analyzed by Student’s t-test. Data for expression of NK1R in basophils of mouse blood were displayed as boxplots, which indicate the median, interquartile range, the largest and smallest values for the number of experiments indicated. For all analyses, P < 0.05 was considered statistically significant.

RESULTS

Upregulated expression of SP in CCR3⁺ and CD123⁺HLA-DR⁻ granulocytes of patients with AR, AA, ARA and HC subjects

The involvement of SP in basophil degranulation and accumulation has been highlighted.13 However, little is known about expression of SP in basophils of allergic airway diseases. We therefore investigated the issue in the present study. Since several gating strategies have been proposed to determine the phenotype of basophils, and it is rather difficult to determine the best strategy, we therefore select 2 gating strategies, CCR3⁺ and CD123⁺HLA-DR⁻ cells (Fig. 1A).27 The results showed that the percentages of SP⁺ cells out of CCR3⁺ (Fig. 1B and C) and CD123⁺HLA-DR⁻ granulocytes (Fig. 1D and E) of AA were increased by 1.4- and 1.33-fold, respectively. Mean fluorescent intensity (MFI) of SP in CCR3⁺ granulocytes of AA enhanced by 24.8% (Fig. 1G).

Fig. 1
Flow cytometric analysis of the expression of SP in peripheral blood granulocytes and PBMC. (A) represents a gating strategy of CCR3⁺ and CD123⁺HLA-DR⁻ cells in peripheral blood granulocytes and PBMCs. (B, C) Proportions of SP⁺ cells in CCR3⁺ granulocytes from AR, AA, ARA and HC subjects. (B) representative graphs of the percentages of SP⁺ cells in CCR3⁺ granulocytes, and (C) The percentages of SP⁺ cells in CCR3⁺ granulocytes. (D, E) proportions of SP⁺ cells in CD123⁺HLA-DR⁻ granulocytes. (D) representative graphs of the percentages of SP⁺ cells in CD123⁺HLA-DR⁻ granulocytes, and (E) the percentages of SP⁺ cells in CD123⁺HLA-DR⁻ granulocytes. (F, G) MFI of SP expression in CCR3⁺ granulocytes. (F) representative graphs and (G) the percentages of SP⁺ cells in CCR3⁺ and CD123⁺HLA-DR⁻ granulocytes or MFI of SP in CCR3⁺ granulocytes, respectively. Each symbol represents the value from one subject. The median value is indicated by a horizontal line. P < 0.05 was taken as statistically significant. Cells were stimulated with or without HDME, ASWE or PPE, all at 0.1 and 1 μg/mL respectively, for 1 hour at 37°C.
SP, substance P; FMO, fluorescence minus one; PBMC, peripheral blood mononuclear cell; AR, allergic rhinitis; AA, allergic asthma; ARA, allergic rhinitis combined with allergic asthma; HC, healthy control; MFI, mean fluorescent intensity; HDME, house dust mite extract; ASWE, Artemisia sieversiana wild allergen extract; PPE, Platanus pollen allergen extract; MFI, mean fluorescent intensity; SSC, side scatter; FSC, forward scatter.

Allergen extract PPE increased the proportions of SP⁺ cells in CCR3⁺ granulocytes of HCs and AR patients (Fig. 1C), and in CD123⁺HLA-DR⁻ granulocytes of HCs, and patients with AR and ARA (Fig. 1E). Similarly, ASWE augmented the proportions of SP⁺ cells in CD123⁺HLA-DR⁻ granulocytes of HCs, AR and ARA patients (Fig. 1E). However, HDME only enhanced the proportions of SP⁺ cells in CD123⁺HLA-DR⁻ granulocytes of patients with AR and ARA (Fig. 1E).

On the other hand, the percentages of SP⁺ cells out of CCR3⁺ and CD123⁺HLA-DR⁻ PBMC and MFI of SP expression on CCR3⁺ and CD123⁺HLA-DR⁻ PBMCs of AR, AA and ARA patients had little changes in comparison to HC subjects (data not shown).

Identification of isolated CCR3⁺ and CD123⁺HLA-DR⁻ cells in peripheral blood granulocytes and PBMC

To visualize CCR3⁺ and CD123⁺HLA-DR⁻ cells under a microscope, we isolated them from human peripheral blood by using flow-cytometric cell sorting technique. The results showed that more than 97.7% CCR3⁺ (Fig. 2A and B) and 99% CD123⁺HLA-DR⁻ cells (Fig. 2C and D) in PBMCs of HC subjects were basophils. On the other hand, up to 13% basophils, 85% eosinophils and 2% neutrophils in CCR3⁺ granulocytes of HC subjects were observed (Fig 2E and F). In terms of CD123⁺HLA-DR⁻ granulocytes of HC subjects, approximately 50% basophils, 4% eosinophils and 46% neutrophils were found (Fig. 2G and H).

Fig. 2
Differential identification of isolated CCR3⁺ and CD123⁺HLA-DR⁻ cells from human peripheral blood. CCR3⁺ and CD123⁺HLA-DR⁻ cells in granulocytes and PBMCs were sorted by using a flow cytometer, and cytocentrifuge slides were prepared. Following staining with Wright Giemsa stain, differential cell counts under the microscope were performed. (A-D) Cells were separated from PBMC. (E-H) Cells were separated from granulocytes: (A, E) Representative images of isolated CCR3⁺ cells; and (B, F) Percentages of cells indicated in CCR3⁺ cells. (C, G) Representative images of isolated CD123⁺HLA-DR⁻ cells; and (D, H) Proportions of cells indicated in CD123⁺HLA-DR⁻ cells. Data are displayed as boxplots, which indicate the median, the interquartile range, the largest value, and the smallest value. Each of the data represented a group of 5–7 separate experiments. P < 0.05 was taken as statistically significant. Magnification was ×100.
PBMC, peripheral blood mononuclear cell.

