Essential Role of IL-4 and IL-4Rα Interaction in Adaptive Immunity of Zebrafish: Insight into the Origin of Th2-like Regulatory Mechanism in Ancient This information is current as Vertebrates of October 1, 2021. Lv-yun Zhu, Ping-ping Pan, Wei Fang, Jian-zhong Shao and Li-xin Xiang J Immunol 2012; 188:5571-5584; Prepublished online 30

April 2012; Downloaded from doi: 10.4049/jimmunol.1102259 http://www.jimmunol.org/content/188/11/5571

Supplementary http://www.jimmunol.org/content/suppl/2012/04/30/jimmunol.110225 http://www.jimmunol.org/ Material 9.DC1 References This article cites 69 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/188/11/5571.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Essential Role of IL-4 and IL-4Ra Interaction in Adaptive Immunity of Zebrafish: Insight into the Origin of Th2-like Regulatory Mechanism in Ancient Vertebrates

Lv-yun Zhu, Ping-ping Pan, Wei Fang, Jian-zhong Shao, and Li-xin Xiang

The roles of IL-4 and IL-4Ra in Th2-mediated immunity have been well characterized in humans and other mammals. In contrast, few reports have been documented in ancient vertebrates. Several putative IL-4– and IL-4Ra–like molecules were identified recently from a few fish species, providing preliminary insight into the occurrence of Th2-type immunity in teleosts. However, functional determination still is required to address this hypothesis. To this end, these two molecules were characterized functionally in zebrafish (Danio rerio). Besides the identification of a full-length IL-4Ra molecule and an isoform lacking most of the cytoplasmic region as predicted previously, two novel alternatively spliced soluble variants with the extracellular domain only also were identified. Zebrafish IL-4Ra (DrIL-4Ra) shared overall conserved structural features of the IL-4Ra family. Immuno- Downloaded from fluorescence staining showed that DrIL-4Ra distributed on B cells. In vitro binding assays demonstrated that zebrafish IL-4 (DrIL-4) can bind specifically to DrIL-4Ra. In vivo administration of DrIL-4 significantly upregulated B cell proliferation and Ab production. These DrIL-4–elicited immune responses were downregulated by the administration of zebrafish soluble IL-4Ra or by DrIL-4Ra blockade using anti–DrIL-4Ra Abs. In addition, Th2-related cytokines or transcription factors were upregulated by DrIL-4. The DrIL-4–DrIL-4Ra interaction promoted CD40 expression on B cells and enhanced the CD154–CD40 costimulatory response, both of which are crucial for the initiation of Th2-type immunity. To our knowledge, this is the first report showing that http://www.jimmunol.org/ a possible Th2-mediated regulatory mechanism may have appeared before the divergence of teleosts and mammals. These results add greater insight into the evolutionary history of adaptive immunity. The Journal of Immunology, 2012, 188: 5571–5584.

n mammals, naive CD4+ T lymphocytes can differentiate into the two Th subsets in humans is associated with the development various Th subsets, such as Th1, Th2, Th3, Th17, and Th25, of autoimmune and allergic diseases (2, 10, 13, 17–19). I with distinct cytokine profiles and functions, in which Th1 IL-4 is an important member of the class I cytokine family with and Th2 are the two well-characterized subsets (1–6). The Th1 four a helix structures that is secreted predominantly by activated subset secretes IFN-g, TNF-a, and IL-2, which are important for Th2 cells (16, 20). It induces Th2-mediated immune responses by by guest on October 1, 2021 Th1-mediated cellular immunity (7, 8). The Th2 subset produces upregulating the expression of MHC class II, CD23, and IL-4R on IL-4, IL-5, and IL-13, which promote further Th2 development of B cells (9, 14, 20, 21). It has an anti-inflammatory effect owing to its stimulated CD4+ Th cells and a variety of Th2-mediated humoral efficient inhibition of the production of proinflammatory cytokines, immune responses, including B cell proliferation and activation, such as TNF-a,IL-1a,IL-1b, IL-6, and IL-8. It also plays a crucial Ig production, and Ig class switching (1, 2, 8–16). Imbalance of role in mediating allergic responses. In this process, IL-4 stimulates the proliferation of mast cells with IL-13 and promotes Ig class switching to IgE that binds to high-affinity Fcε receptors on the College of Life Sciences, Zhejiang University, Hangzhou 310058, People’s Republic of China; Key Laboratory for Cell and Engineering of Zhejiang Province, surfaces of mast cells, , and eosinophils, leading to the de- Hangzhou 310058, People’s Republic of China; and Key Laboratory of Animal granulation and production of inflammatory mediators when it is Virology of Ministry of Agriculture, Hangzhou 310058, People’s Republic of China cross-linked by an Ag (10, 11, 14, 15, 18, 19). IL-4 exerts its bio- Received for publication August 4, 2011. Accepted for publication March 26, 2012. logical functions by interacting with IL-4R (18, 22). The latter is a This work was supported by grants from the Hi-Tech Research and Development heterodimer composed of a specific a-chain (IL-4Ra or IL-4BP) and Program of China (863) (2012AA092202), the National Basic Research Program of acommong-chain (g ). The g also is coutilized by IL-2, IL-7, IL-9, China (973) (2012CB114404, 2012CB114402), the National Natural Science Foun- c c dation of China (30871936, 31072234, 31172436) and the Program for Key Innova- IL-13, IL-15, and IL-21 for the activation of signaling pathways (23, tive Research Team of Zhejiang Province (2010R50026). 24). As a class I , IL-4Ra has specific and high The sequences presented in this article have been submitted to GenBank (http://www. affinities (Kd = 20–300 pM) in the recognition and binding to IL-4 ncbi.nlm.nih.gov/genbank/) under accession numbers EF523375.1, EF523376.1, (14, 20, 24–26). It could be detected on a number of hematopoietic EF523377.1, and EF523378.1. or nonhematopoietic cell surfaces, including endothelial, epithelial, Address correspondence and reprint requests to Prof. Jian-zhong Shao and Assoc. Prof. Li-xin Xiang, Zhejiang University, 866 Yu Hang Tang Road, Hangzhou 310058, muscle, fibroblast, hepatocyte, and brain cells (14, 27). Zhejiang, People’s Republic of China. E-mail addresses: [email protected] (J.-z.S.) The molecular and functional characterization of IL-4, IL-4Rs, and [email protected] (L.-x.X.) and downstream signaling pathways in Th2-mediated immunity The online version of this article contains supplemental material. has been well documented in humans and other mammals (28–32). Abbreviations used in this article: BS3, bis(sulfosuccinimidyl) suberate; gc, common In contrast, there are few reports regarding their occurrence and g-chain; DC, dendritic cell; DrIL-4, zebrafish IL-4; DrIL-4Ra, zebrafish IL-4Ra; DrIL-4Ra-iso, zebrafish IL-4Ra isoform; EST, expressed sequence tag; FN III, fi- functions in adaptive immunity in ancient vertebrates (33–37). In bronectin type III; KLH, keyhole limpet hemocyanin; ORF, open reading frame; a previous study, we identified an IL-4–like gene from pufferfish sCD154, soluble CD154; sDrIL-4Ra, zebrafish soluble IL-4Ra;sDrIL-4Ra-iso, (Tetraodon nigroviridis) (11), and two similar IL-4–like , IL- zebrafish soluble IL-4Ra isoform; UTR, untranslated region. 4/13A and IL-4/13B, were cloned subsequently from zebrafish Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 (Danio rerio) (12). It shows that zebrafish IL-4/13A has a perfect www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102259 5572 ROLE OF IL-4–IL-4Ra INTERACTION IN Th2-LIKE IMMUNITY IN FISH

