Molecular and Functional Characterization of IL-15 in Rainbow Trout ...

The Journal of Immunology

Molecular and Functional Characterization of IL-15 in Rainbow Trout Oncorhynchus mykiss: A Potent Inducer of IFN-␥ Expression in Spleen Leukocytes1,2 Tiehui Wang, Jason W. Holland, Allison Carrington, Jun Zou, and Christopher J. Secombes3 IL-15 is a member of the common ␥-chain family of cytokines that possess a heterogeneous repertoire of activities on various cells of the immune system. We report here the first functional characterization of a fish IL-15 in rainbow trout. The trout IL-15 gene is 6-kb long and contains six exons and five introns that transcribe into a 1.2-kb mRNA containing seven out-of-frame AUG initiation codons and translate into a 193-aa peptide. Potential sites for transcriptional activators and repressors have been identified in the trout IL-15 gene. Like IL-15 from other species, trout IL-15 is closely linked to an INPP4B gene, but there is also a BCL10 gene located between the IL-15 and INPP4B genes. Three alternative splicing variants of the trout IL-15 gene have also been identified and their expression in vivo was studied. Trout IL-15 expression is present in all the tissues and cell lines studied. Recombinant trout IFN-␥ selectively increased IL-15 expression but had little effect on other cytokines such as IL-1␤ and IL-11. Recombinant trout IL-15 preferentially stimulated splenic leukocytes from healthy fish, where it induced a large increase in IFN-␥ expression, with little, if any, effect on IL-1␤ expression. This effect was quite long-lived, and was still apparent 24 h poststimulation. Although the exact cell types being affected have still to be determined, it is clear that once produced IL-15 will have a profound affect on the ability of the fish immune system to activate antimicrobial defenses and genes induced themselves by IFN-␥. The Journal of Immunology, 2007, 179: 1475–1488.

I

nterleukin-15 is a member of the common ␥-chain (␥c)4 family of cytokines including IL-2, IL-4, IL-7, IL-9, and IL-21 that all use receptors containing ␥c, while IL-15 and IL-2 receptors also share the IL-2/15R ␤-chain (1). Since its discovery in 1994, IL-15 has been studied in the context of its structural cousin, IL-2 (2– 4). Although IL-15 shares activities with IL-2 as a result of the use of the common ␤ and ␥ receptor chains that are responsible for downstream cytoplasmic signaling, it has become evident that these cytokines exert differential effects on many cell populations (5). In mammals, the IL-15 gene is not expressed in T cells, which are the prime producer of IL-2, but IL-15 is expressed in a wide variety of tissues and cell types including monocyte/ macrophages (6, 7), dendritic cells, Langerhans cells (8), epithelial

Scottish Fish Immunology Research Centre, School of Biological Sciences, University of Aberdeen, Aberdeen, United Kingdom Received for publication December 6, 2006. Accepted for publication May 8, 2007. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by grants from the European Commission (Stressgenes, Q5RS-2001-002211, and Aquafirst, 513692). 2 The nucleotide sequence(s) presented in this article will appear in the EMBL/DDBJ/ GenBank nucleotide sequence database under the following accession numbers: AJ555868 (IL-15 cDNA) and AJ628345 (genomic sequence for IL-15, BCL10, and partial of INPP4B gene). 3 Address correspondence and reprint requests to Prof. Christopher J. Secombes, Scottish Fish Immunology Research Centre, School of Biological Sciences, University of Aberdeen, Aberdeen AB24 2TZ, U.K. E-mail address: c.secombes@ abdn.ac.uk

Abbreviations used in this paper: ␥c, common ␥-chain; LSP, long signal peptide; SSP, short signal peptide; UTR, untranslated region; ER, endoplasmic reticulum; rt, recombinant trout; Q-PCR, quantitative PCR; EF, elongation factor; SV, splicing variant; NLS, nuclear localization signal; EST, expressed sequence tag; ␥-IRE, IFN␥-responsive element; IPTG, isopropyl ␤-D-thiogalactoside; GR, glucocorticoid receptor. 4

Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 www.jimmunol.org

and endothelial cells, stromal cells, astrocytes and microglia (9), and a number of cell lines (10). IL-15 expression is regulated at many levels, including transcription, translation, and intracellular trafficking. In humans, two IL-15 mRNA isoforms, encoding a long (LSP, 48 aa) and short (SSP, 21 aa) signal peptide, are generated by alternative splicing (11). IL-15 mRNA has been found to be constitutively expressed in several normal tissues and cells, and is up-regulated by inflammatory mediators, activation signals, and infection (9, 12). Despite the broad expression of IL-15 mRNA, it has been difficult to demonstrate the IL-15 protein in the supernatants of many cells that express IL-15 mRNA. This suggested that IL-15 protein production is predominantly regulated posttranscriptionally. The broad array of negative regulatory controls affecting IL-15 expression are the AUG-burdened 5⬘-untranslated region (UTR), the nucleotide or protein sequence of the IL-15 signal peptide and C terminus (13, 14), and the inefficient IL-15 trafficking in the endoplasmic reticulum (ER) (15). The human SSP-IL-15 isoform does not enter the ER and is not secreted, but rather is stored intracellularly and appears in the nuclear and cytoplasmic component (16). In contrast, the LSP isoform enters multiple intracellular pathways resulting in 1) LSP-IL-15 remaining in the cytoplasm unprocessed, unglycosylated and eventually being degraded; 2) LSP-IL-15 entering into the ER, becoming N-glycosylated and being partially processed to yield a proposed 19-aa 3⬘ element of the LSP linked to the mature protein, which is not secreted into the medium; and 3) the classical pathway where LSP-IL-15 is translocated to the ER, fully processed, N-glycosylated, and ultimately secreted by the classical Golgi route (15). The private receptor ␣-chains of IL-2 and IL-15 are structurally similar, have the same intron/exon organization, and are closely linked in mammals. Despite these similarities, there remain four important differences: 1) unlike IL-2R␣, IL-15R␣ has a broad distribution of expression in different cell types (17); 2) IL-15R␣ binds IL-15 with a Ka of 1011/M, a 1000-fold higher affinity than that of IL-2R␣ for IL-2; 3) IL-15 can signal by binding to the

1476

RAINBOW TROUT IL-15

Table I. Primers used for cloning and expression Name

IL-15RF IL-15RR IL-15EF1 IL-15ER1 IL-15F1 IL-15F2 IL-15R1 IL-15R2 IL-15F3 IL-15svF IL-15svR IL-15F IL-15R IL-15sv1R IL-15sv2F IL-15sv3F EF-1aF EF-1aR IFN2F IFN2R IL-1␤F IL-1␤R IL-11F IL-11R

Gene

IL-15

EF-1␣ IFN-␥ IL-1␤ (isoform I) IL-11

Sequence (5⬘ to 3⬘)

GCTGT CGTGT CCATT CGACA TGCTG AGGCG CACGA CACAC CCATT ATTGC CATTT TATTG AAGAT CATAC ACACA GCAGG CAAGG ACAGC CTGAA CCCTG CCTGG GCTGG CTGCT TGGGT

