Authors:

Harianna HacDonald

Department of Biomedical Sciences, Canada

Abstract:

Rich in red wine, peanuts, and grape skin, resveratrol is a polyphenol that has been demonstrated to possess anti-inflammatory, anti-cancer, and antioxidant properties. It may help mitigate allergic inflammation. We looked at how resveratrol and the anti-allergy medication tranilast affected human mast cell activation. Resveratrol and tranilast both prevented substance P, IgE/anti-IgE, and compound 48/80 from inducing degranulation in LAD2 mast cells. When added concurrently to physiological stimuli, resveratrol inhibited degranulation immediately and maintained the effect for up to 24 hours. Although cAMP was not necessary for the inhibitory effect, it might have been caused by calcium modulation, resveratrol, and, to a lesser extent, tranilast, which preceded substance P-induced elevations.
While tranilast had no effect, resveratrol reduced substance P-induced TNF and MCP-1 production as well as the IgE-mediated release of cysteinyl leukotrienes. Moreover, on LAD2 cells, resveratrol and tranilast decreased the production of FcεRI, a high affinity IgE receptor. Human primary CD34+-derived mast cells (HuMC) showed a greater response to resveratrol’s effects on mast cell activation, and the polyphenol was found to be substantially more effective in these cells than tranilast. In summary, resveratrol was as effective as, if not more so than, the anti-allergy medication tranilast in inhibiting important elements of human mast cell activation in response to physiological stimuli. Resveratrol may therefore be a useful medicinal substance for the management of allergic illness. Keywords:
Mast Cells; Resveratrol; Tranilast; IgE;Substance P; Leukotrienes

Introduction :
Certain plants respond to infections by producing resveratrol, an antibacterial substance [1,2]. Red wine, peanuts, and grape skins are the foods highest in polyphenols. This has prompted much investigation into the possible health advantages of red wine, while it is questionable if the resveratrol concentration is sufficient for bioactivity. Numerous health-promoting properties of the polyphenol have been linked to it, most of which have been observed in mice. These properties include the prevention of cancer, antioxidant and anti-inflammatory activity, cardioprotective effects, and dilatation of the airways [3, 4]. As resveratrol inhibits the release of arachidonic acid and the synthesis of lipoxygenase and cyclooxygenase products [5-7], it may also be advantageous for allergic inflammatory reactions.
Key players in the mediation of allergy and anaphylactic responses, mast cells release pro-inflammatory mediators when allergens crosslink IgE coupled to FcεRI [8,9]. In an OVA-induced mouse asthma model, resveratrol decreases airway hyperresponsiveness, inflammation, and allergen-specific IgE levels in vivo [10]. Resveratrol-induced inhibition of degranulation and histamine release in mouse bone marrow mast cells (BMMC) [11], rat basophil-like cells (RBL-2H3) [12,13], and rat peritoneal mast cells [14] has been observed in preliminary in vitro animal studies. This inhibition is most likely caused by suppressing tyrosine phosphorylation of ERK and PLC 1 [12]. Additionally, resveratrol lowers the synthesis of prostaglandin D2, TNF, and cysteine-teinyl leukotriene in mouse BMMC [11]. There aren’t many studies looking at how resveratrol affects human mast cells.

Material and Methods :
The LAD2 cells were cultivated in serum-free media (StemPro-34 SFM, Life Technologies, Burlington, ON) supplemented with 100 ng/ml of stem cell factor (Peprotech Inc., Rocky Hill, NJ), 100 U/ml of penicillin, and 50 µg/ml of streptomycin. The expression of kit and FcεRI was periodically assessed in LAD2 cells using flow cytometry, and the results showed that the expression of these receptors was comparable to that of CD34+ cells obtained from blood. In StemPro-34 SFM supplemented with 2 mM L-glutamine, 50 µg/ml streptomycin, 100 IU/ml penicillin, 100 ng/ml SCF, and 100 ng/ml recombinant human IL-6 and recombinant human IL-3 (PeproTech) (30 ng/ml; first week only), human peripheral blood-derived CD34+ cells were grown. Both LAD2 and HuMC were grown in serum-free medium (StemPro-34 SFM) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 50 µg/ml streptomycin, and 100 ng/ml SCF for investigations that required overnight incubation.
After being suspended in buffer, cells were stimulated for 30 minutes at 37˚C/5% CO2 using either 0.3 μg/ml compound 48/80 or 0.5 μg/ml substance P (Sigma-Aldrich Canada, Oakville, ON). For IgE assays, cells were sensitised with 0.5 μg/mL myeloma IgE (Calbio-chem, Billerica, MA) overnight at 37˚C/5% CO2, then stimulated for 30 minutes with 100 μg/ml rabbit anti-IgE (Dako, Carpinteria, CA) or 0.1 μg/ml IgE-biotin (Abbiotec, San Diego, CA) overnight at 37˚C/5% CO2, and 30 minutes with 0.5 μg/ ml streptavidin. Before being stimulated by an agonist, cells were pretreated for the given amounts of time with resveratrol or tranilast (Sigma-Aldrich). After hydrolyzing sub-stratum p-nitrophenyl N-acetyl-β-D-glucosamide (Sigma-Aldrich) in 0.1 M sodium citrate buffer (pH 4.5) for 90 minutes at 37˚C, the amounts of β-hexosaminidase in the supernatants and cell lysates were measured. The β-hexosaminidase release percentage was computed as a percentage of the overall content.