TATA box and GATA3 binding motif in the proximal promoter free DNase I (Qiagen). Zebrafish IL-4Ra (DrIL-4Ra) and soluble IL-4Ra regions of the gene. However, IL-4/13B lacks such a box and motif (sDrIL-4Ra) cDNAs were cloned using the primers shown in Table I. The in its proximal promoter regions. This result suggests that IL-4/13B 39 and 59 full RACE core sets were used following the manufacturer’s instructions (Takara). Seminested or nested PCR was adopted in RACE. lacks the complete transcription elements. In addition, a search of PCR products were purified using a gel extraction (Qiagen), ligated into the expressed sequence tags (ESTs) in the National Center for a pUCm-T vector (Takara), and transformed into Escherichia coli Top10 Biotechnology Information database revealed several ESTs for competent cells (Invitrogen). Plasmid DNA was extracted by the plasmid zebrafish IL-4/13A, whereas none was found for zebrafish IL-4/ Miniprep method and then sequenced on a MegaBACE 1000 sequencer (GE Healthcare) using a DYEnamic ET dye terminator cycle sequencing 13B. As a consequence, IL-4/13A might be the real zebrafish kit (Pharmacia). Full-length cDNAs were assembled by catabolite gene IL-4, but more functional evidence still is needed. Two putative activator (CAP 3.0) (45). IL-4Ras were identified recently from rainbow trout (Oncorhynchus mykiss) and predicted in zebrafish and Atlantic salmon (Salmo salar) Phylogenetic analysis (38, 39). These observations provide preliminary insights into Th2- Protein sequences of the IL-4Ra family members, including IL-4Ra, type immunity before the divergence of teleost fish and mammals IL2Rb, and IL-21R, from 14 representative vertebrates, including human ∼400 million years ago. However, functional determination still is (H. sapiens), chimpanzee (Pan troglodytes), rhesus monkey (Macaca mulatta), cattle (Bos taurus), horse (Equus caballus), mouse (M. musculus), required to address this hypothesis. rat (Rattus norvegicus), dog (Canis lupus), wild boar (Sus scrofa), chicken In the current study, both structural and functional features of (Gallus domesticus), bird (Taeniopygia guttata), rainbow trout (O. mykiss), IL-4Ra were characterized further in zebrafish. Besides the iden- Atlantic salmon (S. salar), and zebrafish (D. rerio), were identified. The tification of two membrane-bound IL-4Ra–like molecules as pre- neighbor-joining trees were constructed from pairwise Poisson correction dicted previously, two novel alternatively spliced soluble variants distances with 2000 bootstrap replications by the MEGA 3.0 software (46). Downloaded from with only the extracellular domain were identified initially in fish Tissue distribution analysis species. As expected, IL-4 and IL-4Ra are shown to be reciprocally To determine the distribution of DrIL-4Ra and DrIL-4, the liver, gut, regulated in zebrafish. The interaction of these two molecules is kidney, spleen, brain, muscle, skin, gill, ovary, and peripheral lymphocytes essential for B cell proliferation and Ab production, which are the were collected carefully and flash-frozen in liquid nitrogen. Total RNA was two hallmark events in Th2-type immune responses. However, the extracted as described above and reversed-transcribed using the RNA PCR zebrafish soluble IL-4Ra (sDrIL-4Ra) variant plays a negative kit (AMV), version 3.0 (Takara). The PCR program consisted of treatment regulatory role in this process. The reciprocal regulation of IL-4 and at 94˚C for 30 s, 55˚C for 30 s, and 72˚C for 30 s for 36 cycles to amplify http://www.jimmunol.org/ b-actin (as a standard), DrIL-4, and DrIL-4Ras using the corresponding IL-4Ra promotes CD40 expression on B cells and increases B cell pairs of primers (Table I) in parallel tubes, respectively. Then, 5 ml of PCR responses to stimulation by CD154 signals. To our knowledge, this products was loaded on a 1.5% (w/v) agarose gel and visualized by result was the first observation that the IL-4–IL-4Ra interaction staining the gel in 0.1 mg/ml ethidium bromide. To distinguish two variant promotes Th2-type immune responses by upregulating the CD154– forms of DrIL-4Ra, restriction enzymes EcoRV and EcoRI (Takara) were used for specifically cutting at one unique site of DrIL-4Ra to generate CD40-mediated costimulatory signaling pathway in B cells. Results another IL-4Ra–like isoform. PCR products were purified before digestion give functional insights into the existence of a conserved Th2-like using Microcon-PCR (Millipore). Finally, 5 ml of PCR products or enzyme regulatory mechanism in fish, enrich our knowledge of fish im- digestions was loaded on a 1.5% (w/v) agarose gel and visualized by gel munology, and contribute to a better cross-species understanding staining with 0.1 mg/ml ethidium bromide. by guest on October 1, 2021 of the evolutionary history of the IL-4 and IL-4R family, as well as Preparation of recombinant the Th subset and T regulatory mechanisms. Sequences encoding DrIL-4 and sDrIL-4Ra were amplified by RT-PCR with primers (Table I) containing a BamHI or EcoRI site added on the 59 Materials and Methods end and a XhoI site added on the 39 end. The resulting amplifications of Experimental fish DrIL-4 and sDrIL-4Ra were digested and ligated into pET41a and pET32b (Invitrogen Life Technologies), respectively. Plasmid DNAs were D. rerio ∼ One-year old zebrafish ( ) from both sexes, weighing 0.5–1.0 g, transformed into BL21 star cells (DE3; Invitrogen Life Technologies). A with a body length of 1–2 cm, were kept in a circulating water bath at 26˚C single colony of BL21 harboring the expression plasmids was inoculated and fed with commercial pellets at a daily ration of 0.7% of their body into 100 ml of Luria-Bertani medium containing chloramphenicol (100 weight. All of the fish were held in the laboratory for at least 2 wk before mg/l) with kanamycin (25 mg/l) or ampicillin (25 mg/l), and the culture the experiments for acclimatization and evaluation of overall fish health. was shaken and incubated at 37˚C until the OD value reached 0.6. Only healthy fish, as determined by general appearance and level of ac- 600 Isopropyl-b-D-thiogalactoside then was added to a final concentration of 1 tivity, were used for the studies. mM, and the culture was shaken continually at 250 rpm and incubated at Database and bioinformatics analysis 37˚C for 6 h. After ultrasonication, the soluble supernatant liquids were collected. Recombinant Trx-DrIL-4Ra and GST-DrIL-4 proteins in these Genome assemblies, including Homo sapiens, Mus musculus, Gallus gallus, supernatant liquids were purified by nickel-nitrilotriacetic acid agarose and D. rerio, were searched at the University of California at Santa Cruz affinity chromatography according to the QIAexpressionist manual (Qia- genome bioinformatics Web site (http://genome.ucsc.edu). Genome loca- gen) and assessed by 12% SDS-PAGE. tions were retrieved from the National Center for Biotechnology Infor- mation (http://www.ncbi.nlm.nih.gov/mapviewer) and University of Cali- Preparation of Ab fornia at Santa Cruz Web sites as well. Gene organizations were elucidated Six-week-old male New Zealand white rabbits and BALB/c mice were a by comparing IL-4R cDNA with genome sequences. Figures were drawn immunized eight times at biweekly intervals with 100 and 20 mg purified using GeneMaper 2.5. Homologous ESTs were searched by WU-BLAST at recombinant Trx-DrIL-4Ra protein in CFA, respectively. A week after the the Computational Biology and Functional Genomics Laboratory. Gene final immunization, the rabbits and mice were bled when Ab titers were predictions were carried out by the FGENESH program. Sequences were above 1:10,000, as determined by ELISA using recombinant protein compared with the database by the BLASTP algorithm. Multiple align- adsorbed to the solid phase. Anti–DrIL-4Ra Abs were affinity-isolated ments were generated by the Clustal X program (version 1.8) (40). Putative into the IgG isotype using a protein A-agarose column (Qiagen) and pu- signal peptides and transmembrane helices were predicted by the SigP rified further using an immunosorbent-based protocol, as described pre- program (version 3.0) and TMHMM 2.0 (41). Secondary structure was viously with slight modifications (47). In brief, recombinant DrIL-4Ra predicted using the PHD program (42). Structural features of the peptides was electrotransferred (350 mA for 1 h) from a 12% SDS-polyacrylamide were searched in PROSITE (43, 44). gel onto a nitrocellulose membrane. After being washed with PBS, small Sequence cloning sections of the membrane with the target protein were cut and blocked with 2% BSA for 30 min. The membrane was incubated with protein A affinity- Total RNA was isolated from spleen and kidney tissues collected from 20 isolated Ab (500- to 1000-fold dilution with 2% BSA) for 16 h at 4˚C and fish using a TRIzol reagent (Life Technologies BRL) treated with RNase- washed with 0.15 M NaCl and PBS, respectively. Afterward, elution buffer The Journal of Immunology 5573

[0.2 M glycine and 1 M EGTA (pH 2.8)] was added to dissociate the Abs tively. Flow cytometric analysis was performed according to the method from the membrane. The Ab solution was adjusted to pH 7.4, and the titers described above. For real-time PCR, total RNA was isolated from the were assessed by ELISA. Western blot analyses between various tissue/ lymphocytes of both experimental and control fish injected with GST- cell-extracted proteins and Ab, in which 15 mg of each tissue/cell extract DrIL-4 (0.1 mg per fish i.p.) and GST tag, respectively. B cell proliferation was used, were performed to characterize the specificity of the Ab (48). was evaluated by the expression of selected B cell markers, including mIgM, MHC class II, and CD80, using the primers shown in Table I. The Flow cytometric analysis for B cell proliferation total amount of mRNA was normalized to endogenous b-actin mRNA. The 2DDCT Flow cytometric analysis was used to determine the proliferation of mIgM+ related gene expression level was calculated using the 2 method. B cells. For this, whole-blood cell suspensions were collected with hepa- Expression of the control groups was set arbitrarily to 1.0. Each sample rinized capillary tubes. Lymphocytes were separated from the peripheral was from the whole peripheral blood of 10 fish and run in three parallel re- blood of each group by Ficoll-Hypaque (1.077 g/ml) centrifugation at 2500 actions using a Mastercycler ep realplex and detection software (Eppen- rpm for 25 min at room temperature and were washed with PBS. After being dorf). At least three independent experiments were performed for each fixed with 4% paraformaldehyde for 20 min at room temperature and panel. For Ab production analysis, fish were administered different doses blocked with 5% normal goat serum for 30 min at 37˚C, they were incubated (0.01, 0.1, and 1 mg per fish i.p.) of GST-DrIL-4 three times with a 12-h with primary Ab (rabbit anti-IgM, 1:2000) (43, 44) or rabbit IgG as a non- time interval. This was followed by immunization with keyhole limpet related primary Ab for 60 min at 37˚C. The samples then were incubated hemocyanin (KLH) together with CFA at a dose of 10 mg per fish. In with a secondary FITC-labeled anti-rabbit Ab (IgG, 1:100; Chemicon) for negative control groups, fish were administered KLH in combination with 45 min at 37˚C. After the samples were washed, cell fluorescence signals PBS or GST tag protein (1 mg per fish i.p.). After 28 d of immunization, the were determined immediately using a FACScan flow cytometer (BD Bio- serum samples were collected, and IgM in response to KLH in these sciences) equipped with an argon laser with emission at 488 nm. At least samples was measured by ELISA as described previously (43). In brief, 96- 10,000 events were acquired from the lymphocyte gate (49). The analysis well plates were coated with 5 mg/ml KLH overnight at 4˚C. The plates was performed using CellQuest software (BD Biosciences). were washed with PBS containing 0.05% Tween 20 and blocked with 2% BSA for 1 h at 37˚C. After the plates were washed, serially diluted serum Immunofluorescence staining samples and a background control serum sample with PBS administration Downloaded from were incubated for 1 h at 37˚C. After the plates were washed again, rabbit Lymphocytes were separated from the whole-blood cell suspensions as anti-IgM was added and incubated for 1 h at 37˚C. The plates were washed, described above and fixed with cold methanol. They then were incubated and peroxidase-conjugated streptavidin rabbit IgG was added. The color with primary Abs, including rabbit anti-DrIgM (produced in our labora- was developed by adding the tetramethyl benzidine substrate solution. The tory) and mouse anti–DrIL-4Ra, at 37˚C for 1 h. In parallel, the nonrelated plates were analyzed on an ELISA reader at 450 nm. Abs, rabbit IgG and mouse IgG, were used as negative controls. The secondary Abs, including tetramethylrhodamine isothiocyanate-conjugated