ACAAA ACATT ACTAT TGTCC GTGCT ACGTT GCACT ACATG GCTGG GCACA GGACA AGCTG GCAGT AAACA GCCTG ACAAA ATATC GAAAC AGTCC GACTG AGCAT AGAGT CTCGC CTCAT

GCTGA AACTG CCACA TCAAA GCTGA GAAGA AGCTG ACATT TGCTG TCATG CGAGC CCTGA GGTCA GGCAT GAGCT GCTGC CGTCG GACCA ACTAT TGGTG CATGG GCTGT TGCTA CTCAA

AACAC ACAGT CAGCG CAGGA AACTC GCTTC TCACT GTTTG CTGAA TGTTC ACTAG GTGCC TTGTA CTGAT GCCT CTGA TGGCA AGAGG AAGAT TCAC CGTG GGAAG TTGG GGGAG

␤-/␥-chain complex without the ␣-chain; and 4) IL-15R␣ binds, recycles, and presents IL-15 to the opposing cell (18, 19). The interaction of IL-15 with its receptor complex leads to a series of signaling events that include activation of the JAK/STAT pathway, and the PI3K pathway. The ␤-chain is associated with JAK1, whereas the ␥-chain is physically and functionally associated with JAK3, resulting in STAT3 and STAT5 phosphorylation, respectively (20). The PI3K pathway and downstream effectors play a central role in the signaling processes involved in the proliferation and inhibition of apoptosis for every ␥-chain-interacting cytokine. IL-15 also has the ability to activate the transcription factors, NF-␬B and AP-1 (21). The diverse expression of IL-15, IL-15R␣, and the common ␤-/␥-chains has been suggested to reflect a heterogeneous repertoire of activities mediated by this cytokine on various cells of the immune system. Thus, IL-15 promotes extrathymic development of T and NK cells (22), is a growth, differentiation, activation, and survival factor for NK cells that contributes to increased cytolytic activity of NK cells, stimulation of cytokine (IFN-␥) synthesis, and prolongation of NK cell survival (23, 24). IL-15 is also a potent T cell growth factor that enhances the survival, activation, IFN-␥ production, and cytotoxicity of CD8⫹ T cells and has an important role in regulating CD8⫹ T cell homeostasis (25). IL-15 regulates both proliferation and Ab production by activated B cells (26), while mast cells proliferate in response to IL-15 and produce functionally active IL-4 (27). Monocytes, macrophages, and dendritic cells are the major source of IL-15 production. IL-15 exerts an autocrine activity on monocytes by inducing IL-8 and MCP-1 production (28) and monocyte differentiation to dendritic cells (29). The IL-15 gene has been cloned from a number of mammalian species and recently in birds (30). We identified the first fish IL-15 sequence in rainbow trout (AJ555868) in 2003 by surveying SSH sequences prepared from trout tissues challenged by the pathogenic bacterium Aeromonas salmonicida (31). Several fish IL-15 sequences, as well as IL-15-like molecules, have also been described by in silico cloning (32–34), but so far no function has been assigned to any fish IL-15. This article reports the molecular and functional characterization of IL-15 in rainbow trout (On-

ACGGG ATG TTGCC CTATT C TC CAAC

Application

Recombinant cloning RT-PCR 3⬘-racing

C CTAC AC AGGG CTGTC ACT AC GATGT C TTCTC AA

CTCCA

5⬘-racing Screening genomic library Splicing variant study Splicing variant study Real-time PCR (with IL-15R) Real-time PCR (with IL-15EF1 or IL-15F) Real-time PCR (with IL-15EF1) Real-time PCR (with IL-15R) Real-time PCR (with IL-15R) Real-time PCR Real-time PCR Real-time PCR

AACAT ATAG Real-time PCR T

corhynchus mykiss), and analysis of a ␭ genomic clone containing the IL-15 gene which revealed that IL-15 is linked closely to a BCL10 gene, which is essential for NF-␬B activation by TCRs and BCRs (35), and an INPP4B gene which encodes the inositol polyphosphate 4-phosphatase type II, one of the enzymes involved in phosphatidylinositol signaling pathways (36). Trout IL-15 expression is modulated by recombinant trout IFN-␥ (rtIFN-␥) but not rtIL-1␤. We next produced and purified bioactive rtIL-15 from Escherichia coli and found that rtIL-15 can induce IFN-␥ expression in leukocytes from spleen but not from head kidney of normal fish.

Materials and Methods Cell lines and cell culture Four rainbow trout cell lines were used: the mononuclear cell line RTS-11 (37), the gonad cell line RTG-2 (38), the liver cell line RTL (39), and CL-6 (an epithelial liver cell line, a gift from Dr. A. Benmansour, l’Institut National de la Recherche Agronomique, Jovyen en Josas, France). All trout cells were grown in L-15 medium supplemented with 30% FCS for RTS-11 cells and 10% FCS for all the other cell lines.

Bacterial challenge, SSH library construction, and analysis A virulent strain of Aeromonas salmonicida sp. salmonicida, MT 423 (40), was used to challenge female rainbow trout of ⬃300 g. The bacterial preparation, challenge procedure, and tissue sampling were described previously (31). Gill tissues were collected and used for total RNA preparation. The construction of SSH libraries and random sequence analysis of SSH clones were as described before (31). The nucleotide sequences generated were assembled and analyzed with the AlignIR program (LI-COR). A sequence similarity search was performed using FASTA (41) and basic local alignment search tool (42, 43). Direct comparison between two sequences was performed using the GAP program (44). Multiple sequence alignments were generated using CLUSTALW (version 1.7) (45). Phylogenetic trees were created from multiple alignments by the neighbor-joining method (46) within the PIE program at the Human Genome Mapping Project Resource Centre (www.hgmp. mrc.ac.uk/Registered/Webapp/pie/) and were bootstrapped 1000 times.

Trout IL-15 cDNA cloning and analysis Analysis of SSH clones from a gill SSH library revealed a short sequence with homology to mammalian IL-15. 3⬘- and 5⬘-RACE was conducted using total RNA from RTS-11 cells and an RNA ligase-mediated rapid


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FIGURE 1. Nucleotide and deduced amino acid sequences of the compiled trout IL-15 cDNA (Accession No. AJ555868). The upstream ATG codons, the start and stop codons, the two potential N-glycosylation sites (NXS/T), a potential NLS (HRKKH) and the poly(A) signal (AATAAA) are in bold and underlined. The predicted signal peptide is highlighted and in bold italics. The primer binding sites for 3⬘-RACE (F1 and F2) and 5⬘-RACE (R1 and R2) are underlined and directions marked by arrows.

amplification of 5⬘-end (RLM-RACE) (GeneRacer kit; Invitrogen Life Technologies) as described previously (47). 3⬘-RACE using forward primers F1 and F2 (Table I, Fig. 1) resulted in a 0.8-kb product that when sequenced contained the C terminus and the 3⬘-UTR. Additional primers R1 and R2 (Table I, Fig. 1) were designed in the 3⬘-UTR and used for 5⬘-RACE. The resulting 0.9-kb product was cloned and sequenced and found to contain the 5⬘-UTR and complete coding region.