Enhanced expression of NK1R on CCR3⁺ and CD123⁺HLA-DR⁻ peripheral blood cells of patients with AR, AA, ARA and HC subjects

Activation of NK1R can mediate SP-induced migration of basophils14 and consequently amplify the proinflammatory response.28 However, little is known of NK1R expression on blood basophils of allergic airway diseases. In the present study, the percentages of NK1R⁺ cells out of CCR3⁺ granulocytes of AR increased by 1.02-fold (Fig. 3B). In comparison, the percentages of NK1R⁺ cells in CCR3⁺ (Fig. 3B) and CD123⁺HLA-DR⁻ granulocytes (Fig. 3D) of AA and ARA were increased by 1.79- and 2.23-fold and 3.46- and 3.42-fold, respectively.

Fig. 3
Flow cytometric analysis of the expression of NK1R in peripheral blood granulocytes and PBMCs. (A, B) Proportions of NK1R⁺ cells on CCR3⁺ granulocytes from patients with AR, AA, ARA and HC subjects. (C, D) Proportions of NK1R⁺ cells on CD123⁺HLA-DR⁻ granulocytes. (E, F) Proportions of NK1R⁺ cells on CCR3⁺ PBMCs. (G, H) proportions of NK1R⁺ cells on CD123⁺HLA-DR⁻ PBMCs. (A, C, E, G) representative graphs and (B, D, F, H) the percentages of NK1R⁺ cells in CCR3⁺ and CD123⁺HLA-DR⁻ granulocytes or PBMCs, respectively. Each symbol represents the value from one subject. The median value is indicated by a horizontal line. P < 0.05 was taken as statistically significant. Cells were stimulated with or without HDME, ASWE or PPE, all at 0.1 and 1 μg/mL, respectively, for 1 hour at 37°C.
FMO, fluorescence minus one; NK1R, neurokinin-1 receptor; PBMC, peripheral blood mononuclear cell; AR, allergic rhinitis; AA, allergic asthma; ARA, allergic rhinitis combined with allergic asthma; HC, healthy control; HDME, house dust mite extract; ASWE, Artemisia sieversiana wild allergen extract; PPE, Platanus pollen allergen extract; SSC, side scatter.

On the other hand, it was observed that the percentages of NK1R⁺ cells out of CCR3⁺ (Fig. 3F) and CD123⁺HLA-DR⁻ (Fig. 3H) of ARA patients increased by 1.31- and 1.09-fold in comparison with HC subjects. Whilst, the percentage of NK1R⁺ cells in CCR3⁺ PBMCs of AR patients increased by 78% in comparison to HCs subjects (Fig. 3F).

MFI of NK1R expression on CD123⁺HLA-DR⁻ (Fig. 4B) granulocytes of AR and ARA patients were enhanced by 78% and 71% in comparison to HC subjects. MFI of NK1R expression on CCR3⁺ (Fig. 4E) and CD123⁺HLA-DR⁻ PBMCs (Fig. 4F) of AR were augmented by 1.46- and 0.12-fold. Similarly, MFI of NK1R expression on CD123⁺HLA-DR⁻ PBMC of AA (Fig. 4F) were increased by 34%.

Fig. 4
Flow cytometric analysis of MFI of NK1R in granulocytes and PBMCs. (A) representative graph of MFI of NK1R on CD123⁺HLA-DR⁻granulocytes. (B) showed MFIs of NK1R on CD123⁺HLA-DR⁻ granulocytes from patients with AR, AA, ARA and HC subjects, respectively. (C, D) representative graphs of MFIs of NK1R in (C) CCR3⁺ and (D) CD123⁺HLA-DR⁻ PBMC, respectively. (E) and (F) demonstrated MFIs of NK1R on CCR3⁺ PBMC and CD123⁺HLA-DR⁻ PBMC, respectively. Each symbol represents the value from one subject. The median value is indicated by a horizontal line. P < 0.05 was taken as statistically significant. Cells were stimulated with or without HDME, ASWE or PPE, all at 0.1 and 1 μg/mL respectively, for 1 hour at 37°C.
FMO, fluorescence minus one; MFI, mean fluorescent intensity; NK1R, neurokinin-1 receptor; PBMC, peripheral blood mononuclear cell; AR, allergic rhinitis; AA, allergic asthma; ARA, allergic rhinitis combined with allergic asthma; HC, healthy control; HDME, house dust mite extract; ASWE, Artemisia sieversiana wild allergen extract; PPE, Platanus pollen allergen extract.

Allergen extracts ASWE and PPE increased the proportions of NK1R⁺ cells in CCR3⁺ (Fig. 3B) and CD123⁺HLA-DR⁻ granulocytes (Fig. 3D) of HC subjects, and the percentages of NK1R⁺ cells in CCR3⁺ PBMCs of AR patients (Fig. 3F). ASWE and PPE also enhanced the proportions of NK1R⁺ cells in CD123⁺HLA-DR⁻ granulocytes (Fig. 3D) of AR patients. Similarly, HDME augmented the proportions of NK1R⁺ cells in CD123⁺HLA-DR⁻ granulocytes of HC subjects (Fig. 3D), and in CCR3⁺ PBMC of AR patients (Fig. 3F). It was observed that ASWE at 0.1 and 1.0 μg/mL raised MFI of NK1R expression on CCR3⁺ PBMCs of HC subjects by 1.18-fold and 83.73%, respectively (Fig. 4C). PPE enhanced MFI of NK1R expression on CCR3⁺ PBMC of HC subjects and AR patients by up to 1.26- and 1.32-fold (Fig. 4C).