Functional evaluation for the interaction between IL-4 and http://www.jimmunol.org/ goat anti-rabbit IgG and FITC-conjugated goat anti-mouse IgG (Santa Dr Cruz Biotechnology), were used according to the manufacturer’s instruc- DrIL-4Ra tions. Samples were photomicrographed under a confocal laser-scanning In vivo blockade and administration of GST-DrIL-4Ra assays were per- microscope (CLSM 510; Carl Zeiss). formed for the functional evaluation of the interaction between GST- Binding assays between DrIL-4 and DrIL-4Ra DrIL-4 and Trx-DrIL-4Ra. For the administration assay, fish were injected with different combinations of recombinant Trx-sDrIL-4Ra,GST-DrIL-4, GST pull-down and direct binding ELISA assays were performed to and Ag (KLH), including preinjections with Trx-sDrIL-4Ra (0.1 mgperfish measure the interaction between DrIL-4 and DrIL-4Ra. For the pull-down i.p.), GST-DrIL-4 (0.1 mgperfishi.p.),orGST-DrIL-4 (0.1 mg per fish i.p.) assays, purified GST-DrIL-4a protein (or GST protein alone as the nega- with Trx-sDrIL-4Ra (0.1 mg per fish i.p.) three times with a 12-h time tive control) was mixed with glutathione beads (Qiagen) and incubated for interval, respectively. Meanwhile, preinjections with GST tag (1 mgperfish 2 h at 4˚C. The beads then were washed with GST binding buffer [200 mM i.p.), Trx tag (1 mg/fish i.p.), and PBS alone were performed as negative by guest on October 1, 2021 NaCl, 20 mM Tris-HCl, and 1 mM EDTA (pH 7.4)] five times and washed controls. The fish then were immunized with KLH (10 mg per fish i.p.). The again with PBS (pH 7.5) five times. Afterward, equivalent soluble Trx- Ab (IgM) production in response to KLH in combination with GST-DrIL-4, DrIL-4R was incubated with GST-DrIL-4– or GST-coupled glutathione Trx-sDrIL-4Ra, and controls was determined by ELISA at 28 d after im- beads for 1 h at 4˚C. The mixture then was cross-linked using bis(sulfo- munization, followed by the methods described above. B cell proliferation succinimidyl) suberate (BS3) at a final concentration of 1 mM for 30 min in response to the administration of GST-DrIL-4 and Trx-sDrIL-4Ra also at room temperature. The cross-linking reaction was quenched by using was examined by flow cytometric analysis at 72 h poststimulation with PBS, 1 M Tris buffer (pH 7.5), and then the mixture washed with PBS (pH 7.5) Trx (1 mg per fish i.p.), Trx-sDrIL-4Ra (0.1 mgperfishi.p.),GST-DrIL-4 five times. After that, the bound proteins, separated by 12% SDS-PAGE, (0.1 mg per fish i.p.), GST-DrIL-4 (0.1 mg per fish i.p.) with Trx-sDrIL-4Ra were transferred onto a nitrocellulose membrane (Bio-Rad). The mem- (0.1 mg per fish i.p.), and GST-DrIL-4 (0.1 mg per fish i.p.) with Trx-sDrIL- brane was blocked in TBS containing 2% BSA and 0.05% Tween 20 4Ra (0.5 mg per fish i.p.), respectively. For the blockade assay, fish were overnight at 4˚C and then incubated with anti-Trx tag mAb (Sigma- injected with anti– DrIL-4Ra at a dose of 1 mgperfishi.p.followedby Aldrich) at 37˚C for 1 h. After being washed, the membrane was incubated administration of GST-DrIL-4 (0.1 mg per fish i.p.) as described above. In with a HRP-conjugated secondary Ab and visualized with an ECL de- parallel, the same dose of nonspecific rabbit IgG (1:100; Chemicon) in tection system and an x-ray film (Eastman Kodak). In parallel, the purified combination with GST-DrIL-4 was administered as a negative control. After GST-DrIL-4 and Trx-IL-4Ra proteins were used as controls and analyzed 72 h, lymphocytes were collected from the peripheral blood, and B cell by Western blot using anti-GST tag and anti-Trx tag Abs (Sigma-Aldrich), proliferation was examined by flow cytometric analysis as described above. respectively. For direct binding ELISA, recombinant GST-DrIL-4 di- luted to different doses (0.015625, 0.03125, 0.0625, 0.125, 0.25, 0.5, and 1 Expression of Th1- and Th2-related cytokines and mg in 100 ml per well) in carbonate buffer (pH 9.6) was coated on a 96- transcription factors well plate overnight at 4˚C. After being blocked with 2% BSA for 1 h at 37˚C and washed, the wells were incubated with recombinant Trx-DrIL- Total RNA was isolated from the spleen tissues of experimental fish that 4Ra protein (10 mg/ml diluted in 2% BSA) at 37˚C. After 2 h of in- were injected with recombinant GST-DrIL-4 (0.1 mg per fish i.p.) for 72 h cubation at 37˚C, the plates were washed, and anti-Trx tag was incubated or control fish that were injected with GST tag. The PCR primers are for 1 h at 37˚C. After the plates were washed again, peroxidase-conjugated shown in Table I. The expression levels of the selected Th1 and Th2 streptavidin rabbit IgG was added. The color was developed using tetra- cytokines or transcription factors (T-bet, IRF1, STAT1, IFN-g,IL-10, methyl benzidine and analyzed on a plate reader at 450 nm. Recombinant GATA3, c-maf, and STAT6) were quantified by real-time PCR using the mouse IL-4 (Sigma-Aldrich), trout IL-2, and trout IL-15 (produced in our Mastercycler ep realplex and detection software (Eppendorf) according to laboratory) were devised as negative controls using the same procedure. the protocol described above. Each sample was collected from the spleens of 10 fish and was run in three parallel reactions. At least three independent Effect of DrIL-4 on B cell proliferation and IgM production experiments were performed for each panel. Fish were injected i.p. with recombinant GST-DrIL-4 at different doses Effect of DrIL-4 on the CD154–CD40-mediated costimulatory (0.01, 0.1, and 1 mg per fish i.p.), and PBS or GST tag protein alone (1 mg/ pathway fish i.p.) was used as a negative control. Whole-blood cell suspensions were collected after 72 h of stimulation, and GST-DrIL-4–elevated B cells Fish were injected with either recombinant GST-DrIL-4 (0.1 mg per fish were examined by flow cytometric analysis and real-time PCR, respec- i.p.), GST tag (negative control, 1 mg per fish i.p.), PBS (mock control), or 5574 ROLE OF IL-4–IL-4Ra INTERACTION IN Th2-LIKE IMMUNITY IN FISH different combinations of GST-DrIL-4 and various recombinant proteins Results including GST-DrIL-4 (0.1 mg per fish i.p.) plus Trx tag (negative control, Characterization of DrIL-4Ra 1 mg per fish i.p.), GST-DrIL-4 (0.1 mg per fish i.p.) plus Trx-sDrIL-4Ra (0.1 mg per fish i.p.), or GST-DrIL-4 (0.1 mg per fish i.p.) plus soluble Although the IL-4Ra–like gene has been predicted previously in CD154 (sCD154; 1 mg per fish i.p.) (43). After 3 d of stimulation, lym- silico in zebrafish, further molecular cloning, structural, and func- phocytes were separated from peripheral blood as described above and fixed tional characterization of this gene still remains to be performed in 4% paraformaldehyde at room temperature for 20 min. After being (39). In this study, we cloned the full-length DrIL-4Ra cDNA from blocked in 5% normal goat serum for 30 min at 37˚C, cells were incubated spleen and kidney tissues using the primers shown in Table I with primary Abs, mouse-anti-IgM (1:2000) and rabbit-anti-CD40 (1:2000) (GenBank accession number, EF523375.1; http://www.ncbi.nlm. (43), for 60 min at 37˚C. The nonrelated rabbit IgG and mouse IgG Abs used as negative controls were incubated with peripheral lymphocytes in the nih.gov/nuccore/EF523375). It consists of a 1716-bp open reading negative control groups at 37˚C for 1 h. The samples then were incubated frame (ORF), a 100-bp 59-untranslated region (UTR), and a 39-bp with a secondary FITC-labeled goat anti-mouse Ab (IgG, 1:100; Chemicon) 39-UTR (Supplemental Fig. 1A). This cloned sequence is mostly con- and a secondary PE-labeled anti-rabbit Ab (IgG, 1:100; Chemicon) for 45 sistent with the predicted one. The genomic structure of the DrIL- min at 37˚C. After the sampled were washed, a FACSCalibur (BD Bio- 4Ra gene consists of 11 exons and 10 introns, which is consistent sciences) was used for multicolor flow cytometry. At least 10,000 events with that of many other species, including mouse and chicken. were acquired from the lymphocyte gate, and the data were analyzed with However, human IL-4Ra, which possesses 12 exons and 11 introns, CellQuest software (49). is slightly different from the others (Fig. 1A). Statistical analysis DrIL-4Ra is a membrane-bound protein that consists of a 206- aa extracellular region, a single 24-aa transmembrane domain, and Statistical evaluation of differences between means of experimental groups was done by ANOVAs and multiple Student tests. Both p values ,0.05 and a 321-aa cytoplasmic region. Although sequence alignment shows Downloaded from p , 0.01 were considered to be significant. The sample number for each that DrIL-4Ra shares low identity with those of human and other group was .10 fish of equal mean body weight. Data points represented vertebrates (20–25%), including rainbow trout (26%), residues the means of three independent experiments. important for the maintenance of structure and function, such as six