Isolation and sequencing of a genomic clone of IL-15 A rainbow trout genomic library constructed with ␭ GEM-11 was PCR screened with IL-15-specific primer F3 and ER1 (Table I) as described previously (48). A positive ␭ clone was plaque purified and its DNA was prepared using a Wizard ␭ Preps DNA purification system (Promega). After an initial restriction enzyme (all from Promega) analysis with BamHI, EcoRI, SacI, XbaI, and XhoI, the SacI digestion was subcloned in pGEM 7zf(⫹). A subclone with an 8-kb insert containing most of the genomic sequence and 3⬘ flanking region was identified by PCR screening. This clone was fully sequenced from both directions using a transposonbased sequencing primer binding site insertion kit (GeneJumper kit; Invitrogen Life Technologies). The remaining 5⬘-end was sequenced by primer walking. The cDNA sequence was aligned to the genomic sequence and the intron/exon boundary was identified by using the SIM4 program (www.hgmp.mrc.ac.uk/Registered/Webapp/sim4/). The promoter sequence was analyzed by using the program SIGNAL SCAN (49). To get more insight into the regulation of gene expression of IL-15, a comparative promoter analysis was conducted using a web program Possum (http://zlab.bu.edu/⬃mfrith/possum/) which predicts cis elements in DNA sequences using the standard method of position-specific scoring matrices. The first 1000-bp sequences (including the first exon) of IL-15 genes were extracted from BX957281 (zebrafish) and reference assembly NT_016354.18 (human) and analyzed for potential transcription factor binding sites.

Expression of IL-15 transcript in vivo Initial analysis of IL-15 transcript expression by RT-PCR (50) revealed that IL-15 expression is detectable in all the cDNA samples from tissues and cell lines examined. To further quantify the expression of IL-15, six healthy rainbow trout (average, 130 g/fish) were killed and tissues (gills, skin, muscle, liver, spleen, head kidney, intestine, and brain) were removed for RNA preparation using RNA STAT-60 (AMS Biotechnology). The total RNA concentrations were quantified using a Nanodrop Spectrophotometer (NanoDrop Technologies). Thirty micrograms of RNA was reverse transcribed into cDNA using BioScript (Bioline) in 30-␮l reactions. The resulting cDNA was diluted in 500 ␮l of TE buffer (pH 8.0). Four microliters of the resultant cDNA was used for quantitative PCR (Q-PCR) detection of expression of IL-15 (EF1⫹R) and elongation factor-1␣ (EF-1␣ F⫹R) using primers as described in Table I. The Q-PCR was detected with CYBR green (Invitrogen Life Technologies) using DNA Engine Opticon-2 (MJ Research) and quantified by comparing with a serial dilution of standard samples of cloned and linearized plasmid DNA in the same run. The relative expression level in different tissues was obtained by first normalizing the expression level relative to that of EF-1␣ and then comparing against the average expression level of IL-15 in liver (defined as 1) where the expression level was low but detectable in all fish.

rIFN-␥ and IL-1␤ stimulation The preparations of trout rIFN-␥ and rIL-1␤ were as described by Zou et al. (51) and Hong et al. (52), respectively. RTG-2 and RTS-11 cells were prepared 2 days before stimulation by seeding at 2.5 ⫻ 106 cells (RTG-2) or 4 ⫻ 106 cells (RTS-11) in 25-cm2 cell culture flasks (Nunc) in 5 ml of complete medium as described above. The cells were stimulated with different amounts of the trout rIFN-␥ and rIL-1␤, for 4 h with RTS-11 cells and for 5 h with RTG-2 cells, and total RNA was then prepared and cDNA synthesized as described above. The expressions of the IL-15 gene, as well

1478

RAINBOW TROUT IL-15

FIGURE 2. Multiple alignment of the predicted trout IL-15 translation with known IL-15s based on the gene organization of trout, human, mouse, and rat. Gaps introduced to increase identity are shown by dashes. The exons are numbered according to the trout sequence. The highlighted features include the four conserved cysteine residues forming two potential disulphide bonds, the IL-15R␣-binding site, the NLS and the 4-aa repeat/insert (LERL/LERI) in the C terminus of trout IL-15.


1479

FIGURE 3. Phylogenetic tree of the IL-15/IL-2 family. The tree was constructed using the neighbor-joining method, based on the alignment of 15 known IL-15s and IL-15-like molecules, and 23 known IL-2s, performed with Clustal W software using their entire amino acid sequences. Numbers on the branches are bootstrap values of 1000 replicates supporting a given partitioning. The accession number for each molecule analyzed is on the right. The amino acid sequence identity/similarity to trout IL-15, on the furthest right, was calculated using the GAP program.

as the inflammatory cytokine IL-1␤ (isoform I), and the anti-inflammatory cytokine IL-11 (53), were detected by real-time PCR and normalized to the expression of EF-1␣. The relative expression of each gene is arbitrary fold change relative to the control.

Northern blot analysis Total RNA was isolated from the RTS-11 cell line using TRIzol reagent (Invitrogen Life Technologies) according to the manufacturer’s instructions. Northern blot analysis was performed as described previously (54). In each experiment, 10 –25 ␮g of total RNA/lane was transferred from a 1.1% formaldehyde-MOPS agarose gel to nylon membranes by capillary action and hybridized overnight at 65°C with a 32P-labeled 298-bp cDNA probe purified from a trout IL-15 PCR fragment amplified using primers EF1and ER1 (Table I). Following stringent washing, membranes were put into an x-ray cassette with intensifying screens and film (Kodak) and exposed for 2 days.

Alternative splicing variants (SVs) of the trout IL-15 gene and their expression Primers were designed across all the exons of the trout IL-15 gene to amplify PCR products from cDNAs from eight trout tissues (spleen, head kidney, liver, intestine, gills, skin, muscle, and brain) and four trout cell lines (RTG-2, RTS-11, CL-6, and RTL), and the melting curve of the amplified products were examined. The PCR products, amplified using primers IL-15svF and IL-15svR that are located at the first and final exons, respectively, from cell lines CL-6, RTS-11 and liver tissue, have multiple peaks in melting curve analysis. The products were cloned to pGEM-T easy vector (Promega) and four groups of clones with distinct sizes of inserts were sequenced and allowed three alternative SVs to be identified. To study the expression of these SVs, specific primers were synthesized (see Table I) for Q-PCR analysis with the tissue samples described above. IL-15 F⫹R can only amplify the authentic IL-15 transcript as IL-15F crosses intron II and IL-15R crosses intron IV. IL-15sv1R crossing exons

4 and 2, in combination with IL-15EF1 (located in exon 2), can only amplify transcripts without exon 3 (SV-1). IL-15sv2F spanning exon 2⬘ and exon 3, and IL-15sv3F spanning exons 1 and 3, when used in combination with IL-15R, can only amplify SV-2 and SV-3, respectively. A common reference was constructed by mixing equal amounts of plasmids of each variant and was used for Q-PCR quantification.

