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1.
Int J Mol Sci ; 25(13)2024 Jul 06.
Article in English | MEDLINE | ID: mdl-39000527

ABSTRACT

Mast cells are essential immune cells involved in the host's defence against gastrointestinal nematodes. To evade the immune response, parasitic nematodes produce a variety of molecules. Galectin 1, produced by Teladorsagia circumcincta (Tci-gal-1), reduces mast cell degranulation and selectively regulates mediator production and release in an IgE-dependent manner. To uncover the activity of Tci-gal-1, we have examined the effect of the protein on gene expression, protein production, and apoptosis in activated basophilic leukaemia RBL-2H3 cells. Rat RBL-2H3 cells were activated with anti-DNP IgE and DNP-HSA, and then treated with Tci-gal-1. Microarray analysis was used to examine gene expression. The levels of several apoptosis-related molecules and cytokines were determined using antibody arrays and ELISA. Early and late apoptosis was evaluated cytometrically. Degranulation of cells was determined by a ß-hexosaminidase release assay. Treatment of activated RBL-2H3 cells with Tci-gal-1 resulted in inhibited apoptosis and decreased degranulation, although we did not detect significant changes in gene expression. The production of pro-apoptotic molecules, receptor for advanced glycation end products (RAGE) and Fas ligand (FasL), and the cytokines IL-9, IL-10, IL-13, TNF-α, and IL-2 was strongly inhibited. Tci-gal-1 modulates apoptosis, degranulation, and production of cytokines by activated RBL-2H3 cells without detectable influence on gene transcription. This parasite protein is crucial for modulation of the protective immune response and the inhibition of chronic inflammation driven by mast cell activity.


Subject(s)
Apoptosis , Cell Degranulation , Immunoglobulin E , Leukemia, Basophilic, Acute , Animals , Rats , Immunoglobulin E/immunology , Cell Line, Tumor , Leukemia, Basophilic, Acute/metabolism , Leukemia, Basophilic, Acute/immunology , Leukemia, Basophilic, Acute/pathology , Mast Cells/immunology , Mast Cells/metabolism , Cytokines/metabolism , Galectins/metabolism , Helminth Proteins/pharmacology , Helminth Proteins/metabolism , Galectin 1/metabolism , Galectin 1/genetics
2.
Zhonghua Kou Qiang Yi Xue Za Zhi ; 59(7): 721-725, 2024 Jul 09.
Article in Chinese | MEDLINE | ID: mdl-38949141

ABSTRACT

Periodontitis is a chronic inflammatory condition of the periodontal tissues triggered by bacterial biofilm, leading to manifestations such as gingival bleeding, tooth mobility, and eventual exfoliation. Neutrophils exhibit a dual role throughout the course of periodontitis, both in defense against pathogens and in potentially detrimental effects on periodontal tissues. This article elucidates the intricate mechanisms underlying the dual functions of neutrophils in periodontitis, including respiratory burst, neutrophil extracellular traps (NETs) formation, degranulation, and phagocytosis. By providing a comprehensive understanding of neutrophils involvement in periodontitis, this study aims to empower clinicians with insights into the pathogenesis of periodontitis, thereby fostering novel strategies for its prevention and treatment.


Subject(s)
Extracellular Traps , Neutrophils , Periodontitis , Phagocytosis , Neutrophils/immunology , Humans , Extracellular Traps/immunology , Periodontitis/immunology , Periodontitis/microbiology , Respiratory Burst , Biofilms , Inflammation/immunology , Cell Degranulation
3.
Int J Mol Sci ; 25(13)2024 Jun 26.
Article in English | MEDLINE | ID: mdl-39000091

ABSTRACT

Novel (immune) therapies are needed to stabilize remissions or the disease in AML. Leukemia derived dendritic cells (DCleu) can be generated ex vivo from AML patients' blasts in whole blood using approved drugs (GM-CSF and PGE-1 (Kit M)). After T cell enriched, mixed lymphocyte culture (MLC) with Kit M pretreated (vs. untreated WB), anti-leukemically directed immune cells of the adaptive and innate immune systems were already shown to be significantly increased. We evaluated (1) the use of leukemia-specific assays [intracellular cytokine production of INFy, TNFa (INCYT), and degranulation detected by CD107a (DEG)] for a detailed quantification of leukemia-specific cells and (2), in addition, the correlation with functional cytotoxicity and patients' clinical data in Kit M-treated vs. not pretreated settings. We collected whole blood (WB) samples from 26 AML patients at first diagnosis, during persisting disease, or at relapse after allogeneic stem cell transplantation (SCT), and from 18 healthy volunteers. WB samples were treated with or without Kit M to generate DC/DCleu. After MLC with Kit M-treated vs. untreated WB antigen-specific/anti-leukemic effects were assessed through INCYT, DEG, and a cytotoxicity fluorolysis assay. The quantification of cell subtypes was performed via flow cytometry. Our study showed: (1) low frequencies of leukemia-specific cells (subtypes) detectable in AML patients' blood. (2) Significantly higher frequencies of (mature) DCleu generable without induction of blast proliferation in Kit M-treated vs. untreated samples. (3) Significant increase in frequencies of immunoreactive cells (e.g., non-naive T cells, Tprol) as well as in INCYT/DEG ASSAYS leukemia-specific adaptive-(e.g., B, T(memory)) or innate immune cells (e.g., NK, CIK) after MLC with Kit M-treated vs. untreated WB. The results of the intracellular production of INFy and TNFa were comparable. The cytotoxicity fluorolysis assay revealed significantly enhanced blast lysis in Kit M-treated vs. untreated WB. Significant correlations could be shown between induced leukemia-specific cells from several lines and improved blast lysis. We successfully detected and quantified immunoreactive cells at a single-cell level using the functional assays (DEG, INCYT, and CTX). We could quantify leukemia-specific subtypes in uncultured WB as well as after MLC and evaluate the impact of Kit M pretreated (DC/DCleu-containing) WB on the provision of leukemia-specific immune cells. Kit M pretreatment (vs. no pretreatment) was shown to significantly increase leukemia-specific IFNy and TNFa producing, degranulating cells and to improve blast-cytotoxicity after MLC. In vivo treatment of AML patients with Kit M may lead to anti-leukemic effects and contribute to stabilizing the disease or remissions. INCYT and DEG assays qualify to quantify potentially leukemia-specific cells on a single cell level and to predict the clinical course of patients under treatment.