Table I. Primers used for gene cloning and expression http://www.jimmunol.org/

Primer Name Sequence (59–39) Application 3F1 GGAATAGCAGGCTCCAGGGTCAT 39RACE 3F2 CGTCTCCTTTCCCTCCTTCTTATC 39RACE 3AD CTGATCTAGAGGTACCGGATCC 39RACE 5RT PATCCGTTTCCTTC 39RACE DrIL-4-F0 CACTAATCTTTTGAGAAATCC 59RACE DrIL-4-F1 GAAGACACTTCTGCTGCTCGC 59RACE

DrIL-4-Rb CAAATTGTCTTCAGGTAGAAC 59RACE by guest on October 1, 2021 DrIL-4R-Fa TGCCAAGCACTTTGTGACG 59RACE DrIL-4R-Fb CATGTTACCTGGGTTGATAGC 59RACE DrIL-4R-F1 TAGTCAAACTGGTGGTTCCAC 59RACE DrIL-4R-F2 CTACCCAAACCAAGTTGAGG 59RACE DrIL-4R-R1 GGCTGCGTTTTCATATAGC 59RACE DrIL-4R-R2 CCCGAATCTCTGTTAGCACG 59RACE DrIL-4-F CGCGGATCCATGCATAAGACTGTATTAAAGG Gene expression DrIL-4-R CCGCTCGAGTCAACTTGGTCTTGGGCTTTT Gene expression DrIL-4R-LF CCGGAATTCGACGATCCCATTAAGGAC Gene expression DrIL-4R-LR CCGCTCGAGTTAAGTGGTGAAATGAACTGC Gene expression MHCII-F CTCTCATTGAACTATGGATC Real-time MHCII-R CAAAGCTGTCAGTTGCAGTTG Real-time CD80-F GCTGTGACATAGGAGGCTCTG Real-time CD80-R ACCTACTCCAAAGGAATGTGAGC Real-time IgM-F TGAGCACAATAAGCGGAAAG Real-time IgM-R GCCAAGTCACAAACACCT Real-time T-bet-F CTGTCCAGCCAACAAGCG Real-time T-bet-R CACCTCCCTCAAACAAACCA Real-time IRF1-F CCATCAGGAGACGCAAAG Real-time IRF1-R GGAGAATGCTGGAAAGTC Real-time STAT1-F TCAAAGGAGGACCTGAACCGC Real-time STAT1-R CAACACCTCGGACATCTGACTAATC Real-time IFN-g-F AAGATTCTCAGCTACATAATGCACACC Real-time IFN-g-R ATGCTCATCAGTAGATTCTGCTCAC Real-time IL-10-F CTCTGCTCACGCTTCT Real-time IL-10-R TAGGGACTGTTTATGTTATG Real-time GATA3-F TGATACCTGTCCCGCTTAAAAC Real-time GATA3-R GACAGAGTCATTCCACCTTGC Real-time c-maf-F GCTCGCCACTTCTTGAACGAG Real-time c-maf-R GTCAGGGCACTCGCCAAACCAG Real-time STAT6-F CGGTAGTCAGGAAATCAATGC Real-time STAT6-R ATCTGTCCAATAGTCTCGTAGG Real-time CD40-F TTCTCAGAGTCTGTTTGCTGGTCAC Real-time CD40-R TCCCGTCACTACATTCCTCAC Real-time b-actin-F ACACCTTCTACAATGAGCTG Gene expression b-actin-R CTGCTTGCTGATCCACATCT Gene expression The Journal of Immunology 5575

FIGURE 1. Structural characterization of DrIL-4Ra genes and proteins. (A) Comparison of the intron/exon structures of IL-4Ra genes of human, mouse, chicken, and zebrafish. The rectangles represent the exons, in- cluding the alternatively spliced exon shaded in gray, whereas the lines between them indicate the introns. Black (or gray) and white areas indicate the coding regions and UTRs, respectively. The sizes of the exons or introns are shown by the numbers above or below, respectively. (B) Ribbon diagrams of the tertiary structures of the extracellular domains of human (up- per panel) and zebrafish IL-4Ra (lower panel) ob- tained by homology modeling seen from the side and Downloaded from top orientations. The seven antiparallel b strands are designated A, B, C, C9, D, E, F, and G according to human IL-4Ra. The cysteines are displayed as stick models. (C) Schematic representations of cattle IL- 4Ra, horse IL-4Ra, mouse IL-4Ra, human IL-4Ra, trout IL-4Ra1, trout IL-4Ra2, zebrafish IL-4Ra, and http://www.jimmunol.org/ zebrafish IL-4Ra-iso, which are type I transmembrane proteins with intracellular C termini and external N termini. The extracellular region of zebrafish IL-4Ra exhibited an “L” shape organized with two FN III domains that show structural homology with the hu- man FN III domain. by guest on October 1, 2021

cysteines in the extracellular region and three tyrosine residues in residues engaged in three disulfide bridges (Fig. 1B). However, the the intracellular domain, are conserved completely across all of the disulfide bridge derived from Cys49–Cys80 known to be essential aligned species (Supplemental Fig. 2A). However, the “WSXWS” anduniquetoIL-4Ra family members was found to be well motif, which is critical for maintaining ligand binding and signal conservedbothinDrIL-4Ra and other IL-4Ra molecules. transduction in human and other mammalian IL-4Ras(50,51),is Structural feature analyses of DrIL-4Ra showed that the D2 do- not classic in zebrafish (YSXQS) or in rainbow trout and salmon main had no disulfide bridges and contained no classic “WSXWS” (FSXWS) (38). This suggests that the “WSXWS” motif might have motif but contained a “YSXQS” motif. The membrane-bound re- arisen after the divergence of tetrapods from fish. gion of DrIL-4Ra was composed of 20 aa rich in hydrophobic With human IL-4Ra as the template, the tertiary structure of the amino acids. The cytoplasmic region of DrIL-4Ra contained five DrIL-4Ra extracellular domain was modeled. The results show tyrosine residues, three of them (Tyr400,Tyr477, and Tyr566), known that the DrIL-4Ra extracellular domain has an overall tertiary to be binding sites critical for IRS-1/2–, STAT6-, and ITIM/SHP2- structure similar to that of human IL-4Ra (23). The extracellular mediated signaling pathways, were conserved across species. In- region of DrIL-4Ra exhibits an “L” shape and is organized into terestingly, two tyrosine residues (Tyr284 and Tyr369) predicted to two covalently linked domains named D1 (residues 1–91) and D2 be the potential STAT3 and STAT5 binding sites were seen only in (residues 97–197). These two domains are related in overall to- pology to fibronectin type III (FN III) modules, and each of them teleost fish IL-4Ras rather than in tetrapods IL-4Ras, suggesting is folded into a sandwich-like structure composed of antiparallel b that functional diversity may exist between them (Supplemental sheets arranged in a three-strand (strands designated A, B, and E) Fig. 2A). In addition, a short proline-rich sequence in the mem- and a four-strand (strands designated G, F, C, and C9) b pleated brane-proximal region of IL-4Ra, termed the “box1 motif,” which sheet. The D1 domain contained an additional single helical turn is found in a number of hematopoietin receptor family members, in the BC loop and six cysteine residues that are engaged in two also existed in DrIL-4Ra (Fig. 1C). The structural characteristics disulfide bridges (Cys29–Cys39 and Cys49–Cys80), the latter of of DrIL-4Ra and a comparative analysis between DrIL-4Ra and which seems different from human IL-4Ra, which has six cysteine human IL-4Ra are summarized in Table II. 5576 ROLE OF IL-4–IL-4Ra INTERACTION IN Th2-LIKE IMMUNITY IN FISH