Positive correlations between SP⁺ and NK1R⁺ cells of CCR3⁺ and CD123⁺HLA-DR⁻ granulocytes

In order to learn more about the relationships between expressions of SP and NK1R in CCR3⁺ and CD123⁺HLA-DR⁻ granulocytes of AR, AA, ARA patients and HC subjects, Pearson’s correlation test was employed. As shown in Fig. 4, correlation coefficient ranges were defined as follows: R < 0.3 as weak correlation; 0.3 ≤ R ≤ 0.7 as moderate correlation; and R > 0.7 as strong correlation. 29 There were moderate correlations between percentages of SP⁺CCR3⁺ and NK1R⁺CCR3⁺ granulocytes of HC subjects; strong correlations between percentages of SP⁺CCR3⁺ and NK1R⁺CCR3⁺ granulocytes of AR and AA patients (Fig. 5A). Moderate correlations between percentages of SP⁺CD123⁺HLA-DR⁻ and NK1R⁺CD123⁺HLA-DR⁻ granulocytes of HC subjects and AR patients; strong correlations between percentages of SP⁺CD123⁺HLA-DR⁻ and NK1R⁺CD123⁺HLA-DR⁻ granulocytes of AA and ARA patients were also observed (Fig. 5B).

Fig. 5
Scatter plot, linear regression line, and coefficient of determinations between the variables. Correlations between percentages of SP⁺ and NK1R⁺ cells in CCR3⁺ (A) and CD123⁺HLA-DR⁻ (B) granulocytes of patients with AR, AA, ARA and HC subjects were analyzed using Pearson’s correlation test. Correlation coefficient ranges were defined as follows: R < 0.3 as a weak correlation; 0.3 ≤ R ≤ 0.7 as a moderate correlation; and > 0.7 as a strong correlation. P < 0.05 was taken as statistically significant.
SP, substance P; NK1R, neurokinin-1 receptor; AR, allergic rhinitis; AA, allergic asthma; ARA, allergic rhinitis combined with allergic asthma; HC, healthy control.

Upregulated expression of NK1R in KU812 cells induced by allergens, cytokines, mediators and Igs

In order to understand the potential mechanisms of the elevated expression of NK1R in basophils, we investigated actions of allergens including Der p 1, OVA, ASWE, HDME, proinflammatory cytokines including IL-3, IL-31, IL-33, IL-37, TSLP, RANTES, proinflammatory mediators and some of their blockers including SP, tryptase, CI, His, Olo, Olo + His, PGD2, BAY, BAY + PGD2, LTC4, Mon, Mon + LTC4 and Igs including IgE, IgA, IgD, IgM and IgG on KU812 cells. The results showed that compared to the medium alone group, OVA (at the concentrations of 0.03, 0.3, 1.0 and 3.0 μg/mL), Der p 1 (at the concentrations of 0.03, 0.3, 1.0 and 3.0 μg/mL), IL-33 (at the concentrations of 1.0, 3.0 and 10 ng/mL), IL-37 (at the concentrations of 1.0, 3.0 and 10 ng/mL) and SP (at the concentrations of 3.0, 10 and 30 ng/mL) enhanced percentages of NK1R⁺ KU812 cells and MFI of NK1R expression on KU812 cells at 2 hours (Fig. 6A and C) and 16 hours (Fig. 6B and D) following incubation. IgE at 1.0 μg/mL appeared to increase MFI of NK1R expression (Fig. 6C) and NK1R mRNA expression (Fig. 6E) in KU812 cells at 2 hours following incubation, which was blocked by co-incubation with anti-IgE antibody for 30 minutes. IgG at 1.0 μg/mL seemed to enhance MFI of NK1R expression in KU812 cells at 2 hours following incubation (Fig. 6C). IgD and IgM both at 1.0 μg/mL augmented MFI of NK1R expression in KU812 cells at 16 hours following incubation (Fig. 6D). IL-33, IL-37, IL-3, IL-31, SP, PGD2 and IgA, but not OVA and Der p 1 at the concentrations tested elevated NK1R mRNA expression in KU812 cells at 2 hours following incubation (Fig. 6E).

Fig. 6
Expression of NK1R on KU812 cells. The cells were incubated with various concentrations of OVA and Der p 1, IL-33, IL-37 and SP, and ASWE (3.0 μg/mL), HDME (3.0 μg/mL), IL-3 (3.0 ng/mL), IL-31 (10.0 ng/mL), TSLP (10.0 ng/mL), RANTES (0.1 μg/mL), tryptase (1.0 μg/mL), histamine (1.0 μg/mL), Olo (10.0 μg/mL), PGD2 (0.1 μg/mL), BAY (0.35 μg/mL), LTC4 (300 nM), Mon (900 nM), human IgE (1.0 μg/mL), human IgA (1.0 μg/mL), human IgD (1.0 μg/mL), human IgM (1.0 μg/mL), human IgG (1.0 μg/mL), CI (1.0 μM) or PBS (pH 7.4) for 2 or 16 hours at 37°C. (A, B) demonstrated percentages of NK1R⁺ KU812 cells at 2 and 16 hours following incubation. (C, D) showed MFI levelsof NK1R expressed on KU812 cells at 2 and 16 hours following incubation. (E) represented NK1R mRNA expression in KU812 cells at 2 hours following incubation.The data are presented as the mean ± standard error for 4 separate experiments.
SP, substance P; NK1R, neurokinin-1 receptor; OVA, ovalbumin; Der p, Dermatophagoide pteronyssinus; ASWE, Artemisia sieversiana wild allergen extract; HDME, house dust mite extract; IL, interleukin; TSLP, thymic stromal lymphopoietin; RANTES, regulated upon activation, normal T-cell expressed and secreted; Olo, olopatadine hydrochloride; PGD2, prostaglandin D2; BAY, BAY-u-3405 Ramatroban; LTC4, leukotriene C4, Mon, montelukast sodium; Ig, immunoglobulin; CI, calcium ionophore; PBS, phosphate buffered saline; MFI, mean fluorescence intensity.