Subject(s)
Cytokines , Leukemia, Myeloid, Acute , Humans , Leukemia, Myeloid, Acute/immunology , Leukemia, Myeloid, Acute/therapy , Leukemia, Myeloid, Acute/pathology , Leukemia, Myeloid, Acute/drug therapy , Middle Aged , Male , Adult , Female , Cytokines/metabolism , Aged , Dendritic Cells/immunology , Dendritic Cells/metabolism , Cell Degranulation/drug effects , Young Adult
4.
Toxicology ; 506: 153882, 2024 Aug.
Article in English | MEDLINE | ID: mdl-38971550

ABSTRACT

Diazinon is an organophosphorus (OP) insecticides used in agriculture, home gardening and indoor pest control in Japan. It can activate macrophages and induce pro-inflammatory responses and has been reported to cause airway hyper-reactivity, suggesting the possibility of asthma exacerbation from exposure to OP insecticides. Despite the correlation between insecticide use and the pathogenesis of allergic diseases, there have been no reports on the effects of diazinon on mast cell function. Therefore, in this study, we investigated the effects of diazinon on mast cell function in rat basophilic leukemia (RBL)-2H3 cells. Surprisingly, we found that diazinon inhibited mast cell activation, although the degree of inhibition varied with concentration. Diazinon induced reactive oxygen species (ROS) generation and HO-1 expression at a concentration of 150 µM without affecting cell viability. Diazinon inhibited A23187-mediated degranulation and Tnf and Il4 expression in RBL-2H3 cells but did not affect calcium influx. Suppression of degranulation by diazinon was reversed when the culture supernatant was removed. As a signaling event downstream of calcium influx, diazinon inhibited the phosphorylation of extracellular signal-regulated kinase (ERK) induced by A23187, whereas the phosphorylation of p38 had little effect. IgE cross-linking-mediated degranulation as well as the induction of Tnf and IL4 expression was significantly inhibited by diazinon, while diazinon had little effect on calcium influx. In conclusion, diazinon inhibited mast cell activation, including degranulation and cytokine expression. When evaluating the in vivo effects of diazinon, its potential to inhibit mast cell activation should be considered in the pathophysiology and development of allergic diseases in terms of basic and clinical aspects, respectively, although the effect of diazinon varies depending on the cell type.


Subject(s)
Cell Degranulation , Cytokines , Diazinon , Insecticides , Mast Cells , Diazinon/toxicity , Animals , Mast Cells/drug effects , Mast Cells/metabolism , Rats , Cytokines/metabolism , Insecticides/toxicity , Cell Degranulation/drug effects , Reactive Oxygen Species/metabolism , Cell Line, Tumor , Cell Survival/drug effects
5.
Front Immunol ; 15: 1369326, 2024.
Article in English | MEDLINE | ID: mdl-38953022

ABSTRACT

Objectives: Mast cell (MC) degranulation is a key process in allergic reactions and inflammatory responses. Aspartate aminotransferase 1 (AAT1)-derived endogenous sulfur dioxide (SO2) is an important regulator of MC function. However, the mechanism underlying its role in MC degranulation remains unclear. This study aimed to investigate the mechanism by which endogenous SO2 controlled MC degranulation. Methods: HMC-1 and Rat basophilic leukemia cell MC line (RBL-2H3) were used in the cell experiments. SO2 content was detected by in situ fluorescent probe. MC degranulation represented by the release rate of MC ß-hexosaminidase was determined using a colorimetric assay. Sulfenylation of galectin-9 (Gal-9) in MCs and purified protein was detected using a biotin switch assay. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine the exact sulfenylation sites of Gal-9 by SO2. Animal models of passive cutaneous anaphylaxis (PCA) and hypoxia-driven pulmonary vascular remodeling were used to investigate the effect of SO2 on mast cell activation in vivo. Site-directed mutation of Gal-9 was conducted to confirm the exact site of SO2 and support the significance of SO2/Gal-9 signal axis in the regulation of MC degranulation. Results: Degranulation was increased in AAT1-knockdowned MCs, and SO2 supplementation reversed the increase in MC degranulation. Furthermore, deficiency of endogenous SO2 contributed to IgE-mediated degranulation in vitro. Besides, SO2 inhibited IgE-mediated and hypoxia-driven MC degranulation in vivo. Mechanistically, LC-MS/MS analysis and site-directed mutation results showed that SO2 sulfenylated Gal-9 at cysteine 74. Sulfenylation of the 74th cysteine of Gal-9 protein was required in the SO2-inhibited MC degranulation under both physiological and pathophysiological conditions. Conclusion: These findings elucidated that SO2 inhibited MC degranulation via sulfenylating Gal-9 under both physiological and pathophysiological conditions, which might provide a novel treatment approach for MC activation-related diseases.