Table II. Characteristics of zebrafish IL-4Ra and comparison with human IL-4Ra

Characteristics Human IL-4Ra Zebrafish IL-4Ra Gene size 50.8 kb 10.9 kb Exons/introns 12/11 11/10 location No. 16 No. 3 m.w. 89.5 kDa 64.04 kDa Orientation Intracellular C terminus/external N terminus Intracellular C terminus/external N terminus Regions 2207 aa extracellular region 2206 aa extracellular region 224 aa transmembrane region 224 aa transmembrane region 2569 aa cytoplasmic region 2321 aa cytoplasmic region Intramolecular disulfide Cys34–Cys44 Cys29–Cys39 Cys54–Cys84 Cys49–Cys80 Cys74–Cys86 N-linked glycosylation site Asn53, Asn98, Asn128, Asn134, Asn176, Asn209, Asn481 Asn48, Asn167, Functional motif FN III domain FN III domain Potential binding sites for Box/JAK-1, Tyr479/IRS-1/2, Tyr579,Tyr601, Box/JAK-1, Tyr284/STAT-3, Tyr369/STAT-5, signal transduction Tyr631/STAT6, Tyr713/ITIM (SHP2) Tyr400/IRS-1/2, Tyr477/STAT6, Tyr566/ITIM (SHP2) Cysteine location A, B, C, D, and E strands A, B, C9, D, and E strands Tertiary structure An “L” shape comprising two FN III domains An “L” shape comprising two FN III domains Downloaded from A phylogenetic analysis showed that all of the IL-4Ra mole- were variants derived from alternative splicing of the DrIL-4Ra cules examined formed a single well-supported cluster, in which and DrIL-4Ra-iso genes, were cloned from zebrafish. These two the teleost fish IL-4Ras formed an exclusive group, as opposed to variants both contain a 100-bp 59-UTR, a 663-bp ORF, and a 384- human and other higher vertebrates, with high bootstrap proba- bp 39-UTR, encoding a peptide with 220 aa (Supplemental Fig. 4). bility. In addition, another two family members (IL-21R and IL- Exon 7 in the DrIL-4Ra and DrIL-4Ra-iso genes seems to be a

2Rb) were analyzed together with IL-4Ra (Supplemental Fig. “pseudoexon” that usually is spliced out from the precursor mRNA http://www.jimmunol.org/ 2B). These results showed that close phylogenetic relationships during the splicing process (Fig. 1A). In addition, there is a stop existed among these molecules. codon (TAG) at the 59 end of exon 7, and this stop codon could be Characterization of an IL-4Ra isoform spliced into the end of exon 6, resulting in a stop codon that appears much earlier in the alternatively spliced mRNA that encodes We also cloned a unique IL-4Ra–like isoform (designated as sDrIL-4Ra and sDrIL-4Ra-iso, respectively. As a result of this, DrIL-4Ra-iso; GenBank accession number, EF523377.1; http:// sDrIL-4Ra and sDrIL-4Ra-iso retain only a signal peptide and www.ncbi.nlm.nih.gov/nuccore/EF523377) from zebrafish. It extracellular domains but lack the membrane-anchored motif and consists of a sequence of 1872 bp comprising a 101-bp 59-UTR, cytoplasmic region, which may make them soluble with cytokine a 1107-bp ORF, and a 664-bp 39-UTR, encoding a membrane- binding affinity. by guest on October 1, 2021 bound peptide with 368 aa (Supplemental Fig. 1B). The DrIL- 4Ra-iso gene is located ∼36 kb downstream of the IL-4Ra locus Tissue distribution analysis and also consists of 11 exons and 10 introns (Fig. 1A). The most In consideration of the high homology between the two genes significant difference between DrIL-4Ra and DrIL-4Ra-iso genes mentioned above, both DrIL-4Ra and DrIL-4Ra-iso could be is the appearance of an early stop codon in exon 11 of the isoform, amplified using any primers. Moreover, because the PCR products although several nucleotide variations exist in other exons (Sup- are of the same size, it would be difficult to distinguish them. plemental Fig. 3). Exon 11 in the DrIL-4Ra or DrIL-4Ra-iso gene After a careful search over all of the substitutions between the two is the largest one among the 11 exons, consists of 853 bp, and genes, two specific restriction enzyme cutting sites were found in contributes to the encoding of the majority of the intracellular the DrIL-4Ra sequences that disappeared in the corresponding region. The stop codon (UAA) of DrIL-4Ra appears at the 812-bp sites of DrIL-4Ra-iso (an EcoRV site in exon 6 and an EcoRI site locus, which is distributed near the 39 end of exon 11. In DrIL- in exon 11). Thereby, these restriction enzymes were used to 4Ra-iso exon 11, an early stop codon (UGA) appears at the 203- distinguish the transcripts of DrIL-4Ra from PCR products as bp locus, resulting in the early termination of protein translation. well as the alternatively spliced forms. For this purpose, two pairs Therefore, the exact coding region in DrIL-4Ra-iso exon 11 of primers (DrIL-4R-Fb at exon 5 and DrIL-4R-R1 at exon 7 as consists of only 205 bp, and the corresponding 648-bp segment at well as DrIL-4R-F2 at exon 10 and DrIL-4R-R2 at exon 11, as the 39 end of exon 11 actually is unused. For this reason, the DrIL- shown in Table I) were devised to amplify a 312-bp segment, 4Ra-iso gene may encode a receptor protein that lacks a 203-aa representing the membrane-bound DrIL-4Ra and DrIL-4Ra-iso segment of the intracellular tail. Interestingly, an EcoRI site (1034, transcripts, in which DrIL-4Ra–derived transcripts contained the GAATTC) and an EcoRV site (538, CTATAG) were found to exist EcoRI site, as well as a 496-bp segment, representing the sDrIL- in the DrIL-4Ra gene but not in the DrIL-4Ra-iso gene, re- 4Ra and sDrIL-4Ra-iso transcripts, in which sDrIL-4Ra–derived spectively. This makes it possible to distinguish the mRNA from transcripts contained the EcoRV site, respectively (Fig. 2A). As these two genes by digestion with the two restriction enzymes at shown in Fig. 2B, the mixed transcripts of membrane-bound DrIL- the reverse-transcribed cDNA level. 4Ra and DrIL-4Ra-iso could be detected constitutively in most tissues examined, including the liver, gut, kidney, spleen, brain, skin, Identification of soluble IL-4Ra variants gill, ovary, and peripheral lymphocytes, but not in the muscle. In Two soluble forms of IL-4Ra (sDrIL-4Ra;GenBankaccessionnum- contrast, a mixture of the sDrIL-4Ra and sDrIL-4Ra-iso transcripts ber, EF523376.1; http://www.ncbi.nlm.nih.gov/nuccore/EF523376) could be detected constitutively only in limited tissues, such as the and IL-4Ra-iso (sDrIL-4Ra-iso; GenBank accession number, liver, brain, and muscle. These different distribution patterns suggest EF523378.1; http://www.ncbi.nlm.nih.gov/nuccore/EF523378), which the presence of functional diversity between soluble and membrane- The Journal of Immunology 5577 Downloaded from

FIGURE 2. Gene expression analysis of DrIL-4 and DrIL-4Ras. (A) Schematic representations of the strategy used in this study for distinguishing DrIL- 4Ra from DrIL-4Ra-iso transcripts. (B) Gene expression analyses of DrIL-4, DrIL-4Ras, and sDrIL-4Ras in different tissues of zebrafish. The mixed transcripts of the membrane-bound DrIL-4Ra and DrIL-4Ra-iso could be separated by EcoRI digestion. Likewise, the mixture of the sDrIL-4Ra and sDrIL-4Ra-iso transcripts could be distinguished by EcoRV digestion. http://www.jimmunol.org/ bound IL-4Ras. As expected, after being cut by EcoRI, two digested and Trx-DrIL-4Ra was dose-dependent (Fig. 3D). However, in segments from the membrane-bound DrIL-4Ra transcripts with the control groups, recombinant Trx-DrIL-4Ra failed to bind to predicted sizes of 187 and 125 bp could be detected in the liver, mouse IL-4, trout IL-2 and trout IL-15, and three gc cytokine kidney, spleen, brain, skin, gill, and peripheral lymphocytes, indi- family members of other species. These results showed that cating that the tissue distribution pattern of DrIL-4Ra was overall DrIL-4 specifically binds to DrIL-4Ra. consistent with that of DrIL-4Ra-iso, with the exception of several DrIL-4 plays a role in B cell proliferation and Ab production tissues, such as the gut, muscle, and ovary. Accordingly, after being cut by EcoRV, two digested segments from the sDrIL-4Ra tran- To evaluate whether DrIL-4 plays a role in Th2-mediated immune