^*P < 0.05 compared with the medium alone group; ^**P < 0.05 compared with the IgE alone group.

Increased NK1R⁺ blood basophils of allergic mice

In order to confirm that allergen can induce upregulation of expression of NK1R in basophils, we investigated the expression of NK1R in basophils of AR and AA mice sensitized and challenged by OVA. The results showed that the percentages of NK1R expressing basophils were increased in blood of OVA-sensitized as well as OVA sensitized and challenged AR mice (Fig. 7C). However, in AA mice, only OVA sensitized and challenged mice showed increased proportion of NK1R expressing basophils in their blood (Fig. 7E). FcεRI-KO AA mice seemed to have less basophils in their blood than WT AA mice when they were challenged by NS and OVA (Fig. 8B). Similarly, FcεRI-KO AA mice showed less NK1R⁺ basophils in their blood than WT AA mice regardless of whether they were sensitized or challenged by NS and OVA (Fig. 8D). As for BALB/c mice, C57 AA mice also exhibited increased proportion of NK1R expressing basophils in their blood following OVA sensitized and challenged (Fig. 8D).

Fig. 7
Flow cytometric analysis of expression of NK1R on blood basophils of OVA-sensitized or non-sensitized mice. (A) A gating strategy of basophils (Gr1⁻CD49b⁺ FcεRI⁺ cells) in mouse blood. (B, C) Proportions of NK1R⁺ basophils from OVA-OVA, OVA-NS, and NS-NS) mice of AR. (D, E) proportions of NK1R⁺ basophils from AA mice. (B, D) representative graphs and (C, E) percentages of NK1R⁺ cells in basophil populations. A total of 6–7 mice in each group. P < 0.05 was taken as statistically significant.
FMO, fluorescence minus one; NK1R, neurokinin-1 receptor; OVA, ovalbumin; OVA-OVA, ovalbumin sensitized and ovalbumin challenged; OVA-NS, ovalbumin sensitized and normal saline challenged; NS-NS, normal saline injected and normal saline challenged; AR, allergic rhinitis; AA, allergic asthma; SSC, side scatter; FSC, forward scatter.

Fig. 8
Flow cytometric analysis of expression of NK1R on basophils in blood of FcεRI KO and WT mice of AA model. Mice were treated with OVA-OVA, OVA-NS, and NS-NS. (A, B) Percentages of basophils (CD123⁺CD49b⁺ cells) in mouse blood. (C, D) Percentages of NK1R⁺ cells in basophil populations. (A, C) representative graphs and (B, D) the percentages of basophils or NK1R⁺ basophils. A total of 6–7 mice in each group. P < 0.05 was taken as statistically significant.
FMO, fluorescence minus one; NK1R, neurokinin-1 receptor; KO, knock out; WT, wild type; AA, allergic asthma; OVA, ovalbumin; OVA-OVA, ovalbumin sensitized and ovalbumin challenged; OVA-NS, ovalbumin sensitized and normal saline challenged; NS-NS, normal saline injected and normal saline challenged; SSC, side scatter; FSC, forward scatter.

DISCUSSION

Upregulated expression of SP in CCR3⁺ and CD123⁺HLA-DR⁻ granulocytes of patients with AA, but not with AR or ARA, was an unexpected result, which may implicate that these cells may contribute to the pathogenesis of AA via SP. Involvement of SP in AA has been previously reported. For instance, increased amounts of SP are found in sputum and BAL fluid of asthmatics.10, 11 and SP causes many of the typical changes observed in asthmatic airways.14 Our finding suggests CCR3⁺ and CD123⁺HLA-DR⁻ granulocytes may serve as the sources of SP in blood. Since expression of SP in CCR3⁺ and CD123⁺HLA-DR⁻ PBMC of AR, AA or ARA patients had little changes in comparison to HC subjects, and CCR3⁺ cells and CD123⁺HLA-DR⁻ cells 27 are basophils in PBMCs. We anticipate that those upregulated SP expression cells in CCR3⁺, and CD123⁺HLA-DR⁻ granulocytes could be a subtype of basophils which is different from those in PBMC, or includes some other cell types besides basophils. Indeed, it has been reported that AA patients exhibit phenotypically distinct basophil populations in the peripheral blood,5 CCR3⁺ cells in blood granulocytes include eosinophils30 and basophils. While information on CD123⁺HLA-DR⁻ cells in blood granulocytes is not available, CD123⁺ granulocytes consist of eosinophils,31 immature neutrophils32 and basophils. Taking the information together, it is rather difficult to ensure the specific cell type which expresses SP in CCR3⁺ and CD123⁺HLA-DR⁻ granulocytes at this stage.

In contrast, upregulated expression of NK1R is observed in CCR3⁺ and CD123⁺HLA-DR⁻ PBMC of ARA patients, and enhanced expression of NK1R on CCR3⁺ and CD123⁺HLA-DR⁻ PBMC of AR and AA patients is found in the present study, suggesting that basophils are involved in the pathogenesis of AA, AR and ARA via SP-related mechanisms. While information on basophil NK1R expression relating to AA, AR and ARA is not available, the reports that upregulated NK1R expression in asthmatic lung compared to normal controls,15 and that basophils are highly enriched in post-mortem lung tissue of patients who have died from asthma7 may indirectly support the anticipation that upregulated expression of NK1R in basophils could be involved in the pathogenesis of AA, AR and ARA. Therefore, enhanced expression of NK1R in CCR3⁺ and CD123⁺HLA-DR⁻ granulocytes of patients with AR, AA and ARA should be at least in part contributed by basophils. The strong correlations between percentages of SP⁺CCR3⁺ and NK1R⁺CCR3⁺ granulocytes of AR and AA patients, between percentages of SP⁺CD123⁺HLA-DR⁻ and NK1R⁺CD123⁺HLA-DR⁻ granulocytes of AA and ARA patients imply that the increased SP⁺ and NK1R⁺ cells in granulocyte population may originate from the same cell types.