Subject(s)
Cell Degranulation , Cysteine , Galectins , Mast Cells , Sulfur Dioxide , Animals , Cell Degranulation/drug effects , Mast Cells/metabolism , Mast Cells/immunology , Mast Cells/drug effects , Cysteine/metabolism , Rats , Sulfur Dioxide/pharmacology , Sulfur Dioxide/metabolism , Humans , Galectins/metabolism , Mice , Male , Passive Cutaneous Anaphylaxis , Cell Line
6.
Int J Mol Sci ; 25(14)2024 Jul 10.
Article in English | MEDLINE | ID: mdl-39062810

ABSTRACT

Inhibitors of sodium/glucose cotransporter 2 (SGLT2), such as empagliflozin and canagliflozin, have been widely used to block glucose reabsorption in the proximal tubules of kidneys in patients with diabetes. A meta-analysis suggested that SGLT2 inhibitors are associated with a decreased risk of asthma development. Therefore, we investigated whether SGLT2 inhibitors could suppress allergic asthma. Empagliflozin and canagliflozin suppressed the in vitro degranulation reaction induced by antigens in a concentration-dependent manner in RBL-2H3 mast cells. Empagliflozin and canagliflozin were administered to BALB/c mice sensitized to ovalbumin (OVA). The administration of empagliflozin or canagliflozin significantly suppressed OVA-induced airway hyper-responsiveness and increased the number of immune cells and pro-inflammatory cytokine mRNA expression levels in bronchoalveolar lavage fluid. The administration of empagliflozin and canagliflozin also suppressed OVA-induced histopathological changes in the lungs. Empagliflozin and canagliflozin also suppressed serum IgE levels. These results suggested that empagliflozin and canagliflozin may be applicable for the treatment of allergic asthma by suppressing immune responses.


Subject(s)
Asthma , Benzhydryl Compounds , Canagliflozin , Glucosides , Mice, Inbred BALB C , Ovalbumin , Sodium-Glucose Transporter 2 Inhibitors , Animals , Canagliflozin/pharmacology , Canagliflozin/therapeutic use , Sodium-Glucose Transporter 2 Inhibitors/pharmacology , Sodium-Glucose Transporter 2 Inhibitors/therapeutic use , Asthma/drug therapy , Asthma/immunology , Glucosides/pharmacology , Glucosides/therapeutic use , Benzhydryl Compounds/pharmacology , Benzhydryl Compounds/therapeutic use , Mice , Ovalbumin/adverse effects , Lung/pathology , Lung/drug effects , Lung/metabolism , Mast Cells/drug effects , Mast Cells/metabolism , Mast Cells/immunology , Immunoglobulin E/blood , Immunoglobulin E/immunology , Bronchoalveolar Lavage Fluid , Rats , Cytokines/metabolism , Cell Degranulation/drug effects , Disease Models, Animal , Sodium-Glucose Transporter 2/metabolism , Male
7.
Front Immunol ; 15: 1391967, 2024.
Article in English | MEDLINE | ID: mdl-38989281

ABSTRACT

Introduction: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening immune disorder characterized by uncontrolled lymphocyte and macrophage activation and a subsequent cytokine storm. The timely initiation of immunosuppressive treatment is crucial for survival. Methods: Here, we harnessed Vγ9Vδ2 T cell degranulation to develop a novel functional assay for the diagnosis of HLH. We compared the novel assay with the conventional natural killer (NK) cell stimulation method in terms of efficiency, specificity, and reliability. Our analysis involved 213 samples from 182 individuals, including 23 samples from 12 patients with degranulation deficiency (10 individuals with UNC13D deficiency, 1 with STXBP2 deficiency, and 1 with RAB27A deficiency). Results: While both tests exhibited 100% sensitivity, the Vγ9Vδ2 T cell degranulation assay showed a superior specificity of 86.2% (n=70) compared to the NK cell degranulation assay, which achieved 78.9% specificity (n=213). The Vγ9Vδ2 T cell degranulation assay offered simpler technical requirements and reduced labor intensity, leading to decreased susceptibility to errors with faster processing times. Discussion: This efficiency stemmed from the sole requirement of dissolving (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) powder, contrasting with the intricate maintenance of K562 cells necessary for the NK cell degranulation assay. With its diminished susceptibility to errors, we anticipate that the assay will require fewer repetitions of analysis, rendering it particularly well-suited for testing infants. Conclusion: The Vγ9Vδ2 T cell degranulation assay is a user-friendly, efficient diagnostic tool for HLH. It offers greater specificity, reliability, and practicality than established methods. We believe that our present findings will facilitate the prompt, accurate diagnosis of HLH and thus enable rapid treatment and better patient outcomes.


Subject(s)
Cell Degranulation , Killer Cells, Natural , Lymphohistiocytosis, Hemophagocytic , Humans , Lymphohistiocytosis, Hemophagocytic/diagnosis , Lymphohistiocytosis, Hemophagocytic/immunology , Lymphohistiocytosis, Hemophagocytic/genetics , Female , Male , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Receptors, Antigen, T-Cell, gamma-delta/metabolism , Receptors, Antigen, T-Cell, gamma-delta/genetics , Child, Preschool , Child , Infant , Adolescent , rab27 GTP-Binding Proteins/genetics , Membrane Proteins/genetics , Membrane Proteins/metabolism , Adult , T-Lymphocytes/immunology , Reproducibility of Results , Lymphocyte Activation , Sensitivity and Specificity , Munc18 Proteins
8.
Front Immunol ; 15: 1393802, 2024.
Article in English | MEDLINE | ID: mdl-38881896

