Human Mast Cell Line HMC1 Expresses Functional Mas-Related G-Protein Coupled Receptor 2

The Mas-related G-protein-coupled receptor X2 (MRGPRX2) is prominently expressed by mast cells and induces degranulation upon binding by different ligands. Its activation has been linked to various mast cell-related diseases, such as chronic spontaneous urticaria, atopic dermatitis and asthma. Therefore, inhibition of MRGPRX2 activity represents a therapeutic target for these conditions. However, the exact pathophysiology of this receptor is still unknown. In vitro research with mast cells is often hampered by the technical limitations of available cell lines. The human mast cell types LAD2 and HuMC (human mast cells cultured from CD34+ progenitor cells) most closely resemble mature human mast cells, yet have a very slow growth rate. A fast proliferating alternative is the human mast cell line HMC1, but they are considered unsuitable for degranulation assays due to their immature phenotype. Moreover, the expression and functionality of MRGPRX2 on HMC1 is controversial. Here, we describe the MRGPRX2 expression and functionality in HMC1 cells, and compare these with LAD2 and HuMC. We also propose a model to render HMC1 suitable for degranulation assays by pre-incubating them with latrunculin-B (Lat-B). Expression of MRGPRX2 by HMC1 was proven by RQ-PCR and flowcytometry, although at lower levels compared with LAD2 and HuMC. Pre-incubation of HMC1 cells with Lat-B significantly increased the overall degranulation capacity, without significantly changing their MRGPRX2 expression, phenotype or morphology. The MRGPRX2 specific compound 48/80 (C48/80) effectively induced degranulation of HMC1 as measured by CD63 membrane expression and β-hexosaminidase release, albeit in lower levels than for LAD2 or HuMC. HMC1, LAD2 and HuMC each had different degranulation kinetics upon stimulation with C48/80. Incubation with the MRGPRX2 specific inhibitor QWF inhibited C48/80-induced degranulation, confirming the functionality of MRGPRX2 on HMC1. In conclusion, HMC1 cells have lower levels of MRGPRX2 expression than LAD2 or HuMC, but are attractive for in vitro research because of their high growth rate and stable phenotype. HMC1 can be used to study MRGPRX2-mediated degranulation after pre-incubation with Lat-B, which provides the opportunity to explore MPRGRX2 biology in mast cells in a feasible way.

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Disrupted Lipid Raft Shuttling of FcεRI by n-3 Polyunsaturated Fatty Acid Is Associated With Ligation of G Protein-Coupled Receptor 120 (GPR120) in Human Mast Cell Line LAD2

Frontiers in Nutrition ◽

10.3389/fnut.2020.597809 ◽

2020 ◽

Vol 7 ◽

Author(s):

Xiaofeng Wang ◽

Ramses Ilarraza ◽

Brian P. Tancowny ◽

Syed Benazir Alam ◽

Marianna Kulka

Keyword(s):

Mast Cell ◽

Mast Cells ◽

Cell Line ◽

G Protein ◽

Lipid Raft ◽

Human Mast Cell ◽

G Protein Coupled Receptor ◽

Cell Functions ◽

Mast Cell Line ◽

G Protein Coupled

n-3 polyunsaturated fatty acids (PUFA) influences a variety of disease conditions, such as hypertension, heart disease, diabetes, cancer and allergic diseases, by modulating membrane constitution, inhibiting production of proinflammatory eicosanoids and cytokines, and binding to cell surface and nuclear receptors. We have previously shown that n-3 PUFA inhibit mast cell functions by disrupting high affinity IgE receptor (FcεRI) lipid raft partitioning and subsequent suppression of FcεRI signaling in mouse bone marrow-derived mast cells. However, it is still largely unknown how n-3 PUFA modulate human mast cell function, which could be attributed to multiple mechanisms. Using a human mast cell line (LAD2), we have shown similar modulating effects of n-3 PUFA on FcεRI lipid raft shuttling, FcεRI signaling, and mediator release after cell activation through FcεRI. We have further shown that these effects are at least partially associated with ligation of G protein-coupled receptor 120 expressed on LAD2 cells. This observation has advanced our mechanistic knowledge of n-3 PUFA's effect on mast cells and demonstrated the interplay between n-3 PUFA, lipid rafts, FcεRI, and G protein-coupled receptor 120. Future research in this direction may present new targets for nutritional intervention and therapeutic agents.