scripts with predicted sizes of 361 and 135 bp could be detected in responses, an in vivo administration assay with recombinant GST- by guest on October 1, 2021 the liver, brain, and muscle (data not shown for the smaller 135 bp DrIL-4 was performed. In vivo B cell proliferation and Ag- segment). In addition, the tissue distribution of the DrIL-4 tran- stimulated Ab (IgM) production were determined for this pur- + scripts also was examined. The result shows that DrIL-4 was widely pose. Flow cytometric analysis shows that the percentage of mIgM detectable in all of the tissues examined, including the liver, gut, B cells in PBS-injected control fish was 11.04 6 1.8%, whereas it kidney, spleen, brain, muscle, skin, gill, ovary, and peripheral lym- reached 15.89 6 2.66%, 36.64 6 5.18%, and 34.57 6 6.11% in phocytes, in which stronger expression in the gut, kidney, spleen, GST-DrIL-4–administered fish that received 0.01, 0.1, and 1 mg per skin, gill, and peripheral lymphocytes could be detected. fish GST-DrIL-4, respectively. The results demonstrate that B cell proliferation could be upregulated significantly (p , 0.01) with the Preparation of recombinant proteins and Ab increase in administered GST-DrIL-4 in comparison with that of the Recombinant GST-DrIL-4 and Trx-sDrIL-4Ra were expressed PBS control groups (Fig. 4A). In contrast, the percentage of B cells in E. coli BL21 cells as soluble GST or Trx fusion proteins with in GST tag-injected control groups was 13.57 6 2.06%, which was their corresponding molecular weights, respectively (Fig. 3A). Abs not significantly (p . 0.05) different from that of the PBS control for DrIL-4Ra (anti–DrIL-4Ra) were affinity-purified from im- groups. Real-time PCR analysis showed that the expression of munized rabbit serum or mouse serum. ELISA analysis shows that B cell markers (mIgM, MHC class II, and CD80) also was upreg- the purified Ab had an average titer greater than 1:10,000. Western ulated significantly (p , 0.01) in peripheral lymphocytes after blot analysis showed that the purified anti–DrIL-4Ra was bound in vivo administration of recombinant GST-DrIL-4 (Fig. 4B). This specifically to endogenous DrIL-4Ra in different tissues and that result supports the conclusion that DrIL-4 plays a role in B cell no cross-reactions between anti–DrIL-4Ra and other proteins proliferation. Accordingly, Ab (IgM) production in GST-DrIL-4– from tissue extracts were observed (Fig. 3B). stimulated groups also could be upregulated significantly (p , 0.01 in 0.1 and 1 mg per fish groups) with the increase in administered Binding assay between DrIL-4 and DrIL-4Ra GST-DrIL-4 in comparison with that of KLH-immunized control To identify whether DrIL-4Ra did act as a receptor for DrIL-4, groups. In contrast, the production of IgM in GST tag-preinoculated a GST pull-down binding assay and a direct quantitative ELISA control groups did not increase significantly (Fig. 4C). These results were performed. In the GST pull-down assay, a larger band with demonstrated that DrIL-4 plays an important role in B cell prolif- an expected m.w. of 94 kDa, which represents the GST-DrIL-4 eration and Ab production, which are typical characteristics of and Trx-DrIL-4Ra complex, could be detected clearly by Western Th2-mediated immune responses. blot analysis (Fig. 3C). In the ELISA analysis, increased inter- a actions between recombinant GST-DrIL-4 and Trx-DrIL-4Ra DrIL-4R distributes on B cell surfaces could be detected with the increase in GST- DrIL-4 protein coat- To investigate whether DrIL-4Ra distributes on B cell surfaces, a ing, indicating that the specific interaction between GST-DrIL-4 double-immunofluorescence staining experiment was performed. 5578 ROLE OF IL-4–IL-4Ra INTERACTION IN Th2-LIKE IMMUNITY IN FISH

colocalized on these cellular membranes, respectively (Fig. 4D). By contrast, isotype controls utilizing rabbit IgG and mouse IgG as nonrelated primary Abs displayed no fluorescent signals (data not shown). This result suggested that the DrIL-4–upregulated B cell proliferation and activation described above probably were achieved by its interaction with DrIL-4Ra on B cell surfaces. Soluble DrIL-4Ra plays an inhibitory role in B cell proliferation and Ab production To identify the possible role that sDrIL-4Ra may play and to provide further functional observations that DrIL-4 and DrIL-4Ra are two reciprocal molecules, an in vivo recombinant Trx-sDrIL- 4Ra administration assay was performed. As mentioned above, the percentage (36.64 6 5.18%) of mIgM+ B cells in recombinant GST-DrIL-4–treated groups was upregulated significantly (p , 0.01) in comparison with that of PBS-injected control groups (11.04 6 1.8%). In contrast, the amounts of B cells in the group treated with GST-DrIL-4Ra alone (10.43 6 1.33%) and in the control groups administered Trx tag (9.56 6 2.01%) and GST tag (13.57 6 2.06%) did not increase significantly (p . 0.05). Downloaded from However, the percentage of B cells in Trx-sDrIL-4Ra and GST- DrIL-4 coadministered groups (14.80 6 0.95%) was downregu- lated dramatically (p , 0.01) in comparison with that of GST- DrIL-4–treated groups (Fig. 4E). Accordingly, KLH-stimulated Ab (IgM) production in GST-DrIL-4–treated groups was in-

