Initial sensitization and subsequent repeated exposure to antigens are known to compose the pathogenesis of asthma development (Khalaf
In asthma, S1P1 and S1P2 has been extensively studied (Park and Im, 2019). In particular, S1P-induced degranulation of mast cells is mediated through S1P2 receptors (Jolly
CYM50358 was purchased from Tocris (Bristol, UK). Other materials were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Rat RBL-2H3 mast cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). RBL-2H3 cells were cultured at 37°C in a 5% CO2-humidified incubator, and maintained in 10% (v/v) heat-inactivated fetal bovine serum containing high-glucose Dulbecco’s modified Eagle medium (DMEM) with 2 mM glutamine, 100 U/mL penicillin, 1 mM sodium pyruvate, and 50 μg/mL streptomycin (Huang
Female five-week-old BALB/c mice were purchased from Daehan Biolink (Seoul, Korea). They were housed in the laboratory animal facility at Kyung Hee University (Seoul, Korea) and provided
By measuring β-hexosaminidase activity in the medium, degranulation of RBL-2H3 cells was assessed. Monoclonal anti-dinitrophenyl mouse immunoglobulin E and human dinitrophenyl albumin were used to induce degranulation (Huang
Following a simple randomization procedure, 6-week-old female BALB/c mice (22 g) were randomly assigned to one of four treatment groups (n=5): phosphate-buffered saline (PBS)-injected control group, OVA-injected asthma group, CYM50358-treatment before sensitization plus OVA-injected group, and CYM50358-treatment before challenge plus OVA-injected group. Asthma was induced by intraperitoneal injection of 50 μg OVA and 1 mg aluminum hydroxide on D0 and D14 (sensitization). Mice were challenged by exposing to nebulized OVA for D28, D29, and D30 (challenge) (Kim and Im, 2019; Park and Im, 2019). CYM50358 was administered via intraperitoneal injection 30 min before OVA sensitization or OVA challenge. Bronchoalveolar lavage fluids (BALF) were collected from the lungs on D32, and cell population of BALF cells was analyzed after staining.
Using a Cellspin® centrifuge (Hanil Electric, Seoul, Korea), immune cells in BALF were adhered to a glass slide and fixed in methanol for 30 s. Staining with May-Grünwald solution was conducted in the cells on slides for 8 min and subsequently by Giemsa solution for 12 min.
Tissue sections of lungs from mice of each group were prepared. Hematoxylin and Eosin (H&E) staining and periodic acid-Schiff (PAS) staining were conducted to find mucus-secreting goblet cells and eosinophil infiltration, respectively. For PAS staining, Schiff’s regent was used and for H&E staining, hematoxylin and eosin regents were used (Heo and Im, 2019).
Degree of lung inflammation was measured using a subjective scale of 0-3 by a treatment-blind observer. Mucin-secreting cells stained with PAS in the airways were counted from two lung sections per mouse. At the same time we also measured the length of the bronchi basal lamina using ImageJ software (National Institutes of Health, Bethesta, MD, USA). Mucous production was expressed by the number of PAS-positive cells per mm of bronchiole (Kim and Im, 2019).
Mouse IgE levels in the serum were determined using ELISA kits (eBioscience, San Diego, CA, USA). IL-4 and IL-13 levels in BALF were quantitated using ELISA kits (eBioscience). Capture antibodies and biotinylated detection antibodies specific for IL-4 and IL-13 were obtained from eBioscience (IL-4: cat. 14-7041-68 and cat. 33-7042-68C or IL-13: cat no. 14-7043-68 and 33-7135-68B). Avidin-horseradish peroxidase was used and the absorbance was measured at 450 nm (Lee
Results are expressed as means ± standard errors (SEs). For statistical significance analysis of variance (ANOVA) was used, and followed by Turkey’s
Mast cells play a pivotal role in asthma episodes (Prussin and Metcalfe, 2003). Antigen exposure induces cross-linking of IgEs on mast cell membranes, resulting in degranulation (Gilfillan
Next, an OVA-induced mouse model of asthma was employed to verify the inhibitory effect of CYM50358. The total cell number was assessed in the BALF, and the distribution of immune cell populations was calculated. In the BALF, the total cell number increased to 293.1% in the OVA-induced asthma group when compared with that in the PBS-treated control group (Fig. 2). CYM50358 treatment before antigen sensitization or before antigen challenge significantly inhibited the OVA-induced increase in the total cell number by 56.6 and 41.7%, respectively (Fig. 2B). Immune cell populations in the BALF were also assessed, with the eosinophil number increased by the OVA treatment and significantly decreased by CYM50358 treatment before sensitization or before challenge, by 69.3 and 48.5%, respectively (Fig. 2B). Although the lymphocyte number was lower than the eosinophil number, OVA induced an increase and CYM50358 treatment decreased the lymphocyte counts by both treatments (Fig. 2C). Macrophage numbers were not significantly altered by OVA or CYM50358 (Fig. 2B).
