Zebrafish mast cells possess an FcɛRI-like receptor and participate in innate and adaptive immune responses
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.
Section snippets
Zebrafish maintenance
Zebrafish husbandry was carried out according to Westerfield (Westerfield, 1995). The use of zebrafish in these studies has been approved by the Dalhousie University Animal Care Committee.
Histochemical stains
Standard protocols were used for staining 5 μm sections of zebrafish intestine for Periodic Acid Schiff (PAS) and toluidine blue (TB). Slides were visualized using Zeiss Z1 microscope and Axiocam Rev 3.0 camera, Wetzlar, Germany.
Electron microscopy (EM)
Zebrafish intestine was collected immediately following cardiac puncture and fixed
Staining properties of zebrafish intestinal immune cells
MCs are a component of a repertoire of immune cells in the zebrafish intestine. We have previously shown that MC granules are eosinophilic following Periodic Acid Schiff (PAS) staining (Dobson et al., 2008). However, an additional population of cells that we have labeled eosinophils, stain similarly (albeit with more compact and more intensely eosinophilic granules), making the distinction between these two populations extremely challenging under PAS staining (Fig. 1Ai). Conversely, following
Discussion
We previously identified the gastrointestinal submucosa and gills as prominent anatomic sites of zebrafish MC activity (Dobson et al., 2008). These locations correspond to the respective mammalian tissues, where MCs predominate in order to easily function as early effector cells in response to noxious or infectious environmental stimuli. Similarly, mast cell/eosinophilic granule cells (MC/EGCs) were previously described in gastrointestinal tissue sections of a number of other teleost species (
Authorship
S.D. performed research, analyzed data and helped write the paper. E.M.T. performed research and analyzed data. J.T.D. and G.K.N. performed research. E.R.M. analyzed data and provided pathology expertise. H.W. and D.S.N. analyzed data and provided statistical expertise. J.S.M. and T.-J.L. designed research and analyzed data. J.N.B. designed research, analyzed data, and wrote the paper.
Conflict of interest statement
The authors have no conflicts of interest to disclose.
Acknowledgements
We would like to thank Chris Hall and Phil Crosier for providing myd88::GFP embryos, Jeffrey Yoder for providing fcer1g and fcer1gl cDNA clones and critical review of the manuscript. We would like to thank David Traver for critical review of the manuscript. We would like to thank Graham Dellaire for assistance with reviewing gene conservation including assisting with generating phylogenetic trees. Western blots were conducted in the laboratory of Rafael Garduno. We would like to thank Jocelyn
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