rtIL-15 production and purification The cDNA sequence encoding the mature trout IL-15 was amplified from a cDNA clone using Pfu DNA polymerase (Promega) with primers IL15RF: (g ctg tac aaa GCT GAA ACA CAC GGG ATG) and IL15RR (cg tgt aca TTA ACT GAC AGT TTG CCC TAT TC, the BsrGI site was underlined). The resulting amplification was digested with BsrGI and ligated to pET160/DEST (Invitrogen Life Technologies) that was also digested with BsrGI and dephosphorylated. Plasmid DNA was prepared and sequenced to confirm that the construct produced a 162-aa fusion recombinant protein of 18.7 kDa with a N-terminal 6xHis tag for purification and a Lumio tag for specific detection of recombinant protein using Lumio Green Detection kit (Invitrogen Life Technologies). A plasmid pET160/IL15 was subsequently transformed into BL21 star (DE3; Invitrogen Life Technologies) and the expression of rtIL-15 was induced by 1 mM isopropyl ␤-D-thiogalactoside (IPTG) for 4 h at 37°C with vigorous shaking. The rtIL-15 was purified under denaturing conditions using a His-Selected Nickel Affinity Gel (Sigma-Aldrich), refolded on the column (55), and eluted with PBS containing 200 mM imidazol. The buffer of the eluted rtIL-15 was changed to PBS containing 40% glycerol using an iCON concentrator (9 kDa cut-off; Pierce), sterilized by 0.22-␮m filtration and stored at ⫺20°C ready for use. The induction of expression and purification of the rtIL-15 were analyzed using a 4 –12% SDS-PAGE gel and detected first with the Lumio reagent (Invitrogen Life Technologies) and subsequently by staining with Brilliant Blue R (Sigma-Aldrich).

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RAINBOW TROUT IL-15

FIGURE 4. Schematic diagram showing the gene organization between known IL-15 genes and trout IL-15. The coding regions are indicated by solid boxes and noncoding region by hashed boxes. The intron size is shown above the intron in kilobases and is not proportional to the figure intron size. A LSP is encoded across the first three encoding exons in all species. The data are extracted from the database with Ensembl Gene ID: EN SG00000164136 (human), ENSMUSG00000031712 (mouse), ENSRNOG00000003439 (rat), and ENSGAL G00000009870 (chicken), and Accession Nos. DQ0593 88 (Tetraodon nigroviridis), DQ020256 (Takifugu rubripes), and DQ099498 (zebrafish).

Preparation of spleen and head kidney leukocytes and stimulation with rtIL-15 Spleen and head kidney were aseptically removed from freshly killed trout (300 –500 g) and placed in universal tubes containing incomplete medium (L-15 plus 100 U/ml penicillin plus 100 ␮g/ml streptomycin plus 0.5% FBS). The tissues were gently pushed through sterile 100-␮m mesh screens and the screens were rinsed with incomplete medium. The resulting cell suspension was layered on 51% Percoll (Pharmacia) and centrifuged at 400 ⫻ g for 20 min. Cells at the medium/51% Percoll interface were collected, washed twice in incomplete medium by centrifugation at 200 ⫻ g for 5 min, and the leukocytes were then resuspended in complete medium (the same as incomplete medium except with 10% FCS) at 0.5–1 ⫻ 106 cells/ml. Six milliliters of cells were seeded into 25-cm2 cell culture flasks, and incubated at 20°C. For rtIL-15 stimulation, rtIL-15 was added to freshly prepared or in vitro (overnight) cultured leukocytes and incubated at 20°C for different times before total RNA preparation and cDNA synthesis as described above. The expression of IFN-␥, IL-1␤, and IL-11 were detected by real-time PCR as described above.

Statistical analysis Real-time quantitative PCR measurements were analyzed using the nonparametric Mann-Whitney tests within the SPSS package 15.0, with p ⬍ 0.05 between treatment groups and control groups considered significant.

Results Cloning and sequence analysis of trout IL-15 A SSH clone from a gill SSH library, prepared from bacterial challenged rainbow trout, was identified with homology to mammalian IL-15. Based on this clone, primers were designed (Table I) and two overlapping PCR products were obtained using 3⬘- and 5⬘-RACE, that contained the full-length trout IL-15 cDNA (Fig. 1). The transcript contains 1,207 bp with an open reading frame of 193 aa. The 202-bp 5⬘-UTR contains seven out-of-frame AUG initiation codons while the 423-bp 3⬘-UTR contains a polyadenylation signal 16 bp upstream from the poly(A) tail. The translated molecule is 22,715 Da with a theoretical isoelectric point of 6.19, and contains two potential N-linked glycosylation sites (Fig. 1). An

unusually long LSP of 65 aa was predicted (56). Thus, the predicted mature trout IL-15 generated following cleavage of the signal peptide is 128 aa with a calculated molecular mass of 15,050 Da and a theoretical isoelectric point of 5.16. In view of the gene organization of trout IL-15 (described later), a multiple amino acid alignment with other known IL-15 molecules, including mammalian, bird, and other recently identified fish IL-15, was constructed according to the encoding exons (Fig. 2). It showed that good conservation was seen in the mature peptide, including the four cysteine residues forming the two potential disulphide bonds and one of the two IL-15R␣ binding sites identified in the human IL-15 molecule (57) which were conserved in all the IL-15 molecules. Although the mammalian IL-15 molecules are highly homologous, there are apparently several unique features in the other vertebrate IL-15 molecules. These include the even longer signal peptide in fish and birds compared with the unusually long 48-aa mammalian signal peptide, as a result of the longer coding region in the first coding exon. There is also a 4-aa repeat/ insert (e.g., LERL/LERI in the trout sequence) in the C terminus of the fish IL-15s (Fig. 2). Although there is no nuclear localization signal (NLS) found in other IL-15 molecules, a “pat4” type NLS (58) (HRKK or RKKH, Figs. 1 and 2) was identified in the trout IL-15 molecule by PSORT (59). A Hopp-Woods hydrophilicity analysis (60) predicted that the pat4 NLS in the trout IL-15 sequence is in the hydrophilic region and should be accessible (61). The trout IL-15 sequence shows low homology to other IL-15 molecules with an overall amino acid sequence identity/similarity of 24%/ 44% (Fig. 3). It also shows a similar homology to the avian IL-2s and some of the mammalian IL-2s. To define the relationship of trout IL-15 to other IL-15/IL-2 molecules, a phylogenetic tree was constructed by the neighbor-joining method and bootstrapped 1000 times. This tree (Fig. 3) clearly grouped the trout and chicken IL-15s with the mammalian IL-15s and separated from all of the IL-2 molecules.


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Table II. Number of base pairs in the exons and introns of the IL-15 genes in trout, human, mouse, rat, and chicken Intron/Exon Number Rat

Human

1 2 3 4 5b 6 7 8 9

1 2 3 4b 5 6 7 8

Mouse

Chicken

1 2 1 3 2 b 4 3b 5 4 6 5 7 6 8 7 Total size (bp)

Exon Size Trout

1 2b 3 4 5 6

Rat

Human

Mouse

Intron Size Chicken

Trout

Rat

Human

Mouse

Chicken

165a 24,034 122a 624a 271a 4,388 18,961 22,684 171a 122a 122a 46a 25,366 63,058 34,470 95,03 99a/12 87a/12 66a/63 202a/60 1,108 992 1,127 17,944 87a/12 98 98 98 110 95 490 1,357 1,051 1,260 85 85 85 82 118 4,720 5,931 5,628 414 45 45 45 42 48 2,283 1,862 2,960 1,613 138 138 138 144 153 2,616 2,753 2,444 584 108/285a 108/613a 108/288a 120/139a 105/403a 1,316 1,944 1,253 813 1,186 65,005 94,914 70,364 31,128

Trout

Intron Phase

968 3,295 151 268 207

0 I 0 0 0

4,889

a

Numbers refer to the noncoding region of the exons. All numbers without superscripts in the exon size panel refer to the sizes of the coding regions. Numbers in bold and underlined indicate the second coding exon. b The phase I intron in the coding region. Note that all the introns in the coding region are phase 0 except the second intron within the coding region of the gene that is phase I.