creased more significantly (p , 0.01) than those of the control http://www.jimmunol.org/ group and groups treated with GST or Trx tag proteins alone. In contrast, titers of IgM in Trx-sDrIL-4Ra and GST-DrIL-4 coad- ministered groups were decreased significantly (p , 0.05) com- pared with that of the GST-DrIL-4–treated group (Fig. 4F). All of these results demonstrated that administration of Trx-sDrIL-4Ra greatly abrogated GST-DrIL-4–elevated B cell proliferation and Ab production as well as Ab production under the “natural” immuni- zation condition. sDrIL-4Ra may play a negative role in fish Th2- mediated immunity by its competitive binding to DrIL-4. Similar by guest on October 1, 2021 roles also were seen in mammal soluble IL-4Ra molecules. In ad- dition, these results provided further functional evidence that IL-4 FIGURE 3. Binding assays for recombinant GST-DrIL-4 and Trx-sDrIL- and IL-4Ra are two reciprocal molecules in zebrafish. A 4Ra interactions. ( ) SDS-PAGE analysis of purified recombinant GST- a DrIL-4 and Trx-sDrIL-4Ra. Lane 1, purified Trx tag (22 kDa); lane 2, pu- Blockade of DrIL-4R downregulates B cell proliferation rified recombinant Trx-sDrIL-4Ra (47 kDa); lane 3, protein standards; lane To investigate further whether DrIL-4 and DrIL-4Ra were a pair 4, purified recombinant GST-DrIL-4 (51 kDa); lane 5, purified GST tag (35 of reciprocal molecules whose signaling pathway is essential for B kDa). ( ) Western blot showing the effectiveness of the purified DrIL-4Ra Th2-mediated immune responses, an in vivo IL-4Ra blockade as- Ab, which can bind specifically to endogenous DrIL-4Ra in different tis- say with DrIL-4Ra–specific Ab (anti–DrIL-4Ra) was performed. sues. (C) Western blot showing the binding of recombinant GST-DrIL-4 to Flow cytometric analysis shows that the percentage (27.33 6 recombinant Trx-sDrIL-4Ra. After cross-linking using BS3, equal amounts of samples were loaded for Western blot analysis. Sample serial numbers are 3.62%) of B cells in recombinant GST-DrIL-4–treated groups (with , shown on the side. Lane 1, Recombinant GST-DrIL-4; lane 2, recombinant nonspecific rabbit IgG) was upregulated significantly (p 0.01) in Trx-sDrIL-4Ra; lane 3, recombinant GST-DrIL-4 and Trx-sDrIL-4Ra comparison with that of PBS-injected control groups (11.04 6 cross-linked with BS3. Anti-GST tag Ab was used for the detection of 1.8%). In contrast, the amount of B cells in GST tag protein ad- recombinant GST-DrIL-4 in lane 1, and anti-Trx tag Ab was used for the ministered groups (13.57 6 2.06%) did not increase significantly detection of recombinant Trx-sDrIL-4Ra in lanes 2 and 3. GST and Trx (p . 0.05). However, the percentage of B cells in anti–DrIL-4Ra cannot be cross-linked with Trx-sDrIL-4Ra and GST-DrIL-4, respectively blockade groups (13.11 6 1.39%) was downregulated dramatically D (data not shown). ( ) ELISA analysis for the detection of the interaction (p , 0.01) in comparison with that of positive groups (Fig. 4G). between purified recombinant GST-DrIL-4 and Trx-sDrIL-4Ra with anti- These observations demonstrate that blockade of DrIL-4Ra sig- Trx tag Ab. PBS was used as the blank control (upper panel). Recombinant nificantly downregulates B cell proliferation, which suggests that mouse IL-4, trout IL-2, and trout IL-15 were used, instead of GST-DrIL-4, to coat a 96-well plate, because negative controls to show that Trx-sDrIL-4Ra DrIL-4Ra is essential for DrIL-4–upregulated immune responses binds specifically to GST-DrIL-4 (lower panel). All of the data represent and provides further evidence that the DrIL-4–DrIL-4Ra interac- results obtained from at least three independent experiments. tion is essential for Th2-type immunity in zebrafish. DrIL-4 upregulates the expression of Th2 cytokines and The results show that a considerable number of lymphocytes in transcription factors peripheral blood exhibited mIgM and DrIL-4Ra expression To investigate whether DrIL-4 acts as a Th2 cytokine that can (mIgM+IL-4Ra+), as both diffuse green fluorescent and red fluo- upregulate the expression of Th2 cytokines and transcription rescent signals, which indicate that mIgM and DrIL-4Ra were factors and downregulates Th1 cytokines and transcription factors, The Journal of Immunology 5579 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 4. Evaluation of DrIL-4 function and the DrIL-4–DrIL-4Ra interaction involved in Th2-mediated immune responses by in vivo administration assays. (A) Flow cytometric analysis of the percentage of mIgM+ B cells in isolated peripheral lymphocytes separated by Ficoll-Hypaque centrifugation from whole-blood cells after treatment for 72 h with recombinant GST-DrIL-4 (0.01, 0.1, or 1 mg per fish i.p.), GST (1 mg per fish i.p.), or PBS. The gray histograms show background fluorescence on isotype controls. (B) Effect of DrIL-4 on the expression of B cell markers. Transcripts from the peripheral lymphocytes of zebrafish after i.p. inoculation with recombinant GST-DrIL-4 or GST for 72 h were analyzed by real-time quantitative PCR. Relative gene expressions were calculated using the 22DDCT method, with the initial normalization of genes against b-actin within each sample. The expression level of each gene in the GST-treated control groups was set arbitrarily to 1.0, and the levels of recombinant GST-DrIL-4–stimulated groups were adjusted ac- cordingly. The relative expression value was averaged from 10 healthy fish and run in three parallel reactions. The data represent the results obtained from at least three independent experiments. (C) ELISA analysis for KLH-specific IgM Abs in the serum from KLH-immunized (Figure legend continues) 5580 ROLE OF IL-4–IL-4Ra INTERACTION IN Th2-LIKE IMMUNITY IN FISH representative Th2 (IL-10, GATA3, c-maf, and STAT6) and Th1 type) immunity (56). The development of Th1- and Th2-type (T-bet, IRF1, STAT1, and IFN-g) cytokines and transcription immunity is regulated by a number of subset-specific cytokines, in factors in response to the stimulation of DrIL-4 were analyzed which the IL-4–IL-4R–mediated signaling pathway plays a critical (52–56). As expected, administration of recombinant GST-DrIL-4 role in Th2-type immune responses (57). However, little docu- significantly upregulates the expression of IL-10, GATA3, c-maf, mentation exists about whether Th1- and Th2-type immune and STAT6 but downregulates the expression of T-bet, IRF1, mechanisms originate from ancient vertebrates. STAT1, and IFN-g. This result provides the observation that We reported recently on the molecular and functional charac- DrIL-4 acts as a Th2 cytokine that might be involved in Th2 terization of CD154 and CD40 costimulatory molecules from development in fish immunity (Fig. 5). zebrafish (43). It shows that the CD154–CD40-mediated T–B interactions are required for humoral immune responses to DrIL-4 promotes CD40 expression and the CD154–CD40 thymus-dependent Ags in zebrafish and provides a preliminary costimulatory interaction insight into the origin of the Th-like regulatory mechanism in To investigate whether DrIL-4 functions in B cell proliferation early vertebrates. However, further study is needed to elucidate the by upregulating CD40 expression on B cells and by promoting Th1 and Th2 subsets involved in the underlying Th regulatory CD154–CD40-mediated costimulatory signaling in zebrafish, an mechanism. Because a IL-4–IL-4Ra–mediated regulatory mech- in vivo DrIL-4–elicited CD40 expression and CD154–CD40 in- anism is believed to be a hallmark of Th2-type immunity, the teraction assay was performed. Real-time PCR analysis showed functional identification of these two reciprocal molecules becomes that the CD40 transcripts were upregulated dramatically in lym- a prerequisite for the evaluation of Th2-type immunity in fish. For phocytes from the recombinant GST-DrIL-4–treated groups com- this purpose, the biological role that IL-4 and IL-4Ra played in pared with those of the PBS or GST tag-treated control groups (p , B cell proliferation, Ab production, and CD154–CD40-mediated Downloaded from 0.01) (Fig. 6A). This stimulatory effect of GST-DrIL-4 was in- costimulatory Th2-type immunity was characterized carefully in hibited significantly by coadministration of Trx-sDrIL-4Ra (p , zebrafish. 0.01) but enhanced significantly by coadministration of sCD154 In total, four IL-4Ra molecules were identified from zebrafish, (p , 0.01). Flow cytometric analysis demonstrated that the ex- which includes one typical DrIL-4Ra molecule, one DrIL-4Ra-like pression of CD40 proteins on B cells (as determined by the geo- isoform, and two corresponding alternatively spliced soluble var- metric mean of fluorescence intensity of PE-labeled anti-CD40 on iants, the latter of which to our knowledge were first reported in fish. http://www.jimmunol.org/ + + IgM CD40 B cell surfaces) in the GST-DrIL-4–treated groups This result suggests that the composition and function of the fish (75.49 6 3.56) was significantly higher (p , 0.01) than that of the IL-4R system is much more complicated than known previously. PBS- (42.75 6 2.53) or GST tag-treated (44.87 6 2.47) control DrIL-4Ra shares a number of conserved structural features with its group. Significant proliferation of B cells was accompanied by homologues in other species. These include similar chromosomal upregulated expression of CD40 molecules (p , 0.01). These locations and gene organizations, overall conserved protein ho- stimulatory effects of GST-DrIL-4 on CD40 expression and B cell mology domains (such as the FN III domain in the extracellular proliferation also were inhibited significantly by coadministration region), and key residues (such as cysteines) that are important for with Trx-sDrIL-4Ra (58.77 6 2.92) and enhanced by coadminis- the formation of disulfide bonds and the structural integrity of the by guest on October 1, 2021 tration with sCD154 (111.27 6 5.86) (both p , 0.01) (Fig. 6B, 6C). molecules, although sequence alignment between zebrafish and These observations indicate that DrIL-4 promotes CD40 expres- mammal IL-4Ra showed only 20–25% similarity. DrIL-4Ra-iso sion on B cells by interacting with its receptor DrIL-4Ra. The up- seems to be unique in fish species. In this study, a comparison of the regulated CD40 molecules enhance the B cell response to CD154 organizations of the DrIL-4Ra and DrIL-4Ra-iso genes, including stimulation, resulting in the enhanced CD154–CD40 costimula- alternatively spliced exons, is presented. Comparisons between tory interaction critical for B cell proliferation and activation. every counterpart show extremely high identity, including the transcriptional starts, the boundary sites of each intron, and the Discussion polyadenylation signals, implying an obvious gene duplication In humans and other mammals, the adaptive immune system is event occurred between them. Nevertheless, a few remarkable dif- composed of various T and B subsets and cytokines that consist of ferences also were identified in detail. For instance, the lengths of a precise regulatory network for the development of cellular or introns 5, 6a, 6b, and 8 varied between these two genes, and some humoral immunity. The Th cell is one of the most important nucleotides were distinct at several positions, including both syn- populations in such a network, and its two main subsets, Th1 and onymous and nonsynonymous substitutions. The most significant Th2, largely contribute to cellular (Th1-type) and humoral (Th2- nonsynonymous one is the appearance of an early stop codon in

zebrafish treated with recombinant GST-DrIL-4 (0.01, 0.1, or 1 mg per fish i.p.) and GST (1 mg per fish i.p.) and the control group immunized with KLH only. All of the groups were titered from 1:100 to 1:10,000, and the titer was ascertained based on multiple point analysis. The data of the background control group with PBS administration were subtracted from each experimental group. (D) Immunofluorescence staining of lymphocytes with rabbit anti- mIgM and mouse anti–DrIL-4Ra. Nonrelated Abs, including rabbit IgG and mouse IgG, were used as negative controls (data not shown). White arrows indicate the mIgM and DrIL-4Ra double-positive cells. Scale bars, 20 mm (original magnification 3400). (E) B cell proliferation is activated by GST- DrIL-4 that is blocked by Trx-sDrIL-4Ra. Flow cytometric analysis of the percentage of mIgM+ B cells with different treatments in the recombinant Trx- sDrIL-4Ra–injected groups (0.1 mg per fish i.p.), GST-DrIL-4–injected groups (0.1 mg per fish i.p.), GST-DrIL-4/Trx-sDrIL-4Ra (1:1)–injected groups, GST-DrIL-4/Trx-sDrIL-4Ra (1:5)–injected groups, GST (1 mg per fish i.p.)–injected groups, Trx-injected (1 mg per fish i.p.) groups, and control groups. (F) ELISA assays for KLH-specific IgM Abs in the serum from KLH-immunized zebrafish treated with recombinant Trx-sDrIL-4Ra (0.1 mg per fish i.p.), GST-DrIL-4 (0.1 mg per fish i.p.), GST-DrIL-4/Trx-sDrIL-4Ra (0.1/0.1 mg per fish i.p.), GST (1 mg per fish i.p.), and Trx (1 mg per fish i.p.) and the control groups immunized with KLH only. All of the groups were titered from 1:100 to 1:100,000, and the titer was ascertained based on multiple point analysis. The data of the background control group with PBS administration were subtracted from each experimental group. (G) B cell proliferation is inhibited by the Ab of DrIL-4Ra blockade. Peripheral lymphocytes were isolated from fish after treatment with either recombinant GST-DrIL-4 and IgG or recombinant GST-DrIL-4 and rabbit anti–DrIL-4Ra. The gray histograms in (A) and (C) show the background fluorescence of isotype controls. All of the data represent results obtained from at least three independent experiments. *p , 0.05, **p , 0.01. The Journal of Immunology 5581