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Chemokine Production by G Protein-Coupled Receptor Activation in a Human Mast Cell Line: Roles of Extracellular Signal-Regulated Kinase and NFAT

The Journal of Immunology ◽

10.4049/jimmunol.165.12.7215 ◽

2000 ◽

Vol 165 (12) ◽

pp. 7215-7223 ◽

Cited By ~ 54

Author(s):

Hydar Ali ◽

Jasimuddin Ahamed ◽

Cristina Hernandez-Munain ◽

Jonathan L. Baron ◽

Michael S. Krangel ◽

...

Keyword(s):

Mast Cell ◽

Cell Line ◽

G Protein ◽

Receptor Activation ◽

Human Mast Cell ◽

Chemokine Production ◽

Extracellular Signal Regulated Kinase ◽

Extracellular Signal ◽

Mast Cell Line ◽

G Protein Coupled

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In vitro inhibitory effect of rupatadine on histamine and TNF-α release from dispersed canine skin mast cells and the human mast cell line HMC-1

Inflammation Research ◽

10.1007/pl00000216 ◽

2000 ◽

Vol 49 (7) ◽

pp. 355-360 ◽

Cited By ~ 31

Author(s):

M. Queralt ◽

P. Brazís ◽

M. Merlos ◽

F. de Mora ◽

A. Puigdemont

Keyword(s):

Mast Cell ◽

Mast Cells ◽

Cell Line ◽

Inhibitory Effect ◽

Human Mast Cell ◽

Tnf Α ◽

Mast Cell Line

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Dexamethasone suppresses gene expression and production of IL-13 by human mast cell line and lung mast cells

Journal of Allergy and Clinical Immunology ◽

10.1016/s0091-6749(98)70064-8 ◽

1998 ◽

Vol 102 (1) ◽

pp. 134-142 ◽

Cited By ~ 23

Author(s):

Toshiaki Fushimi ◽

Hiroshi Okayama ◽

Sanae Shimura ◽

Hiroki Saitoh ◽

Kunio Shirato

Keyword(s):

Gene Expression ◽

Mast Cell ◽

Mast Cells ◽

Cell Line ◽

Human Mast Cell ◽

Mast Cell Line

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Comparative Cytokine Release from Human Monocytes, Monocyte-Derived Immature Mast Cells, and a Human Mast Cell Line (HMC-1)

Journal of Investigative Dermatology ◽

10.1111/1523-1747.ep12395649 ◽

1994 ◽

Vol 103 (4) ◽

pp. 504-508 ◽

Cited By ~ 48

Author(s):

Jürgen Grabbe ◽

P.i.a. Welker ◽

Annelie Möller ◽

Edgar Dippel ◽

Leonie K Ashman ◽

...

Keyword(s):

Mast Cell ◽

Mast Cells ◽

Cell Line ◽

Human Mast Cell ◽

Human Monocytes ◽

Cytokine Release ◽

Mast Cell Line

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Characterization of human mast cells in long-term culture

Blood ◽

10.1182/blood.v62.6.1251.1251 ◽

1983 ◽

Vol 62 (6) ◽

pp. 1251-1260 ◽

Cited By ~ 31

Author(s):

MA Horton ◽

HA O'Brien

Keyword(s):

Bone Marrow ◽

Mast Cell ◽

Mast Cells ◽

Systemic Mastocytosis ◽

Human Mast Cell ◽

Lymphoid Tissues ◽

Stromal Fibroblasts ◽

Human Mast Cells ◽

Hemopoietic Tissue

Abstract Recent studies in rodents have demonstrated that mast cells derived from lymphoid tissues can be grown in longterm culture, provided that supportive growth factors or stromal fibroblasts are added; such findings have not been reported in man. Furthermore, although a hemopoietic origin for mast cells is supported by transplantation studies in mice, the exact origin of the human mast cell or its relationship to the circulating basophil and other hemopoietic cell lineages is unknown. We have investigated the requirements for in vitro growth of human mast cells derived from the infiltrated bone marrow of a patient with systemic mastocytosis, and have characterized both the mast cells proliferating in these cultures and those obtained from splenic infiltrates. Our data approached two questions: (1) Is there any evidence for the origin of mast cells from a bone-marrow-derived stem cell, and, if so, (2) what lineage relationship is there between mast cells and granulopoietic cells, including basophils? First, we have shown the expression of hemopoietic tissue-specific antigens by mast cells, strongly supporting a bone marrow origin for the mast cell in man (at least for those mast cells analyzed here). Second, the complete lack of granulocyte-monocyte markers contrasts with the phenotype of the basophil and suggests that mast cells diverge considerably from other granulopoietic cells during the acquisition of their differentiated specialized functions.

