Zebrafish mast cells possess an FcɛRI-like receptor and participate in innate and adaptive immune responses

Abstract

We previously identified a zebrafish mast cell (MC) lineage and now aim to determine if these cells function analogously in innate and adaptive immunity like their mammalian counterparts. Intraperitoneal (IP) injection of compound 48/80 or live Aeromonas salmonicida resulted in significant MC degranulation evident histologically and by increased plasma tryptase compared with saline-injected controls (p = 0.0006, 0.005, respectively). Pre-treatment with ketotifen abrogated these responses (p = 0.0004, 0.005, respectively). Cross-reactivity was observed in zebrafish to anti-human high-affinity IgE receptor gamma (FcɛRIγ) and IgE heavy chain-directed antibodies. Whole mount in situ hybridization on 7-day embryos demonstrated co-localization of cpa5, a MC-specific marker, with myd88, a toll-like receptor adaptor, and zebrafish FcɛRI subunit homologs. Zebrafish injected IP with matched dinitrophenyl-sensitized mouse (anti-DNP) IgE and DNP-BSA or trinitrophenyl-sensitized mouse (anti-TNP) IgE and TNP-BSA demonstrated increased plasma tryptase compared with mismatched controls (p = 0.03, 0.010, respectively). These results confirm functional conservation and validate the zebrafish model as an in vivo screening tool for novel MC modulating agents.

Introduction

The zebrafish, a robust model for studying vertebrate hematopoiesis (Berman et al., 2005, Carradice and Lieschke, 2008), has recently been exploited as a versatile organism for better understanding the complexity of both innate and adaptive immune responses (Meeker and Trede, 2008, Traver et al., 2003, Trede et al., 2004). Putative orthologs of eight of ten human toll-like receptors (TLRs), key regulators of innate immune signaling, have been identified in the zebrafish, as have downstream adapter molecules, including Toll/interleukin-1 receptor (TIR)-domain containing adaptor inducing INF-β (TRIF) and myeloid differentiation primary response gene (88) (MyD88) (Jault et al., 2004, Meijer et al., 2004). Similarly, conserved elements required for adaptive immune responses have been described in zebrafish and other teleosts (Fujiki et al., 2000, Stafford et al., 2006, Yoder et al., 2001, Yoder et al., 2004), such as FcRγ and FcRγ-like receptors, found to be syntenic to the gamma subunit of the human high-affinity IgE receptor located at 1q23 (Yoder et al., 2007).

Mast cells (MCs) serve a critical role as sentinels of the immune system, initiating many of the events involved in immune responsiveness, in addition to functioning as the key effector cell in allergic reactions. Our recent discovery of a zebrafish MC equivalent possessing structural and functional similarities to its mammalian counterpart (Dobson et al., 2008) has expanded the repertoire of immune cells in this organism and provided the opportunity to further investigate the conservation of functional genomics as it applies to immunity in general, and MCs in particular.

In this study, we reconcile the conservation of immune cell receptor genes with MC responses in vivo. We demonstrate that zebrafish MC degranulation stimulated by compound 48/80 (c48/80) or Aeromonas salmonicida can be abrogated by the MC stabilizing agent, ketotifen. Myd88 is expressed in a proportion of these mature MCs, suggesting conservation of innate immune responses mediated through TLRs. Most significantly, we present structure–function evidence that zebrafish MCs possess an analogous high-affinity immunoglobulin E (IgE)-like receptor (FcɛRI), which following stimulation, results in reproducible classic passive systemic anaphylactic (PSA) responses.