Gene organization of the trout IL-15 and gene synteny analysis A 10,042-bp sequence containing the complete gene/genomic sequence that is identical with the trout IL-15 cDNA sequence and its 5⬘- and 3⬘-flanking region was fully sequenced and deposited with the EMBL, GenBank, and DDBJ nucleotide sequence databases under Accession No. AJ628345. The trout IL-15 gene is 6,073-bp long and contains six exons and five introns (Fig. 4). All the intron/exon boundaries in the trout sequence conformed strictly to the known GT/AG donor/acceptor site rule and all the introns are phase 0 except intron II that is phase I. The trout IL-15 sequence was compared with the four known IL-15 genes from human (Ensembl Gene ID: ENSG00000164136), mouse (Ensembl Gene ID: ENSMUSG00000031712), rat (Ensembl Gene ID: ENSRNOG00000003439), and chicken (Ensembl Gene ID: ENSGALG00000009870) (Table II and Fig. 4). The IL-15 genes from other species are longer, with ⬎32 kb compared with only 6 kb in trout, and have one to three more exons and large introns in the 5⬘-UTR. Nevertheless, all the IL-15 genes transcribed to mRNAs with comparable size, although different SVs do exist. The IL-15 proteins are encoded by the final six exons in all the known species. Despite the differences of the length of signal peptide in different species, the signal peptide is encoded by the first two and part of the third encoding exons (Fig. 4). Trout IL-15 is closely linked to a BCL10 gene and an IPPN4B gene; 295.4 kb downstream of the human IL-15 gene on chromosome 4 is the INPP4B gene that encodes the inositol polyphosphate 4-phosphatase type II. INPP4B is also linked to IL-15 with a distance of 374.8 kb and 222.6 kb in mouse and rat, respectively. In trout, just 1468 bp downstream of the IL-15 gene is the last exon of the INPP4B gene. This INPP4B genomic sequence (from 7542 to 9292 in AJ628345) contains the last exon and part of the last intron and matches with two trout expressed sequence tags (ESTs; Accession Nos. BX087818 and BX087819) that come from the two ends of the same EST clone (tcav0004.g.08) with sequence identity ⬎99.5%. The EST BX087818 completely overlaps the genomic sequence and contains the 3⬘-end sequence with a poly(A) signal and a poly(A) tail. The EST BX087819 overlaps the genomic sequence with 237 bp and contains 288 bp from the other exons. The translation of this EST showed 77%/85% sequence identities/similarities to mammalian INPP4Bs and lower homology (65%/73%, identity/similarity) to mammalian INPP4As. In the 1468-bp region between the trout IL-15 and INPP4B genes, another gene called BCL10 was identified. This region matches two trout ESTs (CA369968 and BX862375) with 96% nucleotide sequence identity, and with a continuous open read-

ing frame of 203 aa. The trout BCL10 translation showed highest homology to mouse BCL10 with 46.2%/57.9% identity/similarity, and lower homology to BCL10 molecules from rat and humans. There are two poly(A) signals, 240 and 282 bp, respectively, downstream of the TGA stop codon of the BCL10 translation. The human BCL10 gene is located on chromosome 1 and was originally found to be involved in the t(1;14)(p22; q32) MALT lymphoma translocation (62). Human BCL10 has a four exon/three intron structure (Ensembl Gene ID ENSG00000142867). There is no report that BCL10 is linked to the IL-15 gene in other species. The relationship between the IL-15 gene and INPP4B gene is shown schematically in Fig. 5. Thus, the trout IL-15 gene is closely linked to the INPP4B gene compared its mammalian counterparts. In addition, the trout IL-15 gene is also linked with a BCL10 gene that is not linked to mammalian IL-15. These linkages between IL-15 and INPP4B genes, and IL-15 and BCL10 genes have also been confirmed by PCR using primers located in the coding regions of these genes, and genomic DNA from 15 fish (data not shown). Analysis of trout IL-15 promoter A sequence fragment containing 750-bp 5⬘-flanking region, the first exon and part of the first intron was analyzed for transcription

FIGURE 5. Schematic diagram showing the relationship between IL-15 and INPP4B genes. The gene size and distance between genes are shown in kilobase and are not in proportion. The transcriptional direction is indicated by arrow direction. The source data are described as in Fig. 4.

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RAINBOW TROUT IL-15

FIGURE 6. Analysis of the promoters of IL-15 genes. A, Sequence analysis of the promoter of the trout IL-15 gene; 750 bp of the 5⬘-flanking region, the first exon (shaded), and part of the first intron of the trout IL-15 gene are shown. The transcription factor binding sites ␣-IFN.2 (AARK GA), AP-1 (TGANTMA), AP-3 (TGTGGW WW), c-Myb (CMGTTR), c-Myc (TCTCT TA), ␥-IRE (CWKKANNY), GCF (SCGSS SC), GM-CSF (CATTW), GR (TGTCCT), NF-IL-16 (TKNNGNAAK), NF-␬B (GGGR NTYYC) are highlighted and underlined except the GM-CSF site within the ␥-IRE site that is also italicized. Arrow indicates the translation start. B, Comparative analysis of potential transcription factor binding sites among IL-15 genes from trout, humans, and zebrafish. The potential binding sites for NF-␬B (^), ␥-IRE (f), SP1 (u), and NFIL-6 (䡺) in the first 1000 bp of sequence (including the first exon) of trout, zebrafish (BX957281), and human (NT_016354.18) are shown.

factor binding sites using a SIGNAL SCAN program. The important transcription factor binding sites identified include 3 AP-1 and 1 AP-3 sites, 1 ␣-IFN.2 site (63), 13 IFN-␥-responsive element (␥-IRE) sites (64), 10 GM-CSF sites (a repeated sequence CATT(A/T) required for GM-CSF promoter activity) (65), 2 GCF (66), glucocorticoid receptor (GR), NF-IL-6 and NF-␬B binding sites (67), 1 c-Myc and 1 Myb (68) binding site (Fig. 6A). Like mammalian IL-15 genes (69, 70), the trout IL-15 gene also has no conventional TATA box upstream of the transcription start site. Comparative promoter analysis showed that most of these potential transcription factor binding sites found in the trout IL-15 gene are also present in other species (Fig. 6B). Multiple potential binding sites for ␥-IRE, SP1, and NF-IL-6 were present in the promoters of all three species analyzed. There were two potential NF-␬B binding sites present in the 5⬘-flanking region of the human IL-15 gene, but only one in trout and none in zebrafish using the first 1000 bp (including the first exon) of the 5⬘-region (Fig. 6B).