ABSTRACT

Background: Mast cells are critically involved in IgE-mediated diseases, e.g., allergies and asthma. Human mast cells are heterogeneous, and mast cells from different anatomical sites have been shown to respond differently to certain stimuli and drugs. The origin of the mast cells is therefore of importance when setting up a model system, and human lung mast cells are highly relevant cells to study in the context of asthma. We therefore set out to optimize a protocol of IgE-mediated activation of human lung mast cells. Methods: Human lung mast cells were extracted from lung tissue obtained from patients undergoing pulmonary resection by enzyme digestion and mechanical disruption followed by CD117 magnetic-activated cell sorting (MACS) enrichment. Different culturing media and conditions for the IgE-mediated degranulation were tested to obtain an optimized method. Results: IgE crosslinking of human lung mast cells cultured in serum-free media gave a stronger response compared to cells cultured with 10% serum. The addition of stem cell factor (SCF) did not enhance the degranulation. However, when the cells were put in fresh serum-free media 30 minutes prior to the addition of anti-IgE antibodies, the cells responded more vigorously. Maximum degranulation was reached 10 minutes after the addition of anti-IgE. Both CD63 and CD164 were identified as stable markers for the detection of degranulated mast cells over time, while the staining with anti-CD107a and avidin started to decline 10 minutes after activation. The levels of CD203c and CD13 did not change in activated cells and therefore cannot be used as degranulation markers of human lung mast cells. Conclusions: For an optimal degranulation response, human lung mast cells should be cultured and activated in serum-free media. With this method, a very strong and consistent degranulation response with a low donor-to-donor variation is obtained. Therefore, this model is useful for further investigations of IgE-mediated mast cell activation and exploring drugs that target human lung mast cells, for instance, in the context of asthma.


Subject(s)
Cell Degranulation , Immunoglobulin E , Lung , Mast Cells , Humans , Mast Cells/immunology , Mast Cells/metabolism , Immunoglobulin E/immunology , Lung/immunology , Cells, Cultured , Proto-Oncogene Proteins c-kit/immunology , Proto-Oncogene Proteins c-kit/metabolism , Culture Media, Serum-Free/pharmacology , Antibodies, Anti-Idiotypic
9.
Cell Physiol Biochem ; 58(3): 212-225, 2024 May 09.
Article in English | MEDLINE | ID: mdl-38852193

ABSTRACT

BACKGROUND/AIMS: Adrenaline quickly inhibits the release of histamine from mast cells. Besides ß2-adrenergic receptors, several in vitro studies also indicate the involvement of α-adrenergic receptors in the process of exocytosis. Since exocytosis in mast cells can be detected electrophysiologically by the changes in the membrane capacitance (Cm), its continuous monitoring in the presence of drugs would determine their mast cell-stabilizing properties. METHODS: Employing the whole-cell patch-clamp technique in rat peritoneal mast cells, we examined the effects of adrenaline on the degranulation of mast cells and the increase in the Cm during exocytosis. We also examined the degranulation of mast cells in the presence or absence of α-adrenergic receptor agonists or antagonists. RESULTS: Adrenaline dose-dependently suppressed the GTP-γ-S-induced increase in the Cm and inhibited the degranulation from mast cells, which was almost completely erased in the presence of butoxamine, a ß2-adrenergic receptor antagonist. Among α-adrenergic receptor agonists or antagonists, high dose prazosin, a selective α1-adrenergic receptor antagonist, significantly reduced the ratio of degranulating mast cells and suppressed the increase in the Cm. Additionally, prazosin augmented the inhibitory effects of adrenaline on the degranulation of mast cells. CONCLUSION: This study provided electrophysiological evidence for the first time that adrenaline dose-dependently inhibited the process of exocytosis, confirming its usefulness as a potent mast cell-stabilizer. The pharmacological blockade of α1-adrenergic receptor by prazosin synergistically potentiated such mast cell-stabilizing property of adrenaline, which is primarily mediated by ß2-adrenergic receptors.


Subject(s)
Cell Degranulation , Epinephrine , Exocytosis , Mast Cells , Prazosin , Animals , Mast Cells/drug effects , Mast Cells/metabolism , Mast Cells/cytology , Epinephrine/pharmacology , Rats , Prazosin/pharmacology , Cell Degranulation/drug effects , Male , Exocytosis/drug effects , Patch-Clamp Techniques , Adrenergic alpha-1 Receptor Antagonists/pharmacology , Rats, Wistar
10.
J Transl Med ; 22(1): 526, 2024 May 31.
Article in English | MEDLINE | ID: mdl-38822352

ABSTRACT

BACKGROUND: Neutrophils are granulocytes with essential antimicrobial effector functions and short lifespans. During infection or sterile inflammation, emergency granulopoiesis leads to release of immature neutrophils from the bone marrow, serving to boost circulating neutrophil counts. Steady state and emergency granulopoiesis are incompletely understood, partly due to a lack of genetically amenable models of neutrophil development. METHODS: We optimised a method for ex vivo production of human neutrophils from CD34+ haematopoietic progenitors. Using flow cytometry, we phenotypically compared cultured neutrophils with native neutrophils from donors experiencing emergency granulopoiesis, and steady state neutrophils from non-challenged donors. We carry out functional and proteomic characterisation of cultured neutrophils and establish genome editing of progenitors. RESULTS: We obtain high yields of ex vivo cultured neutrophils, which phenotypically resemble immature neutrophils released into the circulation during emergency granulopoiesis. Cultured neutrophils have similar rates of ROS production and bacterial killing but altered degranulation, cytokine release and antifungal activity compared to mature neutrophils isolated from peripheral blood. These differences are likely due to incomplete synthesis of granule proteins, as demonstrated by proteomic analysis. CONCLUSION: Ex vivo cultured neutrophils are genetically tractable via genome editing of precursors and provide a powerful model system for investigating the properties and behaviour of immature neutrophils.


