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 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 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 β‐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 β‐hexosaminidase mRNA is constitutively expressed. Therefore, mast cell researchers should utilize β‐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.

Mast cells promote viral entry of SARS-CoV-2 via formation of chymase/spike protein complex

“…A hypothesis of a potential relationship between mast cell-derived serine proteases and enhanced infectivity of SARS-CoV-2 has emerged. RBL-2H3 cells, the mast cell line used in the present in vitro study, are rat mucosal type-like mast cells that have extremely low expression of ectoenzyme ACE 2 and none of the eight rat tryptase genes are expressed in these cells ( Shim et al, 2019 ). MCP2, a rodent specific β-chymase initially expressed in RBL-2H3 cells, acts as a dominant AT I-hydrolyzing enzyme and generates AT II in these cells ( Caughey et al, 2000 ).…”

IRF3 Activation in Mast Cells Promotes FcεRI-Mediated Allergic Inflammation

“…Connective tissue-type mast cells sensitized with IgE are activated and degranulated by antigen. Tryptase, a major component of granules, is an indicator molecule for detecting mast cell activity As RBL-2H3 cells show low or undetectable tryptase expression [20], only mBMMCs were used for the analysis. Based on the phosphorylation timing of IRF3, we performed IF staining in mBMMCs stimulated for 2 min and 5 min (Figure 1E).…”

Mast cells in goldfish (Carassius auratus) gut: Immunohistochemical characterization

“…In addition, MCs participate also in neurological processes and diseases such as autism (Theoharides et al, 2012) and anxiety disorders (Nautiyal et al, 2008). Morphologically, MCs have characteristic cytoplasmatic granules which are secreted during degranulation, the release process of granular bioactive mediators such as histamine and serotonin (Shim et al, 2019). MCs been characterized in all classes of vertebrates.…”