FIGURE 7. In vivo expression of the IL-15 transcript. The relative expression level of IL-15 in different tissues were normalized first with the expression level of EF-1␣, and then compared with the average expression level in liver where the expression level was low and defined as 1. The results represent the mean ⫹ SEM of six fish.


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FIGURE 8. Up-regulation of the IL-15 expression by rtIFN-␥ and IL-1␤ in RTG-2 (A and B) and RTS-11 (C and D) cell lines. Different concentrations of rtIFN-␥ and IL-1␤ were added to RTG-2 cells for 5 h and to RTS-11 cells for 4 h and total RNA prepared. The relative expression of the trout IL-15 gene, as well as IL-1␤ and IL-11, were detected by real-time PCR and normalized to the expression of EF-1␣. The mean of three replicates is shown and bars indicate the SEMs. ⴱ, the difference between the treatment group and the control group is significant (p ⬍ 0.05).

Further analysis revealed a single potential NF-␬B binding site 3.5 kb upstream of the first exon of the zebrafish IL-15 gene (data not shown). Multiple sites for AP-1, GR, and GCF are also present in the human and zebrafish promoters. Trout IL-15 is broadly expressed in tissues and cell lines RT-PCR was initially used to examine the expression of the trout IL-15 gene in tissues and cell lines and revealed that IL-15 expression was detectable in all the tissues and cell lines examined. The expression of IL-15 in vivo was further quantified by real-time PCR using tissue samples from six individual fish. The expression of the housekeeping gene EF-1␣ among all the tissues was almost constant in the same tissue among individuals but its expression

was lower in muscle and brain as indicted by a higher crossing point (ct) (of 13–14) relative to the other tissues (count of ⬃11). The expression of IL-15 was detectable by Q-PCR in all the tissues examined. IL-15 was most highly expressed in spleen, with considerable levels in gills, head kidney, and intestine (Fig. 7). Because the expression of IL-15 was normalized to the expression of EF-1␣ and EF-1␣ expression in muscle and brain was low, this explains why the relative expression of IL-15 in muscle and brain appears high. Trout IL-15 is up-regulated by rIFN-␥ but not by IL-1␤ Multiple ␥-IRE and NF-␬B sites present in the promoter region of the trout IL-15 gene imply that trout IL-15 could be modulated by IFN-␥ and IL-1␤, as IL-1␤ can activate NF-␬B in trout cells (47).

FIGURE 9. Alternative SVs of the trout IL-15 gene and their expression. A, Schematic diagram of the trout IL-15 gene producing an authentic IL-15 mRNA and three alternative SVs. B, The expression of transcripts of trout IL-15 gene. The expression of each transcript was normalized against the expression level of EF-1␣. C, The relative abundance of each alternative SV was compared with the authentic IL-15 mRNA. The mean of three individual fish is shown in B and C, and bars indicate the SEMs. D, Northern blot analysis of IL-15 transcript in three independent samples of RTS-11 cells.

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FIGURE 10. SDS-PAGE analysis of rtIL-15 expressed in E. coli BL21 Star (DE3) detected by Lumio green reagent (Invitrogen Life Technologies) (A) and stained with Brilliant Blue R (Sigma-Aldrich) (B). 1, sample without IPTG induction; 2, sample induced with 1 mM IPTG for 4 h; 3, purified expression product; and 4, BenchMark Fluorescent Protein standard (Invitrogen Life Technologies).

To test this hypothesis, rtIFN-␥ and IL-1␤ (isoform I) were added to RTS-11 cells for 4 h and to RTG-2 cells for 5 h and the expression of IL-15, as well as IL-1␤ and IL-11, were detected by real-time PCR. rtIFN-␥ increased the expression of IL-15 ⫻ 5- to 6-fold at all the doses (1–100 ng/ml) tested in both cell lines (Fig. 8, A and C). No difference in the expression of IL-1␤ was detectable in either cell line stimulated with IFN-␥ (Fig. 8, A and C); however, IFN-␥ decreased the expression of IL-11 in the monocyte/macrophage RTS-11 cells (Fig. 8C). rtIL-1␤ only had a limited effect on the expression of IL-15, with a 2-fold increase of IL-15 expression seen in RTG-2 cells, and ⬍2-fold increase in RTS-11 cells at all the tested concentrations (Fig. 8, B and D). The effectiveness of IL-1␤ stimulation was confirmed by the up-regulation of the expression of IL-1␤ itself and IL-11 seen in both cell lines (Fig. 8, B and D). Thus, IFN-␥ selectively up-regulates the expression of IL-15 but IL-1␤ only has a limited role on IL-15 expression in RTG-2 and RTS-11 cells. Isolated head kidney leukocytes showed the same pattern of IFN-␥ and IL-1␤ action on the expression of the IL-15 transcript (data not shown). Alternative SVs of the trout IL-15 gene and their expression Although there is always a major band amplified by PCR from the cDNAs from tissues and cell lines examined, melting curve analysis revealed that additional peaks were amplified from some cDNAs, e.g., cDNAs from CL-6, RTS-11, and liver. Sequence analysis of the PCR products using primer pair IL-15svR and R revealed three alternative SV existed for the trout IL-15 gene (Fig. 9A). IL-15 SV-1 has the third exon spliced out and thus the transcript is 118 bp shorter. IL-15 SV-2 has an additional exon located in the second intron (E2⬘). This exon has 94% identity to the cloned genomic sequence reported in this article and also present in other unrelated fish genes with similar identities (e.g., AY78595, Salmo salar zonadhesin-like gene). This sequence may represent an old retroposition. The IL-15SV-2 cDNA sequence only shares 97% identity to the IL-15 cDNA when the additional E2⬘ is removed. Thus, the transcript of IL-15SV-2 may be from a pseudogene of IL-15. IL-15 SV-3 has the second exon spliced out but the fifth intron retained. All the alternative splicings result in a premature stop of translation of the putative IL-15 protein. The authentic IL-15 mRNA is expressed at a higher level relative to the alternative SVs in all eight tissues examined, although the three alternative SVs are all detectable in the tissue samples

RAINBOW TROUT IL-15

FIGURE 11. rtIL-15 selectively up-regulates IFN-␥ in splenic leukocytes. Leukocytes prepared from spleen and head kidney from the same fish were stimulated with 0 or 200 ng/ml rtIL-15 for 24 h. The relative expression of the trout IFN-␥ gene, as well as the IL-1␤ gene, was detected by real-time PCR and normalized to the expression of EF-1␣. The fold increase is expressed by comparison to the controls in each individual fish. The mean of three individual fish is shown and bars indicate the SEMs. Similar experiments repeated more than three times showed the same pattern of rtIL-15 action. ⴱ, The increase of IFN-␥ expression is significant (p ⬍ 0.05).