Subject(s)
Antigens, CD34 , Neutrophils , Humans , Neutrophils/metabolism , Neutrophils/cytology , Antigens, CD34/metabolism , Cells, Cultured , Reactive Oxygen Species/metabolism , Proteomics , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/cytology , Gene Editing , Cell Degranulation , Stem Cells/metabolism , Stem Cells/cytology , Cytokines/metabolism , Phenotype
11.
J Pharmacol Exp Ther ; 390(2): 240-249, 2024 Jul 18.
Article in English | MEDLINE | ID: mdl-38902033

ABSTRACT

Sympathetic hyperinnervation is the leading cause of fatal ventricular arrhythmia (VA) after myocardial infarction (MI). Cardiac mast cells cause arrhythmias directly through degranulation. However, the role and mechanism of mast cell degranulation in sympathetic remodeling remain unknown. We investigated the role of oxytocin (OT) in stabilizing cardiac mast cells and improving sympathetic innervation in rats. MI was induced by coronary artery ligation. Western blotting, immunofluorescence, and toluidine staining of mast cells were performed to determine the expression and location of target protein. Mast cells accumulated significantly in peri-infarcted tissues and were present in a degranulated state. They expressed OT receptor (OTR), and OT infusion reduced the number of degranulated cardiac mast cells post-MI. Sympathetic hyperinnervation was attenuated as assessed by immunofluorescence for tyrosine hydroxylase (TH). Seven days post-MI, the arrhythmia score of programmed electrical stimulation was higher in vehicle-treated rats with MI than in rats treated with OT. An in vitro study showed that OT stabilized mast cells via the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling pathway. Further in vivo studies on OTR-deficient mice showed worsening mast cell degranulation and worsening sympathetic innervation. OT pretreatment inhibited cardiac mast cell degranulation post-MI and prevented sympathetic hyperinnervation, along with mast cell stabilization via the PI3K/Akt pathway. SIGNIFICANCE STATEMENT: This is the first study to elucidate the role and mechanism of oxytocin (OT) in inflammatory-sympathetic communication mediated sympathetic hyperinnervation after myocardial infarction (MI), providing new approaches to prevent fatal arrhythmias.


Subject(s)
Cell Degranulation , Mast Cells , Myocardial Infarction , Oxytocin , Rats, Sprague-Dawley , Receptors, Oxytocin , Sympathetic Nervous System , Animals , Oxytocin/pharmacology , Oxytocin/metabolism , Myocardial Infarction/drug therapy , Myocardial Infarction/metabolism , Mast Cells/drug effects , Mast Cells/metabolism , Rats , Sympathetic Nervous System/drug effects , Sympathetic Nervous System/metabolism , Male , Cell Degranulation/drug effects , Receptors, Oxytocin/metabolism , Receptors, Oxytocin/antagonists & inhibitors , Mice , Signal Transduction/drug effects , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Arrhythmias, Cardiac/metabolism , Arrhythmias, Cardiac/drug therapy , Arrhythmias, Cardiac/etiology
12.
Drug Discov Ther ; 18(3): 143-149, 2024 Jul 09.
Article in English | MEDLINE | ID: mdl-38839286

ABSTRACT

This study aims to investigate the antiallergic effects of Shiikuwasha (Citrus depressa Hayata) leaf and peel extracts by examining the regulation of degranulation and inflammatory cytokine production from rat basophilic leukemia (RBL-2H3) cells and antigen-specific antibody production in sensitized mouse spleen lymphocytes. In vivo antiallergic activity was evaluated using the passive cutaneous anaphylaxis (PCA) reaction model. Extracts of Shiikuwasha leaves and peel were prepared using 80% methanol and dissolved in dimethylsulfoxide. The dinitrophenyl-human serum albumin-induced ß-hexosaminidase levels in immunoglobulin (Ig) E-sensitized RBL-2H3 cells were assessed using enzymatic assays. Cytokine production was measured by enzyme-linked immunosorbent assay. Antibody production capacity was evaluated using lymphocytes isolated from spleens of type I allergy model mice. Lymphocytes were cultured for 72 h with Shiikuwasha extracts, and ovalbumin-specific IgE, IgG1, and IgG2a levels were measured. Shiikuwasha leaf and peel extract significantly reduced ß-hexosaminidase release and suppressed interleukin-4 and tumor necrosis factor-α production from RBL-2H3 cells. Ovalbumin-specific IgE and IgG1 production decreased in Shiikuwasha extract-treated lymphocytes. These extracts also significantly suppressed the PCA reaction. Shiikuwasha leaf and peel extract reduce degranulation in RBL-2H3 cells and antibody production in spleen-derived lymphocytes and therefore exhibit antiallergic effects.


Subject(s)
Anti-Allergic Agents , Cell Degranulation , Immunoglobulin E , Plant Extracts , Plant Leaves , Spleen , Animals , Plant Extracts/pharmacology , Rats , Spleen/drug effects , Spleen/immunology , Spleen/cytology , Plant Leaves/chemistry , Mice , Cell Line, Tumor , Cell Degranulation/drug effects , Immunoglobulin E/blood , Anti-Allergic Agents/pharmacology , Anti-Allergic Agents/therapeutic use , Lymphocytes/drug effects , Lymphocytes/immunology , Lymphocytes/metabolism , beta-N-Acetylhexosaminidases/metabolism , Passive Cutaneous Anaphylaxis/drug effects , Mice, Inbred BALB C , Leukemia, Basophilic, Acute/immunology , Cytokines/metabolism , Disease Models, Animal , Immunoglobulin G , Male , Interleukin-4/metabolism
13.
Int Immunopharmacol ; 137: 112490, 2024 Aug 20.
Article in English | MEDLINE | ID: mdl-38897121

ABSTRACT

BACKGROUND: Vitamin K3 (VK3), a fat-soluble synthetic analog of the vitamin K family, has coagulant, anti-inflammatory, antibacterial, and anticancer properties. Pseudo allergy is a IgE-independent immune response associated with mast cells. This study investigated the role of VK3 in IgE-independent mast cell activation. METHODS: Substance P (SP) was used to induce LAD2-cell activation in order to analyze the effects of VK3 in vitro. Cutaneous allergy and systemic allergy mouse models were used to analyze the anti-pseudo-allergic effects of VK3. Proteome microarray assays were used to analyze VK3-binding protein. Biolayer interferometry and immunoprecipitation were used to verify interaction between VK3 and its key targets. RNA interference was used to determine the role of GAB1 in LAD2cell activation. RESULTS: VK3 inhibited SP-induced LAD2-cell activation, and resulted in the release of ß-hexosaminidase, histamine and cytokines; VK3 inhibited SP-induced pseudo allergic reactions in mice, and serum histamine and TNF-α levels decreased. Degranulation of skin mast cells was reduced; GAB1 in mast cells was stably bound to VK3. GAB1 participated in SP-induced LAD2-cell activation. GAB1 knockdown in LAD2 cells prevented SP-induced ß-hexosaminidase release, calcium mobilization and cell skeletal remodeling. VK3 directly binds to GAB1 and reduces its expression to inhibited SP-induced LAD2 cell activation. CONCLUSION: The anti-pseudo-allergic activity of VK3 was confirmed in vitro and in vivo. VK3 can inhibit SP-induced mast cell activation by directly targeting GAB1. This study provides new insights on the activity of VK3 and the mechanism of pseudoallergic reaction.


