Teleost Basophils Have IgM-Dependent and Dual Ig-Independent Degranulation Systems Tomoyuki Odaka, Hiroaki Suetake, Tomoki Maeda and Toshiaki Miyadai This information is current as of September 28, 2021. J Immunol published online 7 March 2018 http://www.jimmunol.org/content/early/2018/03/07/jimmun ol.1701051 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2018/03/07/jimmunol.170105 Material 1.DCSupplemental Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published March 7, 2018, doi:10.4049/jimmunol.1701051 The Journal of Immunology Teleost Basophils Have IgM-Dependent and Dual Ig-Independent Degranulation Systems Tomoyuki Odaka, Hiroaki Suetake, Tomoki Maeda, and Toshiaki Miyadai Recently, mammalian basophils have been highlighted as having roles in allergy and antiparasitic immunity; however, there is little information about the functions and evolutionary origin of basophils, because they are the least abundant leukocyte in most vertebrates. In this study, we characterized the teleost basophils that are abundant in the peripheral blood of fugu (Takifugu rubripes). Fugu basophils have two distinct granules: reddish-purple and dark violet ones. Teleost fish do not have IgG and IgE, but we found that fugu IgM bound on the surface of the basophils, and the cross-linked IgM induced degranulation of both types of granules. This indicates that teleost basophils can be activated in an Ab-dependent manner. Furthermore, papain induced the degranulation of the reddish-purple granules, which contain histamine, and the released granules stimulated the migration of + various leukocytes. In contrast, chitin elicited the degranulation of the dark violet granules, which resulted in CD4 T cell–specific Downloaded from migration. Thus, fugu basophils control immune responses via two distinct Ab-independent mechanisms. In addition, fugu basophils endocytosed soluble Ag and expressed MHC class II and B7-H1/DC. These findings suggested that fugu basophils can interact with T cells as APCs. Thus, the Ab-dependent basophil activation predates the emergence of IgG and IgE, and fish basophils exhibit different dynamics and features of degranulation to distinct stimuli compared with mammalian basophils. Some features of teleost basophils are more similar to those of mammalian mast cells than to those of mammalian basophils. The Journal of Immunology, 2018, 200: 000–000. http://www.jimmunol.org/ asophils and mast cells are closely related granulocytes that is absent in teleost fish (6). Therefore, we wonder whether teleost contribute to allergy and antiparasitic immunity in mammals fish have an Ig-dependent basophil-degranulation mechanism. B (1, 2). However, in contrast to well-characterized mast cells, Basophils also have an Ig-independent degranulation system the functions of basophils in the immune system have not been fully (8–10). Some allergens, such as chitin, are structural components understood, because basophils are the least abundant leukocyte in of parasites and fungi, and others are proteases that are secreted by most vertebrate species. To understand the functions of basophils, parasites. However, little is known about the differences among some turtles and teleosts have been proposed to represent unique the cellular responses activated by these different types of aller- animal models that have a natural abundance of basophils in their gens in basophils. by guest on September 28, 2021 circulating blood (3, 4). In this study, we focused on teleost basophils, To characterize the features of basophils in lower vertebrates, we which are more evolutionarily primitive than turtle basophils. This selected fugu (Takifugu rubripes), which is a teleost fish species study will yield important insights into the functions of basophils and whose genome has been sequenced (11). Fugu has a natural the difference between mast cells and basophils from an evolutionary abundance of basophils in peripheral blood (4), and it is easy to perspective. isolate the basophils because of their high density. In this study, Basophils release inflammatory mediators, such as histamine, by we characterized fish basophils and provide insights into the degranulation and induce inflammation (5). Mammalian basophils evolutionary history of vertebrate basophils. Teleost fish basophils have activation mechanisms mediated