Originally, the flow cytometer system was used to separate unfixed and unstained human leukocyte cells into morphologically distinct populations based only on the intensity of 488-nm wavelength laser light simultaneously scattered by each cell at 2 different angles. Three populations were observed as distinct peaks in a 2-parameter pulse-height distribution. The 3 groups consisted of lymphocytes, monocytes and neutrophils.33 Later, the 2-parameters 90 degrees light scatter (SSC) reflecting primarily cell granularity vs. forward angle light scatter (FSC) correlating with cell size were found to clearly identify PBMCs including lymphocytes and monocytes, and granulocytes in white blood cell suspensions. It was observed that proportions of lymphocytes and granulocytes obtained by the flow cytometer correlated well with those obtained by both microscopic and automatic differential.34 Moreover, the granulocyte population obtained by sorting on a plot of FSC/SSC was a mixture of eosinophils and neutrophils. Eosinophils were concentrated above the neutrophil cluster, but they are frequently found distributed throughout the neutrophil cluster.35

For the treatment regimen of allergen extracts, a 60-minute incubation period was adopted on the basis of our previous work.36 Sonneck et al.37 previously reported that blood basophils from allergic patients were exposed to various concentrations of recombinant grass pollen (Phl p 1, Phl p 5), birch pollen (Bet v 1) or HDM (Der p 2) allergens for 15 minutes. The concentrations of HDM,38 ASWE and PPAE36 used were based upon published papers.

It is difficult to obtain large quantities of highly purified inactive basophils. It has been reported that KU812 cells are a suitable model for studying the activation and degranulation of human basophils.39 Elevated percentages of NK1R⁺ KU812 cells and MFI of NK1R expression on KU812 cells induced by OVA and Der p 1, but not ASWE or HDME, suggest that purified allergens may be more efficient in induction of NK1R expression on basophils than allergen extracts. Induction of upregulated NK1R expression in KU812 cells by IgE, IgG, IgD and IgM implicates that Igs-associated mechanisms may be capable of contributing to regulate NK1R expression in basophils. Nevertheless, further work is required to prove the issue. The ability to induce enhanced NK1R expression on KU812 cells suggests that proinflammatory cytokines IL-33 and IL-37, and SP are likely to be involved in the modification of NK1R expression in basophils.

The influence of allergens on the expression of SP and NK1R in basophils has not previously been investigated. In the present study, allergen extract PPE upregulated SP expression in CCR3⁺ and CD123⁺HLA-DR⁻ granulocytes of AR and ARA patients. ASWE and HDME augmented the proportions of SP⁺ cells in CD123⁺HLA-DR⁻ granulocytes of AR and ARA patients, implicating that allergen extracts may affect more SP expression in AR than in AA patients. Unexpectedly, PPE also upregulated SP expression in CCR3⁺ and CD123⁺HLA-DR⁻ granulocytes as well as ASWE augmented SP⁺ cells in CD123⁺HLA-DR⁻ granulocytes of the HC subjects. Since these allergen extracts failed to induce upregulated SP expression in CCR3⁺ and CD123⁺HLA-DR⁻ PBMCs of HC subjects, AR, AA and ARA patients, the elevated SP expression may not be in basophils. Considering that allergen extract can alter behavior of peripheral blood eosinophils during allergen-induced late-phase airway inflammation in patients with AA,40 that bronchial HDME challenge elicits significant increases in sputum eosinophils and ECP in AA patients,41 and that eosinophils express CCR3,30 we believe that these SP⁺CCR3⁺ granulocytes could be eosinophils. Nevertheless, these are rather confusing results, but we believe that the experiment using purified allergens and isolated cell types may help understand this issue.

It is observed that allergen extracts ASWE and PPE enhanced NK1R expression in CD123⁺HLA-DR⁻ granulocytes of AR patients, as well as in CCR3⁺ and CD123⁺HLA-DR⁻ granulocytes of HC subjects. HDME augmented NK1R expression in CD123⁺HLA-DR⁻ granulocytes of HC subjects. Since PPE and HDME enhanced NK1R expression on CCR3⁺ PBMCs, ASWE and PPE raised NK1R expression on CCR3⁺ and CD123⁺HLA-DR⁻ PBMC of HC subjects and AR patients, the enhanced NK1R expression in CCR3⁺ and CD123⁺HLA-DR⁻ granulocytes of HC subjects and AR patients is at least partially on basophils.