Subject(s)
Adaptor Proteins, Signal Transducing , Mast Cells , Mast Cells/immunology , Mast Cells/drug effects , Animals , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Signal Transducing/genetics , Mice , Humans , Substance P/metabolism , Cell Degranulation/drug effects , Mice, Inbred BALB C , Hypersensitivity/immunology , Hypersensitivity/drug therapy , Anti-Allergic Agents/pharmacology , Anti-Allergic Agents/therapeutic use , Female , Cell Line , beta-N-Acetylhexosaminidases/metabolism , Disease Models, Animal
14.
Sci Rep ; 14(1): 13540, 2024 06 12.
Article in English | MEDLINE | ID: mdl-38866832

ABSTRACT

Mast cells are immune cells minimally present in normal tendon tissue. The increased abundance of mast cells in tendinopathy biopsies and at the sites of tendon injury suggests an unexplored role of this cell population in overuse tendon injuries. Mast cells are particularly present in tendon biopsies from patients with more chronic symptom duration and a history of intensive mechanical loading. This study, therefore, examined the cross talk between mast cells and human tendon cells in either static or mechanically active conditions in order to explore the potential mechanistic roles of mast cells in overuse tendon injuries. A coculture of isolated human tenocytes and mast cells (HMC-1) combined with Flexcell Tension System for cyclic stretching of tenocytes was used. Additionally, human tenocytes were exposed to agonists and antagonists of substance P (SP) receptors. Mast cell degranulation was assessed by measuring ß-hexosaminidase activity. Transwell and cell adhesion assays were used to evaluate mast cell migration and binding to tendon extracellular matrix components (collagen and fibronectin), respectively. Gene expressions were analyzed using real time qRT-PCR. Our results indicate that mechanical stimulation of human tenocytes leads to release of SP which, in turn, activates mast cells through the Mas-related G-protein-coupled receptor X2 (MRGPRX2). The degranulation and migration of mast cells in response to MRGPRX2 activation subsequently cause human tenocytes to increase their expression of inflammatory factors, matrix proteins and matrix metalloproteinase enzymes. These observations may be important in understanding the mechanisms by which tendons become tendinopathic in response to repetitive mechanical stimulation.


Subject(s)
Mast Cells , Receptors, G-Protein-Coupled , Receptors, Neuropeptide , Substance P , Tendons , Tenocytes , Humans , Substance P/metabolism , Substance P/pharmacology , Mast Cells/metabolism , Tenocytes/metabolism , Receptors, G-Protein-Coupled/metabolism , Receptors, Neuropeptide/metabolism , Receptors, Neuropeptide/genetics , Tendons/metabolism , Tendons/pathology , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/genetics , Cell Degranulation , Tendinopathy/metabolism , Tendinopathy/pathology , Inflammation/metabolism , Inflammation/pathology , Male , Coculture Techniques , Cells, Cultured , Adult , Cell Movement
15.
Front Immunol ; 15: 1399459, 2024.
Article in English | MEDLINE | ID: mdl-38812508

ABSTRACT

Initiation of the bradykinin generation cascade is responsible for the occurrence of attacks in some types of angioedema without wheals. Hereditary angioedema due to C1 inhibitor deficiency (HAE-C1-INH) is one such clinical entity. In this paper, we explore the existing evidence that mast cells (MCs) degranulation may contribute to the activation of the kallikrein-kinin system cascade, followed by bradykinin formation and angioedema. We present the multidirectional effects of MC-derived heparin and other polyanions on the major components of the kinin-kallikrein system, particularly on the factor XII activation. Although, bradykinin- and histamine-mediated symptoms are distinct clinical phenomena, they share some common features, such as some similar triggers and a predilection to occur at sites where mast cells reside, namely the skin and mucous membranes. In addition, recent observations indicate a high incidence of hypersensitivity reactions associated with MC degranulation in the HAE-C1-INH patient population. However, not all of these can be explained by IgE-dependent mechanisms. Mast cell-related G protein-coupled receptor-X2 (MRGPRX2), which has recently attracted scientific interest, may be involved in the activation of MCs through a different pathway. Therefore, we reviewed MRGPRX2 ligands that HAE-C1-INH patients may be exposed to in their daily lives and that may affect MCs degranulation. We also discussed the known inter- and intra-individual variability in the course of HAE-C1-INH in relation to factors responsible for possible variability in the strength of the response to MRGPRX2 receptor stimulation. The above issues raise several questions for future research. It is not known to what extent a prophylactic or therapeutic intervention targeting the pathways of one mechanism (mast cell degranulation) may affect the other (bradykinin production), or whether the number of mast cells at a specific body site and their reactivity to triggers such as pressure, allergens or MRGPRX2 agonists may influence the occurrence of HAE-C1-INH attacks at that site.