(Fig. 9B). SV-1 and SV-2 are expressed at 1.8 ⫾ 0.3, and 4.8 ⫾ 1.6(mean ⫾ SD)‰ of the level of the authentic IL-15 mRNA (Fig. 9C). SV-3 is expressed at a relatively higher level and closer to the authentic IL-15 mRNA expression level. Northern blot analysis revealed that only one major band was detectable using RNA prepared from RTS-11 cells (Fig. 9D). Production and purification of rtIL-15 To study the function of trout IL-15, a fusion recombinant protein, containing a N-terminal 6 his-tag for purification, a Lumio tag for detection, and the predicted mature trout IL-15 in the C-terminal was produced in E. coli as an inclusion body. The rtIL-15 was highly induced by IPTG induction, purified under denaturing conditions and then renatured on the column (Fig. 10). rtIL-15 selectively up-regulates the expression of IFN-␥ in leukocytes from spleen but not head kidney Mammalian IL-15 stimulates macrophages and NK cells to produce IFN-␥ (10). rtIL-15 (200 ng/ml) was added to the macrophage cell line RTS-11, and head kidney leukocytes enriched for macrophages, but no effect on the IFN-␥ transcript was detectable (data not shown). However, addition of rtIL-15 to leukocytes prepared from spleen consistently showed an increase of IFN-␥ expression (Fig. 11). In contrast, rtIL-15 had no

FIGURE 12. Time course of rtIL-15 up-regulated expression of IFN-␥ in splenic leukocytes. Overnight cultured splenic leukocytes were stimulated with rtIL-15 (200 ng/ml) for different times and total RNA was prepared. The relative expression of IFN-␥ was detected by real-time RT-PCR and normalized to the expression of EF-1␣. The mean of three fish is shown. Bars indicate the SEMs. Similar experiments were repeated in eight individual fish with similar results. ⴱ, The increase of IFN-␥ expression is significant (p ⬍ 0.05).


FIGURE 13. Dose-dependent up-regulation of IFN-␥ expression by rtIL-15. Splenic leukocytes were incubated with different amounts of rtIL-15 for 6 h and total RNA was prepared for real-time RT-PCR analysis as described in Fig. 11. The relative expression of three individual fish is shown.

significant effect on the expression of IL-1␤ in spleen or head kidney leukocytes. The up-regulation of IFN-␥ by rtIL-15 was studied in detail in splenic leukocytes. Overnight cultures of splenic leukocytes were stimulated with 200 ng/ml rtIL-15 and the stimulation was terminated at 1, 3, 6, 9, 12, and 24 h after addition of rtIL-15. The up-regulation of IFN-␥ expression by rtIL-15 was seen after 1 h stimulation with rtIL-15 and reached a plateau at 3 h that lasted for at least a day after stimulation (Fig. 12). The modulation of IFN-␥ expression varied among individuals, as shown in a dose response study where cells from different fish were stimulated individually with rtIL-15 for 6 h (Fig. 13). Nevertheless, splenic leukocytes from all the fish (17 fish) tested showed an increase of IFN-␥ expression after rtIL-15 stimulation. The increase of IFN-␥ expression was also confirmed at the protein level by Western blotting using a mAb to rtIFN-␥, where IFN-␥ was only detected in rtIL-15-stimulated splenic leukocytes (our unpublished data). rtIL-15 was also found to up-regulate the expression of the IL-12␤ chain in splenic leukocytes (our unpublished data).

Discussion In this study, we have first characterized the IL-15 gene in rainbow trout (O. mykiss). We then investigated the expression and modulation of IL-15 transcript expression in different tissues and cell lines. We have also identified three alternative SVs of the trout IL-15 gene and studied their expression. Finally, we have produced the bioactive rtIL-15 and show it can up-regulate the expression of IFN-␥ in splenic leukocytes. Although the translation of the cloned sequence only showed low homology (23⬃30% sequence identities) with the IL-15s from other species, six lines of evidence presented here suggest that the gene cloned in this study is a fish homolog of IL-15. First, There are seven out-of frame AUG initiation codons in the 202 bp 5⬘UTR of the trout sequence (Fig. 1). IL-15s in mammals contain multiple upstream AUGs (5 in mouse, 12 in human) that have been proposed as negative regulators of gene expression (13). The chicken IL-15 reported also contains at least two upstream AUGs (30). Second, the trout translation has a predicted extraordinarily LSP of 65 aa. The IL-15s in mammals and chicken also have a LSP of 48 aa (in mammals) or 66 aa (in chicken). In contrast to most signal peptides that are encoded by a single exon, the signal peptides of IL-15 in trout, chicken, and mammals are all encoded across three exons (Figs. 2 and 4). Third, there are well-conserved residues of defined function in the multiple alignment of the IL-15 sequences (Fig. 2), including four highly conserved cysteine residues, which have been postulated in mammals to be important in the formation of correctly folded molecules (13), and one of the two IL-15R␣ binding sites identified in the human

1485 IL-15 molecule (57). Fourth, all the IL-15 proteins, including the trout IL-15, are encoded by six exons, and all the introns in the coding region are phase 0 except the second one that is phase I (Table II). Fifth, the trout IL-15 has a broad tissue distribution of expression and could be detected in all the tissues and cell lines examined. The mammalian and chicken IL-15 molecules also show a wide distribution of expression in tissues and cell types (2, 30). Lastly, an unrooted phylogenetic tree (Fig. 3) clearly grouped the trout IL-15 with the IL-15 sequences from other species and separate from the IL-2 molecules. The unique characteristics of trout IL-15 include a 4-aa repeat (LERL/LERI) encoded by the final exon (Fig. 2) and a putative NLS in the fifth exon. The (LERL/LERI) repeat might be a fish IL-15-specific feature as this repeat can be identified in all the fish IL-15 molecules (Fig. 2). Classical NLS sequences incorporate regions enriched in basic aa and generally conform to one of three motifs (58). The pat4-type NLS consists of a continuous stretch of four basic amino acids (K or R) or three basic amino acids associated with histidine (H) or proline (P). Although there is no NLS found in other IL-15 molecules, a pat4-type NLS (HRKK or RKKH, Figs. 1 and 2) was identified in the trout IL-15 molecule by PSORT. A protein containing NLS sequences may remain cytoplasmic, especially if the NLS sequence is blocked or buried within the protein (61). Hydrophobicity analysis revealed that this NLS is in a hydrophilic region and could be accessed and functional. NLSs are short polypeptide sequences that affect nuclear targeting of the proteins carrying them, and have been identified in cytokines such as IFN-␥ (71), IL-5 (72), and other STAT using ligands or receptors (73). It was proposed that putative NLS-bearing ligands and/or their receptors might play a direct role in nuclear translocation of transcription factors such as STATs that do not contain a polycationic NLS (73). The NLS of IFN-␥ is involved in complexing STAT1␣ with nuclear importin ␣ for nuclear translocation of STAT1␣, and is crucial for the biological properties of IFN-␥ (73). It is known that STAT3/5/6 are involved in IL-15 signaling in mammals (74) and thus the NLS in trout IL-15 might function to assist the translocation of the STAT3/5/6 transcription factors. Multiple SVs have been reported in both human and mouse resulting in two isoforms of IL-15 molecules being translated (75). The LSP isoform of human IL-15 is secreted through the ER/Golgi pathway (76) and is regulated by a different intracellular trafficking pathway (15). In contrast, the SSP isoform is not secreted but rather is stored intracellularly and appears in the nuclear and cytoplasmic components. It is speculated that the intracellular form may serve as a reservoir of IL-15 protein, being released on damage, or that it plays other novel as yet undefined roles within the cells that produce it (16). The NLS in the trout IL-15 might also function to translocate itself to the nucleus after being translated. RLM-RACE uses T4 RNA ligase to join cellular RNA with a defined ribo-oligonucleotide that marks and preserves the termini of cellular RNA molecules and can be used to analyze the 5⬘ terminus of a mRNA (77). Using the same batch of cDNA prepared in this article, we have successfully obtained the full-length 5⬘ end of the trout IL-1␤ and defined the transcription start site (47). The cloned IL-15 cDNA sequence matched perfectly with the genomic sequence, and Northern blot analysis revealed that a 1.2-kb transcript was detectable (Fig. 9D). This suggests that the cDNA sequence reported in this article is the full-length trout IL-15. Comparison of the cDNA sequence with the genomic sequence obtained, revealed that the trout IL-15 has a unique six exon/five intron organization, compared with IL-15s from other vertebrates which have nine (rat), eight (human and mouse), or seven (chicken) exons. The fish IL-15 gene is the smallest among all the

