Zebrafish Mast Cells Demonstrate Conserved Innate and Adaptive Immune Responses

Mast cells (MCs) are multifunctional immune cells derived from hematopoietic stem cells that uniquely complete maturation where they take up residence, namely in tissues exposed to the external environment. These anatomic locations position them to play a critical primary regulatory role in eliciting both innate and adaptive immune responses. The zebrafish has emerged as a powerful new model system for studying infection and immunity owing to conserved cell biology, and ease of manipulation and phenotypic analysis due to ex-utero embryonic development. We were the first (Dobson et al., Blood 2008) to identify MCs in zebrafish gills and intestine and carboxypeptidase A5 (cpa5) as a developmental marker of both embryonic progenitors and mature MCs. Intraperitoneal injection of compound 48/80, a MC activator, results in MC degranulation and elevated plasma tryptase levels as measured by chromogenic assay. Interestingly, we found that imatinib mesylate (Gleevec), an inhibitor of the C-KIT receptor, appears to block this MC activation. Pathogenic activation of MCs can occur through various well-conserved Toll-like receptors. We have demonstrated evidence of these innate immune pathways in zebrafish by infection with heat-inactivated A. salmonicida and the fungal wall constituent, zymosan. Each of these infectious stimuli results in zebrafish MC degranulation observed by light microscopy and by increased plasma tryptase levels. Mammalian MCs are better known for adaptive immune responses mediated through IgE/FcεRI signaling. We are characterizing an analogous pathway in the zebrafish and have shown that zebrafish MCs sensitized with mouse anti-DNP IgE followed by injection of DNP-BSA respond by degranulation, seen both by electron microscopy and tryptase assay. Equally interesting is the recruitment of eosinophils observed following MC stimulation by mouse anti-DNP/DNP-BSA. We are currently evaluating whether ketotifen, a MC stabilizer can attenuate this response as seen in mammalian systems. Moreover, we are interested to see whether imatinib mesylate or other tyrosine kinase inhibitors may play a role in abrogating this response, on account of cross-talk between C-KIT and IgE signaling cascades in mammals. The importance of proper MC function has been demonstrated in humans as well as various animal models where dysregulation results in disorders such as allergy, autoimmunity and mastocytosis. Our studies effectively establish the zebrafish as a novel model for evaluating vertebrate MC responses, which will be further enhanced through the fluorescent labeling of zebrafish MCs. These transgenic lines expressing green fluorescent protein (GFP) under the zebrafish cpa5 or c-kit promoters are being generated and germline screening is currently underway. Ultimately, we will be able to exploit the zebrafish system as an in vivo platform for high-throughput screening of potential MC stabilizing/inhibiting agents, with a goal of identifying new effective therapeutic strategies for use in allergic, inflammatory, and malignant diseases.