Subject(s)
Bradykinin , Cell Degranulation , Mast Cells , Receptors, G-Protein-Coupled , Receptors, Neuropeptide , Humans , Mast Cells/immunology , Mast Cells/metabolism , Bradykinin/metabolism , Receptors, G-Protein-Coupled/metabolism , Receptors, Neuropeptide/metabolism , Animals , Angioedema/metabolism , Angioedema/immunology , Angioedema/etiology , Nerve Tissue Proteins/metabolism , Kallikrein-Kinin System/physiology
16.
Curr Opin Allergy Clin Immunol ; 24(4): 195-202, 2024 Aug 01.
Article in English | MEDLINE | ID: mdl-38814742

ABSTRACT

PURPOSE OF REVIEW: Patients with mast cell disorders frequently experience symptoms from excessive mediator release like histamine and tryptase, ranging from mild flushing to severe anaphylactic responses. Hypersensitivity reactions (HRs) to drugs are a major cause of anaphylaxis in these patients, who often worry about triggering mast cell degranulation when taking medications. The aim of this review is to explore the complex interactions between mast cell disorders and drug HRs, focusing on the clinical challenges of managing these conditions effectively to enhance understanding and guide safer clinical practices. RECENT FINDINGS: Among the drugs most commonly associated with hypersensitivity reactions in patients with mast cell disorders are non-steroidal anti-inflammatory drugs, antibiotics, and perioperative agents. Recent studies have highlighted the role of Mas-related G-protein coupled receptor member X2 (MRGPRX2) - a receptor involved in non-immunoglobulin E mediated mast cell degranulation - in exacerbating HRs. Investigations reveal varied drug tolerance among patients, underscoring the need for individual risk assessments. SUMMARY: Tailored diagnostic approaches are crucial for confirming drug allergies and assessing tolerance in patients with mastocytosis, preventing unnecessary medication avoidance and ensuring safety before acute situations arise.


Subject(s)
Drug Hypersensitivity , Mast Cells , Receptors, G-Protein-Coupled , Humans , Mast Cells/immunology , Drug Hypersensitivity/diagnosis , Drug Hypersensitivity/immunology , Drug Hypersensitivity/therapy , Receptors, G-Protein-Coupled/immunology , Receptors, G-Protein-Coupled/metabolism , Anaphylaxis/immunology , Anaphylaxis/diagnosis , Receptors, Neuropeptide/immunology , Receptors, Neuropeptide/metabolism , Cell Degranulation/immunology , Mastocytosis/immunology , Mastocytosis/diagnosis , Anti-Inflammatory Agents, Non-Steroidal/adverse effects , Animals , Anti-Bacterial Agents/adverse effects , Nerve Tissue Proteins
17.
Curr Opin Allergy Clin Immunol ; 24(4): 183-188, 2024 Aug 01.
Article in English | MEDLINE | ID: mdl-38743470

ABSTRACT

PURPOSE OF REVIEW: Perioperative anaphylaxis has historically been attributed to IgE/FcεRI-mediated reactions; there is now recognition of allergic and nonallergic triggers encompassing various reactions beyond IgE-mediated responses. This review aims to present recent advancements in knowledge regarding the mechanisms and pathophysiology of perioperative anaphylaxis. RECENT FINDINGS: Emerging evidence highlights the role of the mast-cell related G-coupled protein receptor X2 pathway in direct mast cell degranulation, shedding light on previously unknown mechanisms. This pathway, alongside traditional IgE/FcεRI-mediated reactions, contributes to the complex nature of anaphylactic reactions. Investigations into the microbiota-anaphylaxis connection are ongoing, with potential implications for future treatment strategies. While serum tryptase levels serve as mast cell activation indicators, identifying triggers remains challenging. A range of mediators have been associated with anaphylaxis, including vasoactive peptides, proteases, lipid molecules, cytokines, chemokines, interleukins, complement components, and coagulation factors. SUMMARY: Further understanding of clinical endotypes and the microenvironment where anaphylactic reactions unfold is essential for standardizing mediator testing and characterization in perioperative anaphylaxis. Ongoing research aims to elucidate the mechanisms, pathways, and mediators involved across multiple organ systems, including the cardiovascular, respiratory, and integumentary systems, which will be crucial for improving patient outcomes.


Subject(s)
Anaphylaxis , Mast Cells , Perioperative Period , Anaphylaxis/immunology , Anaphylaxis/diagnosis , Anaphylaxis/physiopathology , Anaphylaxis/etiology , Humans , Mast Cells/immunology , Animals , Immunoglobulin E/immunology , Cell Degranulation/immunology , Tryptases/blood , Tryptases/metabolism , Receptors, IgE/immunology , Microbiota/immunology , Nerve Tissue Proteins , Receptors, Neuropeptide , Receptors, G-Protein-Coupled
18.
Int Immunopharmacol ; 134: 112256, 2024 Jun 15.
Article in English | MEDLINE | ID: mdl-38744172

ABSTRACT

The incidence of allergic reactions has risen steadily in recent years, prompting growing interest in the identification of efficacious and safe natural compounds that can prevent or treat allergic diseases. Phellodendron amurense Rupr. has long been applied as a treatment for allergic diseases, whose primary component is phellodendrine. However, the efficacy of phellodendrine as a treatment for allergic diseases remains to be assessed. Mast cells are the primary effectors of allergic reactions, which are not only activated by IgE-dependent pathway, but also by IgE-independent pathways via human MRGPRX2, rat counterpart MRGPRB3. As such, this study explored the effect and mechanism of phellodendrine through this family receptors in treating allergic diseases in vitro and in vivo. These analyses revealed that phellodendrine administration was sufficient to protect against C48/80-induced foot swelling and Evans blue exudation in mice, and suppressed C48/80-induced RBL-2H3 rat basophilic leukemia cells degranulation, and ß-HEX, HIS, IL-4, and TNF-α release. Moreover, phellodendrine could reduce the mRNA expression of MRGPRB3 and responsiveness of MRGPRX2 by altering its structure. It was able to decrease Ca2+ levels, phosphorylation levels of CaMK, PLCß1, PKC, ERK, JNK, p38, and p65, and inhibit the degradation of IκB-α. These analyses indicate that berberine inhibits the activation of PLC and downregulates the release of Ca2+ in the endoplasmic reticulum by altering the conformation of MRGPRB3/MRGPRX2 protein, thereby inhibiting the activation of PKC and subsequently inhibiting downstream MAPK and NF-κB signaling, ultimately suppressing allergic reactions. There may thus be further value in studies focused on developing phellodendrine as a novel anti-allergic drug.