by IgE and IgG, which are have an Ig-dependent degranulation system and have two distinct exclusively present in mammalian species (6). The degranulation types of granules with distinct roles whose release is activated by of basophils or mast cells in nonmammalian species, such as different stimuli. reptiles and avians, has also been observed and is likely to be mediated by IgY, which is analogous to mammalian IgG and IgE Materials and Methods (3, 7). IgY emerged from amphibians, and an IgY/IgG/IgE analog Animals Fugu weighing between 300 and 800 g were bred at the Research Center for Marine Bioresources of Fukui Prefectural University. The fish were reared Faculty of Marine Science and Technology, Fukui Prefectural University, Obama, in a tank with seawater kept at 20˚C and were fed dry food pellets once a Fukui 917-0003, Japan day (Nippai). All experiments were done in accordance with the approved Received for publication July 21, 2017. Accepted for publication February 10, 2018. guidelines and regulations of Fukui Prefectural University. This work was supported in part by Yakult Grants for Research on Lactic Acid Bacteria (to T. Miyadai) and the Fukui Prefectural Government’s Grant-in-Aid for Cell preparation Collaboration Research Projects (to H.S.). Fish were anesthetized with 2-phenoxyethanol (Wako), and peripheral Address correspondence and reprint requests to Dr. Hiroaki Suetake, Fukui Prefec- blood was collected immediately using heparinized syringes and needles tural University, 1-1 Gakuen-cho, Obama, Fukui 917-0003, Japan. E-mail address: and centrifuged at 500 3 g for 20 min. After centrifugation, the peripheral [email protected] blood was separated into four layers: plasma, peripheral blood leukocytes The online version of this article contains supplemental material. (PBLks; including lymphocytes, monocytes, and nonhigh-density gran- Abbreviations used in this article: EIA, enzyme immunoassay; HDG, high-density ulocytes), erythrocytes, and high-density granulocytes (HDGs). HDGs pre- granulocyte; KLH, keyhole limpet hemocyanin; KLH-FITC, KLH coupled to FITC; cipitating at the tube bottom were collected and washed with PBS following MGG, May–Gru¨nwald–Giemsa; PBLk, peripheral blood leukocyte. the removal of plasma, PBLks, and erythrocytes. The PBLk layer excluding HDGs (described above) was further sepa- Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 rated from erythrocytes using Percoll (GE Healthcare). In brief, PBLks were www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701051 2 TELEOST BASOPHILS HAVE A NOVEL DEGRANULATION SYSTEM layered on 53% Percoll diluted with PBS and centrifuged at 2465 3 g for was detected (final concentration 0.016 EU/ml). It has long been estab- 35 min at 4˚C. After centrifugation, PBLks in the intermediate layer of lished that fish are resistant to the toxic effect of LPS (14), and there is no the Percoll gradient were collected and washed with PBS. tlr4 ortholog in the fugu genome (15). Thus, it is reasonable that the effect For the endocytosis and phagocytosis assays, monocytes and erythro- of LPS in the chitin was excluded in our experiments. cytes were collected, respectively. Monocytes were collected as follows. The isolated PBLks were suspended in RPMI 1640 medium (Nissui Chemotaxis assay Pharmaceutical) and cultured at 22˚C for 1 h in tissue culture flasks Fugu HDGs were cultured for 4 h with chitin, papain, or heat-inactivated (Sumitomo Bakelite) coated with fugu plasma to enhance monocyte ad- papain (100 mg/ml). The culture medium was collected by centrifugation hesion to the flasks. Adherent monocytes were washed thoroughly several at 500 3 g for 10 min and was filtered using a 0.2-mm syringe filter times with PBS to remove nonadherent cells. Following the wash steps, the (Sartorius). This medium was used in the chemotaxis assay as the che- monocytes were stimulated with 50 mg/ml LPS from Escherichia coli O26 motactic solution. Chemotaxis was evaluated using a microchamber (Wako) at 22˚C for 24 h. The peripheral monocytes were collected for technique. Briefly, the chemotactic solution (600 ml per well) was placed further use. To isolate erythrocytes, after centrifugation of the peripheral in a 24-well plate (Sumitomo Bakelite), and Chemotaxicell chambers with blood, as described above, the erythrocyte layer was collected