Additionally, histological analysis of the lung samples was performed. In the H&E staining, eosinophils in the lung sections were present as small, navy-blue dots (Fig. 3). Although substantially few eosinophils were detected in the PBS control group, numerous eosinophils densely surrounded bronchioles in the OVA group (Fig. 3). CYM50358 treatment before sensitization or before challenge reduced eosinophil numbers (Fig. 3). On using a subjective scale of 0-3, semi-quantitative evaluation of lung inflammation indicated an average inflammation score of 2.2 in the OVA-treated group, and CYM50358 treatment before sensitization or before challenge significantly reduced the score (Fig. 3).
PAS staining was also performed to reveal mucins and mucous glycoproteins produced by goblet cells. As shown in Fig. 4, secreted or stored mucins appeared as dark violet. Mucins were stained in cells surrounding bronchioles in the OVA group. However, mucin production was suppressed following CYM50358 treatment before sensitization or before challenge (Fig. 4). Furthermore, a semi-quantitative analysis of mucin production was performed by counting PAS-positive cells in bronchioles (Fig. 4). Stained cells were scarce in the PBS-treated group. However, in the OVA-treated group, approximately 100 PAS-positive cells/mm were detected, and CYM50358 treatment before sensitization or before challenge significantly suppressed the number of PAS-positive cells (Fig. 4).
Serum IgE levels were assessed to confirm the immunological effects of OVA and CYM50358. IgE production was increased in the sera of OVA-treated mice (Fig. 5A). An OVA-induced increase in serum IgE levels was significantly repressed by CYM50358 treatment, both before antigen sensitization and challenge.
Th2 cytokines, such as IL-4, play major roles in the progression of allergic asthma (Romagnani, 2002). Th2 cytokines induce eosinophil recruitment and activation, hypersecretion of mucus in epithelial cells, metaplasia of goblet cells, and proliferation of smooth muscle cells (Tagaya and Tamaoki, 2007). The protein levels of Th2 cytokines, IL-4 and IL-13 in BALF were measured by ELISA. The IL-4 levels were increased in the OVA-induced group compared to the vehicle-treated control group, and the increase in IL-4 levels was significantly suppressed by both treatments of CYM50358 (Fig. 5B). We were not able to detect IL-13 levels in the BALF, because they were below the detectable ranges.
In the present study, two new findings were revealed using the S1P4 antagonist, CYM50358. First, the S1P4 receptor was involved in mast cell degranulation in RBL-2H3 cells and OVA-induced allergic asthma. Following the administration of CYM50358 before antigen challenges, immunological responses such as increase of IgE levels, immune cell accumulation in BALF, and increased mucin-secreting cells in the lungs were significantly suppressed. CYM50358-mediated inhibition of mast cell degranulation might contribute to the suppressive actions of the CYM50358
The results with S1P4 deficient mice are in contrast with the present results. In the present study, inhibition of S1P4 using CYM50358 suppressed allergic responses, while S1P4 gene deficiency increased the magnitude of Th2-dominated immune responses, including the aggravation of passive systemic anaphylaxis to IgE/anti-IgE in mice (Schulze
An abundant expression of S1P4 has been observed in cultured mouse mast cells (Kulinski
Previously, the significance of S1P and its receptors has been reported in animal models, such as antigen-induced allergic asthma and airway inflammation (Roviezzo
This research was supported by the Basic Research Laboratory Program (BRL) and the Basic Science Research Program of the Korean National Research Foundation funded by the Korean Ministry of Science, ICT and Future Planning (NRF-2020R1A4A1016142 and NRF-2019R1A2C1005523).
The authors declare that there is no conflict of interest.