Subject(s)
Cell Degranulation , Hypersensitivity , Mast Cells , Receptors, G-Protein-Coupled , Animals , Rats , Mast Cells/drug effects , Mast Cells/immunology , Cell Degranulation/drug effects , Mice , Humans , Hypersensitivity/drug therapy , Hypersensitivity/immunology , Receptors, G-Protein-Coupled/metabolism , Anti-Allergic Agents/pharmacology , Anti-Allergic Agents/therapeutic use , Cytokines/metabolism , p-Methoxy-N-methylphenethylamine , Male , Phellodendron/chemistry , Cell Line, Tumor , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/genetics , Mice, Inbred BALB C , NF-kappa B/metabolism , Signal Transduction/drug effects , Receptors, Neuropeptide
19.
Int Immunopharmacol ; 133: 112113, 2024 May 30.
Article in English | MEDLINE | ID: mdl-38657498

ABSTRACT

BACKGROUND: Phytosphingosine and its derivative are known for their skin-protective properties. While mYG-II-6, a phytosphingosine derivative, has shown anti-inflammatory and antipsoriatic effects, its potential antipruritic qualities have yet to be explored. This study aimed to investigate mYG-II-6's antipruritic properties. METHODS: The calcium imaging technique was employed to investigate the activity of ion channels and receptors. Mast cell degranulation was confirmed through the ß-hexosaminidase assay. Additionally, in silico molecular docking and an in vivo mouse scratching behavior test were utilized. RESULTS: Using HEK293T cells transfected with H1R and TRPV1, we examined the impact of mYG-II-6 on histamine-induced intracellular calcium rise, a key signal in itch-mediating sensory neurons. Pretreatment with mYG-II-6 significantly reduced histamine-induced calcium levels and inhibited TRPV1 activity, suggesting its role in blocking the calcium influx channel. Additionally, mYG-II-6 suppressed histamine-induced calcium increase in primary cultures of mouse dorsal root ganglia, indicating its potential antipruritic effect mediated by histamine. Interestingly, mYG-II-6 exhibited inhibitory effects on human MRGPRX2, a G protein-coupled receptor involved in IgE-independent mast cell degranulation. However, it did not inhibit mouse MrgprB2, the ortholog of human MRGPRX2. Molecular docking analysis revealed that mYG-II-6 selectively interacts with the binding pocket of MRGPRX2. Importantly, mYG-II-6 suppressed histamine-induced scratching behaviors in mice. CONCLUSIONS: Our findings show that mYG-II-6 can alleviate histamine-induced itch sensation through dual mechanisms. This underscores its potential as a versatile treatment for various pruritic conditions.


Subject(s)
Cell Degranulation , Histamine , Mast Cells , Receptors, G-Protein-Coupled , TRPV Cation Channels , Animals , Humans , Male , Mice , Antipruritics/pharmacology , Antipruritics/therapeutic use , Calcium/metabolism , Cell Degranulation/drug effects , Ganglia, Spinal/drug effects , Ganglia, Spinal/metabolism , HEK293 Cells , Histamine/metabolism , Mast Cells/drug effects , Mast Cells/immunology , Mice, Inbred C57BL , Molecular Docking Simulation , Nerve Tissue Proteins/metabolism , Pruritus/drug therapy , Receptors, G-Protein-Coupled/metabolism , Receptors, Neuropeptide/metabolism , Sphingosine/analogs & derivatives , Sphingosine/pharmacology , Sphingosine/metabolism , TRPV Cation Channels/metabolism
20.
Exp Mol Med ; 56(5): 1164-1177, 2024 May.
Article in English | MEDLINE | ID: mdl-38689088

ABSTRACT

Recent evidence of gut microbiota dysbiosis in the context of psoriasis and the increased cooccurrence of inflammatory bowel disease and psoriasis suggest a close relationship between skin and gut immune responses. Using a mouse model of psoriasis induced by the Toll-like receptor (TLR) 7 ligand imiquimod, we found that psoriatic dermatitis was accompanied by inflammatory changes in the small intestine associated with eosinophil degranulation, which impaired intestinal barrier integrity. Inflammatory responses in the skin and small intestine were increased in mice prone to eosinophil degranulation. Caco-2 human intestinal epithelial cells were treated with media containing eosinophil granule proteins and exhibited signs of inflammation and damage. Imiquimod-induced skin and intestinal changes were attenuated in eosinophil-deficient mice, and this attenuation was counteracted by the transfer of eosinophils. Imiquimod levels and the distribution of eosinophils were positively correlated in the intestine. TLR7-deficient mice did not exhibit intestinal eosinophil degranulation but did exhibit attenuated inflammation in the skin and small intestine following imiquimod administration. These results suggest that TLR7-dependent bidirectional skin-to-gut communication occurs in psoriatic inflammation and that inflammatory changes in the intestine can accelerate psoriasis.


Subject(s)
Cell Degranulation , Disease Models, Animal , Eosinophils , Imiquimod , Intestine, Small , Psoriasis , Toll-Like Receptor 7 , Animals , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 7/genetics , Psoriasis/pathology , Psoriasis/metabolism , Mice , Eosinophils/metabolism , Eosinophils/immunology , Humans , Intestine, Small/pathology , Intestine, Small/metabolism , Skin/pathology , Skin/metabolism , Inflammation/pathology , Inflammation/metabolism , Mice, Knockout , Caco-2 Cells , Membrane Glycoproteins
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