It has previously been reported that allergens ASWE and HDME, but not PPE enhanced NK1R expression on CD14⁺ blood leukocytes regardless of atopic dermatitis (AD) or HC subjects.36 Since AD, AA and AR all belong to atopic disease, it is likely that these allergens affect NK1R expression in AA and AR. Indeed, B cells appeared to play a major role in the adaptive immune response to inhaled HDM allergen in HDM-driven asthma by expanding allergen-specific T cells,42 and HDM allergen Der p 1 catalytically inactivated alpha 1-antitrypsin and promoted airway inflammation and asthma.43 It has been noticed that the production of IL-25, IL-33 and TSLP from airway epithelial cells was induced by the protease activity of an HDMs in HDM-induced asthma mouse model.44 Because IL-33 is one of the most highly replicated susceptibility loci for asthma,45 HDM-induced asthma may occur via IL-33-associated mechanisms. In a mouse asthma model, mice sensitized with ASWE had significantly increased IL-4, IL-5, IL-13 and allergen-specific IgE levels in the plasma.46 Moreover, Halogen immunoassay analysis demonstrated IgE binding to Platanus pollen in all Platanus-sensitized allergy subjects.47 and Pla a 1 and Pla a 2 were responsible for 79% of the IgE-binding capacity against PPE.48 These findings may help understand differences between the pro-asthma mechanisms of the allergen extracts. It was reported that FcεRI-activated mast cells upregulated NK1R transcripts and protein synthesis, without modifying SP expression,49 suggesting that the allergens may affect some transcriptional factors and upregulate NK1R transcripts in mast cells as allergens are likely to activate mast cells via FcεRI.

Upregulated NK1R expression in AR and AA mouse blood basophils upon OVA-sensitization and challenge confirms that allergen is capable of inducing AR and AA and upregulation of NK1R expression. A report that basophils play a direct role in promoting optimal Th2 cytokine responses in a murine model of HDM-induced airway inflammation may support the above observation.17 Considering that OVA can enhance NK1R expression in the basophil line of KU812, and a study has shown that the development of bronchial hyperreactivity was completely inhibited after 4 hours in mice treated with the NK1R antagonist L-732138 and stimulated with OVA,50 we anticipate that allergens may mediate AR and AA through a SP-NK1R mediated mechanism. FcεRI KO AA mice seemed to have less basophils and NK1R⁺ basophils in their blood than WT AA mice, when they were sensitized and challenged by NS and OVA. The result may be caused by the fact that the deletion of FcεRI gene in mice leads to the inability of IgE binding to their basophils or mast cells, and subsequently diminishing formation of sensitized basophils or mast cells,50 resulting in a significant decrease in NK1R expression in basophils.

In conclusion, upregulated expression of SP and NK1R in CCR3⁺ and CD123⁺HLA-DR⁻ cells of patients with AA and AR implicates that these cells may contribute to the pathogenesis of AA and AR via SP-NK1R-related mechanisms. Allergens can modify behavior of these cells, particularly basophils, suggesting further that SP-NK1R related mechanisms are involved in these airway allergic diseases. Induction of upregulated NK1R expression in basophils by OVA and IgE in vitro as well as deletion of the FcεRI gene in mice decrease NK1R expression in basophils in vivo implicate that IgE-related mechanisms may be involved in upregulation of NK1R expression. Understanding SP-NK1R-related mechanisms will add novel content for discovery of roles of basophils in allergy.

Notes

Disclosure:Shaoheng He is a cofounder and employee of Huiallergy Co. LTD. The other authors declare no competing interests.

ACKNOWLEDGMENTS

This project was sponsored by Shenyang Science and Technology Plan Project (19-109-4-17), Key Laboratory of Research on Pathogenesis of Allergen provoked Allergic Disease, Liaoning Province (2018-30), the grants from the Climbing Scholar Project in Liaoning province (2018-38), Science and Technology Innovation “double hundred project” in Shenyang (Z17-5-044), Innovation and Entrepreneurship Projects for High-Level Talent in Shenyang (2017CXCY-C-01), Leader Team Project in Jinzhou Medical University (2017-2020), Liaoning Province Science and Technology Planning project (2021JH2/10300022, 2019JH8/10300053), Natural Science Foundation of Liaoning Province (LJKZ0796; 2019-ZD-0801); 2021 Youth Science and Technology Talents Support Plan from Boze Project of Jinzhou Medical University (JYBZQT2110).

References

1. Bousquet J, Jeffery PK, Busse WW, Johnson M, Vignola AM. Asthma. From bronchoconstriction to airways inflammation and remodeling. Am J Respir Crit Care Med 2000;161:1720–1745.
  PubMed
  
  CrossRef
1. Bousquet J, Van Cauwenberge P, Khaltaev N. Aria Workshop Group; World Health Organization. Allergic rhinitis and its impact on asthma. J Allergy Clin Immunol 2001;108:S147–S334.
  PubMed
  
  CrossRef
1. Guerra S, Sherrill DL, Martinez FD, Barbee RA. Rhinitis as an independent risk factor for adult-onset asthma. J Allergy Clin Immunol 2002;109:419–425.
  PubMed
  
  CrossRef
1. Heinemann A, Hartnell A, Stubbs VE, Murakami K, Soler D, LaRosa G, et al. Basophil responses to chemokines are regulated by both sequential and cooperative receptor signaling. J Immunol 2000;165:7224–7233.
  PubMed
  
  CrossRef
1. Youssef LA, Schuyler M, Gilmartin L, Pickett G, Bard JD, Tarleton CA, et al. Histamine release from the basophils of control and asthmatic subjects and a comparison of gene expression between “releaser” and “nonreleaser” basophils. J Immunol 2007;178:4584–4594.
  PubMed
  
  CrossRef
1. Shiraishi Y, Jia Y, Domenico J, Joetham A, Karasuyama H, Takeda K, et al. Sequential engagement of FcεRI on mast cells and basophil histamine H₄ receptor and FcεRI in allergic rhinitis. J Immunol 2013;190:539–548.
  PubMed
  
  CrossRef
1. Kepley CL, McFeeley PJ, Oliver JM, Lipscomb MF. Immunohistochemical detection of human basophils in postmortem cases of fatal asthma. Am J Respir Crit Care Med 2001;164:1053–1058.
  PubMed
  
  CrossRef
1. Zheng W, Wang J, Zhu W, Xu C, He S. Upregulated expression of substance P in basophils of the patients with chronic spontaneous urticaria: induction of histamine release and basophil accumulation by substance P. Cell Biol Toxicol 2016;32:217–228.
  PubMed
  