ELISA Analysis of MCP-1 and TNF :
After a three-hour pretreatment at 37˚C with 5% CO2, the cells were stimulated for twenty hours with 0.5 μg/ml substance P. Centrifugation was used to separate the cell-free supernatants, and commercial TNF and MCP-1 ELISA kits (eBiosciences, San Diego, CA) were used to measure human cytokine expression.
The cells were treated with resveratrol or tranilast for three hours at 37˚C and 5% CO2 after being sensitised with 0.5 μg/ml IgE for the previous night. After that, cells were stimulated with 100 μg/ml of anti-IgE. Cysteinyl leukotriene (cysLT) levels were assessed using commercial competitive enzyme immunoassays (Assay Designs) after cell free supernatants were separated by centrifugation.
After treating the cells for 30 minutes with 10 μg/ml resveratrol, tranilast, or 10 μM forskolin, the process was halted by adding 5 M HCL and waiting 20 minutes before using a pipette to triturate the mixture. Centrifugation was used to extract cell-free supernatants, and a commercial competitive immunoassay (Cayman Chemicals, Ann Arbour, MI) was utilised to quantify cAMP.

Flow Cytometry-Based Expression of FcεRI :
For 20 hours at 37˚C and 5% CO2, cells were treated with resveratrol or tranilast. After being cleaned and resuspended in 0.1% BSA/PBS, they were kept on ice for ten minutes. After that, the cells were treated for one hour at 4˚C with PE-conjugated anti-FcεRI antibody or isotype control antibody (eBiosciences, San Diego, CA). The cells underwent two rounds of washing in 0.1% BSA/PBS, were resuspended in the same buffer, and then examined using a BD Biosci- ences, Mississauga, ON, FACSArray flow cytometer. The expression for FcεRI levels is mean fluorescence intensity (MFI).
The means ± standard errors were used to describe the results. One-way ANOVA or the Student’s t test were used to analyse the data. for every experimental condition, with a minimum of three repetitions of each experiment, and data analysis using a repeated measures ANOVA combined with the Tukey Post Hoc test. Using GraphPad Prism software (GraphPad Software Inc., San Diego, CA), non-linear regression and 4-parametric analysis were used to calculate the IC50 values. After accounting for the effects of the vehicle (DMSO) on all data, a P value of less than 0.05 was considered significant.

Result
LAD2 cells were pre-treated with varying concentrations of resveratrol and tranilast for 30 minutes before being stimulated with either substance P (0.5 μg/ml), compound 48/80 (0.3 μg/ ml), or IgE/anti-IgE (0.5 μg/ml/100 ng/ml) (Figure 1). This was done to ascertain the impact of the two compounds on human mast cell degranulation. The degranulation caused by substance P (68% and 52%, respectively), compound 48/80 (80% and 51%), and IgE/anti-IgE (37% and 27%, respectively) was suppressed by both resveratrol and tranilast (100 μg/ml).
In order to ascertain the ideal resveratrol inhibitory effects, multiple treatment intervals were examined. Before being activated with 0.5 μg/ml substance P, 0.3 μg/ml compound 48/80, or 0.5 μg/ml streptavidin (in 0.1 μg/ml IgE-biotin sensitised cells), LAD2 cells were pretreated with resveratrol for 0, 30, 3, and 24 hours. At every time point examined, resveratrol dramatically reduced substance P- and IgE-mediated human mast cell degranulation (Figures 2(a) and (c), respectively). Additionally, compound 48/80-induced degranulation was blocked by resveratrol, albeit less successfully and at earlier times needing greater doses to block reactions. IC50 values varied from 1.34 to 12.5 ug/ml according on the stimulant used and length of resveratrol treatment, as determined by centration response curves.

Citation:

Harianna HacDonaldEffects of phytoalexin on human leukocyte release, hormone, chemical, and mast cell proliferation. Journal of Immunology 2024.