the interaction of natural or administered recombinant GST-DrIL-4 with Trx-DrIL-4Ra. This resulted in the inhibition of GST-DrIL-4– enhanced B cell proliferation and Ab production in response to Ag (KLH) stimulation. As expected, GST-DrIL-4–enhanced in vivo B cell proliferation and Ab (IgM) production could be downregu- lated significantly by administration of Trx-sDrIL-4Ra. This not only indicates that DrIL-4 and DrIL-4Ra are two reciprocal mol- ecules but also shows that sDrIL-4Ra may be used as a negative regulator to downregulate the hyperimmune responses elicited by excessive DrIL-4. Such regulators have potential application in the control in immune-related diseases. To optimize the concentration of IL-4 for in vivo administration assays, different dosages of the reagent were used in the experiments. The optimized concentration was determined by in vivo B cell proliferation by flow cytometric analysis. The results show that the most appropriate concentration of GST-DrIL-4 is 0.1 mg per fish. The FIGURE 5. Effects of DrIL-4 on the expression of Th1- and Th2-type optimized time point for in vivo B cell proliferation assays was cytokines and transcription factors. The transcripts from the spleen of chosen as 72 h poststimulation of GST-DrIL-4 based on our previous zebrafish after i.p. inoculation with recombinant GST-DrIL-4 or GST for investigation. We found that the activation of APCs (as determined 72 h were analyzed by real-time quantitative PCR. Relative gene expres- by the expression of DC-SIGN/CD209) could be upregulated dra- Downloaded from 2DDCT sion was calculated using the 2 method with initial normalization of matically, even after 5 d of in vivo inoculation with recombinant genes against b-actin within each sample. The expression level of each DrIL-4 (44). Likewise, the time point used around 72 h also had been gene in the GST-treated control group was set arbitrarily at 1.0, and the reported in other animal models, including mammals. For example, levels of the recombinant GST-DrIL-4–stimulated group were adjusted correspondingly. The relative expression value was averaged from 10 in vivo treatment with recombinant IL-4 for 72 h directly increased healthy fish and run in three parallel reactions, and the data represent dendritic cell (DC) maturation and recruitment independent of http://www.jimmunol.org/ results obtained from at least three independent experiments. *p , 0.05, innate immune signals in a mouse model (63). According to these **p , 0.01. observations, it could be considered that the function of IL-4 is a critical driver in the priming and initiation of Th2-type adaptive exon 11 of DrIL-4Ra-iso, making the cytoplasmic region of DrIL- immunity and is crucial for the recruitment, maturation, and acti- 4Ra-iso much shorter than that of DrIL-4Ra. As a result, a number vation of DCs and T lymphocytes by upregulating a number of key of potential phosphorylation sites that are involved in various sig- regulatory factors, such as CD40, CD154, CD80/86, CD83, and DC- naling pathways, such as Tyr369/STAT-5, Tyr400/IRS-1/2, Tyr477/ SIGN/CD209. As a result, although the half-life of recombinant IL-4 STAT6, and Tyr566/ITIM (SHP2), may be lost, resulting in func- in vivo is expected to be very short, a quick initial response could be tional diversity between these two receptors. The alternatively induced considerably by this cytokine at the early stage of Th2-type by guest on October 1, 2021 spliced soluble variants also are seen in humans and other mam- immunity. This reaction may be powerful enough to trigger subse- mals (58–60). In these species, exon 8 in the IL-4Ra genes is quent immune response cascades, including the maturation and ac- a “pseudoexon” that contains a stop codon (TAG) at its 59 end. tivation of DCs and T and B lymphocytes, the latter of which may When exon 8 is spliced into the end of the upstream exon, the stop occur in 72 h, at least in fish species. codon may appear much earlier in the alternatively spliced mRNA, To provide further functional observations that DrIL-4 plays a resulting in the production of a soluble IL-4Ra variant that lacks role in adaptive humoral immunity as a Th2 cytokine and that the transmembrane and cytoplasmic regions. This soluble splicing the DrIL-4–DrIL-4Ra interaction is essential for this process, is similar to that in DrIL-4Ra genes. Notably, DrIL-4Ra-iso and in vivo administration of recombinant GST-DrIL-4 and in vivo sDrIL-4Ra-iso presented here are two novel molecules of the IL- Trx-DrIL-4Ra blockade assays were performed. The B cell pro- 4Ra family, and their functions had never been reported previously. liferation and Ab production in response to GST-DrIL-4 were Their in-depth study may lead to the discovery of new features of examined for this purpose. The results show that B cell prolifer- the IL-4R family ation is upregulated dramatically after stimulating the fish with The distribution pattern of the sDrIL-4Ras, expressed only in GST-DrIL-4, because the percentages of mIgM+ B lymphocytes in the liver, brain, and muscle, is quite different from that found for the stimulated fish and the expression of B cell markers in the membrane-bound DrIL-4Ra. To our knowledge, the liver is gen- stimulated lymphocytes were elevated significantly (p , 0.01). erally an immune-relevant organ that produces various immune- Accordingly, the production of Ab also was found to be enhanced related cytokines and their receptors in teleost fish (38, 61, 62). significantly (p , 0.01) by GST-DrIL-4. However, blockade of From this point of view, the liver produces sDrIL-4Ra for relevant DrIL-4Ra by specific anti–DrIL-4Ra Ab could decrease B cell immune responses. Considering the different distribution patterns proliferation significantly (p , 0.05). Immunofluorescence stain- between sDrIL-4Ra and membrane-bound DrIL-4Ra, we suggest ing showed that DrIL-4Ra and mIgM were colocated on B cell that sDrIL-4Ra may play different roles aside from its antagonist- surfaces. This, together with the observation that B cell prolifer- related functions to membrane-bound DrIL-4Ra. Further investi- ation and Ab production could be downregulated by in vivo ad- gation would be needed to clarify this suggestion. ministration of Trx-sDrIL-4Ra, demonstrates that DrIL-4 plays an To provide functional evidence that IL-4 and IL-4Ra in zebrafish important role in B cell proliferation and Ab production by in- are two reciprocal molecules, the interaction between these two teraction with DrIL-4Ra on B cells, which is a typical charac- molecules was investigated initially both in vitro and in vivo. The teristic of Th2-mediated immune responses in mammals. In results from the in vitro binding assay showed that recombinant addition, the changes in expression levels of the key cytokines or GST-DrIL-4 could bind specifically to recombinant Trx-DrIL- transcription factors involved in Th1 and Th2 differentiation were 4Ra. The in vivo binding assay was evaluated functionally by investigated after stimulating the fish with recombinant GST- administering the fish with recombinant Trx-sDrIL-4Ra to inhibit DrIL-4. As expected, GST-DrIL-4 was able to enhance the ex- 5582 ROLE OF IL-4–IL-4Ra INTERACTION IN Th2-LIKE IMMUNITY IN FISH

FIGURE 6. Effect of recombinant GST-DrIL-4 on the CD154–CD40-mediated costimulatory pathway. (A) Real-time quantitative PCR analyses of zebrafish CD40 transcripts in isolated peripheral lymphocytes. PCRs were run in combination with the endogenous b-actin control. The graphs represent the means ob- tained from 30 fish. Bars represent SE. Data are from at least three independent experiments. (B) Flow cytometric analysis measuring the percentage of mIgM+CD40+ B cells and the expression of CD40 on mIgM+ B cells. Different treatments are presented, including injection with PBS (control), GST (1 mg per Downloaded from fish i.p.), recombinant GST-DrIL-4 (0.1 mg per fish i.p.), or coinjection with GST-DrIL-4 (0.1 mg per fish i.p.) and Trx (1 mg per fish i.p.), GST-DrIL-4 (0.1 mg per fish i.p.) and Trx-sDrIL-4Ra (0.1 mg per fish i.p.), or GST-DrIL-4 (0.1 mg per fish i.p.) and sCD154 (0.1 mg per fish i.p.). Numbers in the quadrants (upper) http://www.jimmunol.org/ indicate the percentage of double-positive cells in each group, and the numbers in parentheses (below) indicate the geometric mean fluorescence intensities of FITC–anti-mIgM (left) and PE–anti-mCD40 (right). (C) Statistical analyses of the geometric mean of the fluorescence intensities of FITC–anti-mIgM and PE–anti-mCD40 indicate the expression of mIgM and mCD40 on B cells (left panel) and the percentage of B cells in isolated lymphocytes (right panel) in each treatment group. Each result was obtained from 40 by guest on October 1, 2021 fish. Bars represent SE. Data are from three indepen- dent experiments for each panel. *p , 0.05, **p , 0.01.

pression of Th2 cytokines and transcription factors (IL-10, a report showing that IL-4 has no effect on CD40 expression in GATA3, c-maf, and STAT6) but inhibit the expression of Th1 human monocytes (68). At the same time, IL-4 can suppress IFN- transcription factors (T-bet, IRF1, STAT1, and IFN-g), as its g–induced CD40 gene expression in both mice macrophages and counterparts did in the adaptive immunity of humans and other microglia (69). In the current study, we demonstrated that the mammals (2–4). Therefore, it seems reasonable to suggest that DrIL-4–DrIL-4Ra interaction promotes B cell proliferation and DrIL-4 may play a role as a Th2 cytokine and that a Th2-mediated activation by upregulating CD40 expression on B cells. These regulatory mechanism might be developed in adaptive immunity B cells with upregulated CD40 were more responsive to CD154 as early as fish during vertebrate evolution. stimulation. This result suggests the existence of a close regula- The essential roles of IL-4–IL-4Ra signaling and the CD154– tory relationship between the DrIL-4–DrIL-4Ra signaling path- CD40-mediated costimulatory pathway in Th2-type immunity way and the CD154–CD40-mediated costimulatory pathway in have been well documented in mammals (28–32, 64–66). How- zebrafish, which may delineate a signal network that is essential ever, the regulatory relationship between these two signaling for modulating Th2 immune responses. Further study of this pathways was still controversial. CD154–CD40 costimulatory model will lead to a better understanding of IL-4–regulated co- signaling can induce effector T cell-derived IL-4 (67), but whether stimulatory signaling pathways in Th2-type immunity. IL-4–IL-4Ra signaling can promote a CD154–CD40-mediated In conclusion, this study shows the role that zebrafish IL-4 and costimulatory pathway in B cells still lacks evidence. 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