  CrossRef
1. Cima K, Vogelsinger H, Kähler CM. Sensory neuropeptides are potent chemoattractants for human basophils in vitro. Regul Pept 2010;160:42–48.
  PubMed
  
  CrossRef
1. Nieber K, Baumgarten CR, Rathsack R, Furkert J, Oehme P, Kunkel G. Substance P and beta-endorphin-like immunoreactivity in lavage fluids of subjects with and without allergic asthma. J Allergy Clin Immunol 1992;90:646–652.
  PubMed
  
  CrossRef
1. Tomaki M, Ichinose M, Miura M, Hirayama Y, Yamauchi H, Nakajima N, et al. Elevated substance P content in induced sputum from patients with asthma and patients with chronic bronchitis. Am J Respir Crit Care Med 1995;151:613–617.
  PubMed
  
  CrossRef
1. Chaen T, Watanabe N, Mogi G, Mori K, Takeyama M. Substance P and vasoactive intestinal peptide in nasal secretions and plasma from patients with nasal allergy. Ann Otol Rhinol Laryngol 1993;102:16–21.
  PubMed
  
  CrossRef
1. Lundberg JM, Martling CR, Saria A. Substance P and capsaicin-induced contraction of human bronchi. Acta Physiol Scand 1983;119:49–53.
  PubMed
  
  CrossRef
1. Laitinen LA, Heino M, Laitinen A, Kava T, Haahtela T. Damage of the airway epithelium and bronchial reactivity in patients with asthma. Am Rev Respir Dis 1985;131:599–606.
  PubMed
  
  CrossRef
1. Adcock IM, Peters M, Gelder C, Shirasaki H, Brown CR, Barnes PJ. Increased tachykinin receptor gene expression in asthmatic lung and its modulation by steroids. J Mol Endocrinol 1993;11:1–7.
  PubMed
  
  CrossRef
1. Phillips C, Coward WR, Pritchard DI, Hewitt CR. Basophils express a type 2 cytokine profile on exposure to proteases from helminths and house dust mites. J Leukoc Biol 2003;73:165–171.
  PubMed
  
  CrossRef
1. Hill DA, Siracusa MC, Abt MC, Kim BS, Kobuley D, Kubo M, et al. Commensal bacteria-derived signals regulate basophil hematopoiesis and allergic inflammation. Nat Med 2012;18:538–546.
  PubMed
  
  CrossRef
1. Boulet LP, Reddel HK, Bateman E, Pedersen S, FitzGerald JM, O’Byrne PM. The Global Initiative for Asthma (GINA): 25 years later. Eur Respir J 2019;54:1900598
  PubMed
  
  CrossRef
1. Seidman MD, Gurgel RK, Lin SY, Schwartz SR, Baroody FM, Bonner JR, et al. Clinical practice guideline: Allergic rhinitis. Otolaryngol Head Neck Surg 2015;152:S1–43.
  PubMed
  
  CrossRef
1. Brożek JL, Bousquet J, Agache I, Agarwal A, Bachert C, Bosnic-Anticevich S, et al. Allergic Rhinitis and its Impact on Asthma (ARIA) guidelines-2016 revision. J Allergy Clin Immunol 2017;140:950–958.
  CrossRef
1. Dombrowicz D, Flamand V, Brigman KK, Koller BH, Kinet JP. Abolition of anaphylaxis by targeted disruption of the high affinity immunoglobulin E receptor alpha chain gene. Cell 1993;75:969–976.
  PubMed
  
  CrossRef
1. Wang SB, Deng YQ, Ren J, Xiao BK, Liu Z, Tao ZZ. Exogenous interleukin-10 alleviates allergic inflammation but inhibits local interleukin-10 expression in a mouse allergic rhinitis model. BMC Immunol 2014;15:9.
  PubMed
  
  CrossRef
1. Pauwels RA, Brusselle GJ, Kips JC. Cytokine manipulation in animal models of asthma. Am J Respir Crit Care Med 1997;156:S78–S81.
  PubMed
  
  CrossRef
1. Zhong Q, Zhan M, Wang L, Chen D, Zhao N, Wang J, et al. Upregulation of the expression of Toll-like receptor 9 in basophils in patients with allergic rhinitis: an enhanced expression by allergens. Scand J Immunol 2021;93:e13003
  PubMed
1. Ost V, Neukammer J, Rinneberg H. Flow cytometric differentiation of erythrocytes and leukocytes in dilute whole blood by light scattering. Cytometry 1998;32:191–197.
  PubMed
  
  CrossRef
1. Zhang H, Yang X, Yang H, Zhang Z, Lin Q, Zheng Y, et al. Modulation of mast cell proteinase-activated receptor expression and IL-4 release by IL-12. Immunol Cell Biol 2007;85:558–566.
  PubMed
  
  CrossRef
1. Weinstock JV. Substance P and the regulation of inflammation in infections and inflammatory bowel disease. Acta Physiol (Oxf) 2015;213:453–461.
  PubMed
  
  CrossRef
1. Lee TH, McCully BH, Underwood SJ, Cotton BA, Cohen MJ, Schreiber MA. Correlation of conventional thrombelastography and rapid thrombelastography in trauma. Am J Surg 2013;205:521–527.
  PubMed
  
  CrossRef
1. Hausmann OV, Gentinetta T, Fux M, Ducrest S, Pichler WJ, Dahinden CA. Robust expression of CCR3 as a single basophil selection marker in flow cytometry. Allergy 2011;66:85–91.
  PubMed
  
  CrossRef
1. Kim Z, Choi BS, Kim JK, Won DI. Basophil markers for identification and activation in the indirect basophil activation test by flow cytometry for diagnosis of autoimmune urticaria. Ann Lab Med 2016;36:28–35.
  PubMed
  
