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Searching for tryptase in the RBL-2H3 mast cell model: Preparation for comparative mast cell toxicology studies with zebrafish.

ABSTRACT: Mast cells comprise a physiologically and toxicologically important cell type that is ubiquitous among species and tissues. Mast cells undergo degranulation, in which characteristic intracellular granules fuse with the plasma membrane and release many bioactive substances, such as enzymes ?-hexosaminidase and tryptase. Activity of mast cells in the toxicology model organism, zebrafish, has been monitored via tryptase release and cleavage of substrate N-?-benzoyl-dl-Arg-p-nitroanilide (BAPNA). An extensively used in vitro mast cell model for studying toxicant mechanisms is the RBL-2H3 cell line. However, instead of tryptase, granule contents such as ?-hexosaminidase have usually been employed as RBL-2H3 degranulation markers. To align RBL-2H3 cell toxicological studies to in vivo mast cell studies using zebrafish, we aimed to develop an RBL-2H3 tryptase assay. Unexpectedly, we discovered that tryptase release from RBL-2H3 cells is not detectable, using BAPNA substrate, despite optimized assay that can detect as little as 1 ng tryptase. Additional studies performed with another substrate, tosyl-Gly-Pro-Lys-pNA, and with an enzyme-linked immunosorbent assay, revealed a lack of tryptase protein released from stimulated RBL-2H3 cells. Furthermore, none of the eight rat tryptase genes (Tpsb2, Tpsab1, Tpsg1, Prss34, Gzmk, Gzma, Prss29, Prss41) is expressed in RBL-2H3 cells, even though all are found in RBL-2H3 genomic DNA and even though ?-hexosaminidase mRNA is constitutively expressed. Therefore, mast cell researchers should utilize ?-hexosaminidase or another reliable marker for RBL-2H3 degranulation studies, not tryptase. Comparative toxicity testing in RBL-2H3 cells in vitro and in zebrafish mast cells in vivo will require use of a degranulation reporter different from tryptase.

SUBMITTER: Shim JK

PROVIDER: S-EPMC6822387 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Searching for tryptase in the RBL-2H3 mast cell model: Preparation for comparative mast cell toxicology studies with zebrafish.

Shim Juyoung K JK Kennedy Rachel H RH Weatherly Lisa M LM Abovian Andrew V AV Hashmi Hina N HN Rajaei Atefeh A Gosse Julie A JA

Journal of applied toxicology : JAT 20181030 3

Mast cells comprise a physiologically and toxicologically important cell type that is ubiquitous among species and tissues. Mast cells undergo degranulation, in which characteristic intracellular granules fuse with the plasma membrane and release many bioactive substances, such as enzymes β-hexosaminidase and tryptase. Activity of mast cells in the toxicology model organism, zebrafish, has been monitored via tryptase release and cleavage of substrate N-α-benzoyl-dl-Arg-p-nitroanilide (BAPNA). An ...[more]

PMID: 30374992

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Project description:Mast cells and basophils are central players in allergic reactions triggered by immunoglobulin E (IgE). They have intracellular granules containing allergic mediators (e.g., histamine, serotonin, inflammatory cytokines, proteases and β-hexosaminidase), and stimulation by IgE-allergen complex leads to the release of such allergic mediators from the granules, that is, degranulation. Mast cells are residents of mucosal surfaces, including those of nasal and oral cavities, and play an important role in the innate defense system. Members of the mitis group streptococci such as Streptococcus oralis, are primary colonizers of the human oral cavity. They produce hydrogen peroxide (H2O2) as a by-product of sugar metabolism. In this study, we investigated the effects of streptococcal infection on RBL-2H3 mast cell/basophil cell line. Infection by oral streptococci did not induce degranulation of the cells. Stimulation of the RBL-2H3 cells with anti-dinitrophenol (DNP) IgE and DNP-conjugated human serum albumin triggers degranulation with the release of β-hexosaminidase. We found that S. oralis and other mitis group streptococci inhibited the IgE-triggered degranulation of RBL-2H3 cells. Since mitis group streptococci produce H2O2, we examined the effect of S. oralis mutant strain deficient in producing H2O2, and found that they lost the ability to suppress the degranulation. Moreover, H2O2 alone inhibited the IgE-induced degranulation. Subsequent analysis suggested that the inhibition of degranulation was related to the cytotoxicity of streptococcal H2O2. Activated RBL-2H3 cells produce interleukin-4 (IL-4); however, IL-4 production was not induced by streptococcal H2O2. Furthermore, an in vivo study using the murine pollen-induced allergic rhinitis model suggested that the streptococcal H2O2 reduces nasal allergic reaction. These findings reveal that H2O2 produced by oral mitis group streptococci inhibits IgE-stimulated degranulation by inducing cell death. Consequently, streptococcal H2O2 can be considered to modulate the allergic reaction in mucosal surfaces.

Dataset Information

Searching for tryptase in the RBL-2H3 mast cell model: Preparation for comparative mast cell toxicology studies with zebrafish.

Publications

Searching for tryptase in the RBL-2H3 mast cell model: Preparation for comparative mast cell toxicology studies with zebrafish.

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