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CAFs and TGF-β Signaling Activation by Mast Cells Contribute to Resistance to Gemcitabine/Nabpaclitaxel in Pancreatic Cancer

Cancers ◽

10.3390/cancers11030330 ◽

2019 ◽

Vol 11 (3) ◽

pp. 330 ◽

Cited By ~ 34

Author(s):

Letizia Porcelli ◽

Rosa Iacobazzi ◽

Roberta Di Fonte ◽

Simona Serratì ◽

Angelica Intini ◽

...

Keyword(s):

Mast Cell ◽

Mast Cells ◽

Cell Line ◽

Tumor Invasion ◽

Tumor Stroma ◽

Ductal Adenocarcinoma ◽

Human Mast Cell ◽

Mast Cell Line ◽

Tgf Β1 ◽

Immunosuppressive Cytokines

Tumor–stroma interactions are of key importance for pancreatic ductal adenocarcinoma (PDAC) progression. Our aim was to investigate whether cancer associated fibroblasts (CAFs) and mast cells (MC) affected the sensitivity of PDAC cells to gemcitabine/nabpaclitaxel (GEM/NAB). For this purpose, the combination cytotoxicity and the effect on tumor invasion and angiogenesis were evaluated with or without a conditioned medium from the mast cell line HMC-1 (human mast cell line-1 cells) and CAFs. Beside the clinical outcome of a homogenous population of PDAC patients, receiving GEM/NAB, was correlated to the circulating levels of mast cell tryptase and to a panel of inflammatory and immunosuppressive cytokines. CAFs neither affected drugs’ cytotoxicity nor the inhibition of angiogenesis, but promoted tumor cell invasion. The MC instead, caused resistance to drugs by reducing apoptosis, by activating the TGF-β signalling and by promoting tumor invasion. Indeed, the inhibition of TβRI serine/threonine kinase activity by galunisertib restored drugs cytotoxicity. Moreover, MC induced the release of TGF-β1, and increased expression of PAR-2, ERK1/2 and Akt activation. Accordingly, TGF-β1, tryptase and other pro-inflammatory and immunosuppressive cytokines increased in the unresponsive patients. In conclusion, MC play a pivotal role in the resistance to GEM/NAB. A correlation between high level of circulating pro-inflammatory/ immunosuppressive cytokines and unresponsiveness was found in PDAC patients.

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Characterization of human mast cells in long-term culture

Blood ◽

10.1182/blood.v62.6.1251.bloodjournal6261251 ◽

1983 ◽

Vol 62 (6) ◽

pp. 1251-1260

Author(s):

MA Horton ◽

HA O'Brien

Keyword(s):

Bone Marrow ◽

Mast Cell ◽

Mast Cells ◽

Systemic Mastocytosis ◽

Human Mast Cell ◽

Lymphoid Tissues ◽

Stromal Fibroblasts ◽

Human Mast Cells ◽

Hemopoietic Tissue

Recent studies in rodents have demonstrated that mast cells derived from lymphoid tissues can be grown in longterm culture, provided that supportive growth factors or stromal fibroblasts are added; such findings have not been reported in man. Furthermore, although a hemopoietic origin for mast cells is supported by transplantation studies in mice, the exact origin of the human mast cell or its relationship to the circulating basophil and other hemopoietic cell lineages is unknown. We have investigated the requirements for in vitro growth of human mast cells derived from the infiltrated bone marrow of a patient with systemic mastocytosis, and have characterized both the mast cells proliferating in these cultures and those obtained from splenic infiltrates. Our data approached two questions: (1) Is there any evidence for the origin of mast cells from a bone-marrow-derived stem cell, and, if so, (2) what lineage relationship is there between mast cells and granulopoietic cells, including basophils? First, we have shown the expression of hemopoietic tissue-specific antigens by mast cells, strongly supporting a bone marrow origin for the mast cell in man (at least for those mast cells analyzed here). Second, the complete lack of granulocyte-monocyte markers contrasts with the phenotype of the basophil and suggests that mast cells diverge considerably from other granulopoietic cells during the acquisition of their differentiated specialized functions.

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