  CrossRef
1. Toba K, Koike T, Shibata A, Hashimoto S, Takahashi M, Masuko M, et al. Novel technique for the direct flow cytofluorometric analysis of human basophils in unseparated blood and bone marrow, and the characterization of phenotype and peroxidase of human basophils. Cytometry 1999;35:249–259.
  PubMed
  
  CrossRef
1. Meghraoui-Kheddar A, Chousterman BG, Guillou N, Barone SM, Granjeaud S, Vallet H, et al. Two new neutrophil subsets define a discriminating sepsis signature. Am J Respir Crit Care Med 2022;205:46–59.
  PubMed
  
  CrossRef
1. Salzman GC, Crowell JM, Martin JC, Trujillo TT, Romero A, Mullaney PF, et al. Cell classification by laser light scattering: identification and separation of unstained leukocytes. Acta Cytol 1975;19:374–377.
  PubMed
1. Sucić M, Kolevska T, Kopjar B, Kosanović M, Drobnjak M, Zalud I, et al. Accuracy of routine flow-cytometric bitmap selection for three leukocyte populations. Cytometry 1989;10:442–447.
  CrossRef
1. Gopinath R, Nutman TB. Identification of eosinophils in lysed whole blood using side scatter and CD16 negativity. Cytometry 1997;30:313–316.
  PubMed
  
  CrossRef
1. Zhang Z, Zheng W, Xie H, Chai R, Wang J, Zhang H, et al. Up-regulated expression of substance P in CD8⁺ T cells and NK1R on monocytes of atopic dermatitis. J Transl Med 2017;15:93.
  PubMed
  
  CrossRef
1. Sonneck K, Baumgartner C, Rebuzzi L, Marth K, Chen KW, Hauswirth AW, et al. Recombinant allergens promote expression of aminopeptidase-n (CD13) on basophils in allergic patients. Int J Immunopathol Pharmacol 2008;21:11–21.
  PubMed
  
  CrossRef
1. Ueda Y, Nakagome K, Kobayashi T, Noguchi T, Soma T, Ohashi-Doi K, et al. Effects of β2-adrenergic agonists on house dust mite-induced adhesion, superoxide anion generation, and degranulation of human eosinophils. Asia Pac Allergy 2020;10:e15
  PubMed
  
  CrossRef
1. Falcone FH, Haas H, Gibbs BF. The human basophil: a new appreciation of its role in immune responses. Blood 2000;96:4028–4038.
  PubMed
  
  CrossRef
1. Lavinskiene S, Malakauskas K, Jeroch J, Hoppenot D, Sakalauskas R. Functional activity of peripheral blood eosinophils in allergen-induced late-phase airway inflammation in asthma patients. J Inflamm (Lond) 2015;12:25.
  PubMed
  
  CrossRef
1. Lopuhaä CE, Out TA, Jansen HM, Aalberse RC, van der Zee JS. Allergen-induced bronchial inflammation in house dust mite-allergic patients with or without asthma. Clin Exp Allergy 2002;32:1720–1727.
  CrossRef
1. Dullaers M, Schuijs MJ, Willart M, Fierens K, Van Moorleghem J, Hammad H, et al. House dust mite-driven asthma and allergen-specific T cells depend on B cells when the amount of inhaled allergen is limiting. J Allergy Clin Immunol 2017;140:76–88.e7.
  PubMed
  
  CrossRef
1. Kalsheker NA, Deam S, Chambers L, Sreedharan S, Brocklehurst K, Lomas DA. The house dust mite allergen Der p1 catalytically inactivates alpha 1-antitrypsin by specific reactive centre loop cleavage: a mechanism that promotes airway inflammation and asthma. Biochem Biophys Res Commun 1996;221:59–61.
  PubMed
  
  CrossRef
1. Yasuda Y, Nagano T, Kobayashi K, Nishimura Y. Group 2 innate lymphoid cells and the house dust mite-induced asthma mouse model. Cells 2020;9:1178.
  PubMed
  
  CrossRef
1. Cayrol C, Girard JP. Interleukin-33 (IL-33): a nuclear cytokine from the IL-1 family. Immunol Rev 2018;281:154–168.
  PubMed
  
  CrossRef
1. Zhang Q, Xi G, Yin J. Artemisia sieversiana pollen allergy and immunotherapy in mice. Am J Transl Res 2021;13:13654–13664.
  PubMed
1. Sercombe JK, Green BJ, Rimmer J, Burton PK, Katelaris CH, Tovey ER. London Plane Tree bioaerosol exposure and allergic sensitization in Sydney, Australia. Ann Allergy Asthma Immunol 2011;107:493–500.
  PubMed
  
  CrossRef
1. Asturias JA, Ibarrola I, Amat P, Tella R, Malet A, Cisteró-Bahíma A, et al. Purified allergens vs. complete extract in the diagnosis of plane tree pollen allergy. Clin Exp Allergy 2006;36:1505–1512.
  PubMed
  
  CrossRef
1. Sumpter TL, Ho CH, Pleet AR, Tkacheva OA, Shufesky WJ, Rojas-Canales DM, et al. Autocrine hemokinin-1 functions as an endogenous adjuvant for IgE-mediated mast cell inflammatory responses. J Allergy Clin Immunol 2015;135:1019–1030.e8.
  PubMed
  
  CrossRef
1. Hens G, Raap U, Vanoirbeek J, Meyts I, Callebaut I, Verbinnen B, et al. Selective nasal allergen provocation induces substance P-mediated bronchial hyperresponsiveness. Am J Respir Cell Mol Biol 2011;44:517–523.
  PubMed
  
  CrossRef