1486 known IL-15s, being 6 kb compared with 32 to 97 kb in other species. However, the large sizes of IL-15 genes in other species are mainly contributed by additional exons and large introns in the 5⬘-UTR. Although the sizes of all the common introns in the coding region are generally larger in other species, all the IL-15s have a transcript with comparable size (Table II). Multiple transcripts exist for mammalian IL-15 genes. The human IL-15 gene is transcribed into at least four transcripts that encode two functional IL-15 proteins with a LSP and SSP, respectively. Three alternative transcripts have also been identified for the trout IL-15 gene but all the alternative transcripts result in a premature stop of translation, and thus no functional protein is expected. Although expressed at a low level, all the alternative transcripts are detectable in the eight tissues examined, and perhaps represent components of complex regulatory mechanisms to generate the functional protein. PI3Ks catalyze the phosphorylation of the 3-OH position of the inositol head groups of the PI lipids, namely phosphatidylinositol (PtdIns), PtdIns(4)P, and PtdIns(4,5)P2, resulting in the formation of PtdIns(3)P, PtdIns(3,4)P2, and PtdIns(3,4,5)P3, respectively. PtdIns(3,4)P2 and PtdIns(3,4,5)P3 are generally absent from resting cells but increase intracellularly upon stimulation via a variety of receptors and function as second messengers (78). The PI3K pathway has been well-documented in the signaling of IL-15 (79). Two isoforms of the INPP4 gene, namely INPP4A and B, which encode the inositol polyphosphate 4-phosphatase with similar enzymatic properties, have been isolated in mammals. This enzyme removes the phosphate group at position 4 of the inositol ring, preferentially from PtdIns(3,4)P2 (36). INPP4A has been reported as a regulator of cell proliferation, linked to its ability to dephosphorylate PtdIns(3,4)P2 (80). Despite the difference of intergene distance, the linkage between IL-15 and INPP4B genes and transcription direction is well-conserved. This conservation suggests that these two genes might be linked functionally, with INPP4B acting as a regulator of the PI3K pathway during IL-15 signaling. The human BCL10 gene resides on chromosome 1, has a four exon/three intron organization (Ensembl Transcript ID ENST00000271015), and is not linked to IL-15 or INPP4B. BCL10 genes from other species also have multiple exons and no clear physical linkage to IL-15 or INPP4B, as analyzed using the Ensembl database. The trout translation is from a continuous sequence without introns. Although there is a good conservation in the CARD domain with a sequence identity/similarity of ⬃58%/ 75%, the overall sequence homology is low, especially in the C terminus, with two deletions apparent. The ESTs only have 96% sequence identities with the genomic sequence and might be transcribed from a related gene at a different locus or from a BCL homolog with multiple exon/intron structure. Thus, it is possible that the trout BCL10 homolog that reside between the IL-15 and INPP4B genes could be due to a retroposition event and may not be functional. Computer analysis identified a number of potential sites for transcriptional activators (AP-1 and 3, anti-IFN.2, ␥-IRE, GM-CSF, NF-IL-6, NF-␬B, c-Myc, and Myb), as well as for repressors (GCF and GR), in the 5⬘-flanking region and 5⬘ end of the trout IL-15 gene. Indeed, ␥-IRE may be involved in the up-regulation of IL-15 by IFN-␥ as seen in this study. Comparative promoter analysis revealed that most of the sites are also present in the promoters of human and zebrafish IL-15 genes, suggesting that these transcription factors and pathways leading to IL-15 expression may be conserved across vertebrates. GCF is a transcriptional regulator that was found to repress transcription of the epidermal growth factor receptor and other genes and binds to GC-rich sequences (SCGSSC) (66). Two GCF-binding sites just upstream of the tran-

RAINBOW TROUT IL-15 scription start site of the trout IL-15 gene imply that GCF could be a repressor of IL-15 transcription in fish also. Many different cell types express IL-15, although mammalian IL-15 is probably mainly produced by activated macrophages (6, 12). Although IL-15 is constitutively expressed in cells of the monocyte/macrophage lineage, the expression of IL-15 mRNA has been shown to be up-regulated by LPS, poly I:C, as well as bacterial (12, 81, 82) and viral (83) infection. The present results show clearly that IFN-␥ is an important immune regulator of IL-15 in fish. rtIFN-␥ selectively increased IL-15 expression but had little effect on other cytokines such as IL-1␤ and IL-11. In contrast, rtIL-1␤ had little effect on IL-15 expression while increasing expression of itself dramatically. Mammalian IL-15 mRNA is broadly expressed, however, it is extremely difficult to detect IL-15 in cell culture supernatants. The production of bioactive membrane bound or secreted IL-15 in mammals appears to be tightly controlled (81, 84). Enhanced IL-15 protein production results from either increased transcription and/or stability of IL-15 mRNA, posttranslational modification, or altered cellular trafficking, and enhanced release of already preformed intracellular IL-15 protein. The specific factors capable of inducing biologically meaningful IL-15 protein production have still to be identified in fish, as IL-15 is mainly regulated in a posttranscriptional manner in mammals. Nevertheless, in this study, we produced the rIL-15 protein and studied its biological activity. Mammalian IL-15 is a pleiotropic cytokine that has a broad spectrum of biologic activities on different types of cells, including NK, NKT, T, B cells, macrophage/dendritic cells, neutrophils, and mast cells, and other nonimmune cells (10). Thus, it was expected that rtIL-15 would affect most mixed leukocyte primary cultures. However, unexpectedly the rtIL-15 preferentially stimulated splenic leukocytes from healthy fish, where it induced a large increase in IFN-␥ expression, with little, if any, effect on IL-1␤ expression. This effect was quite long-lived and was still apparent 24 h poststimulation. Although the exact cell types being affected have still to be determined, it is clear that once produced IL-15 will have a profound affect on the ability of the fish immune system to activate antimicrobial defenses and genes induced themselves by IFN-␥.

Disclosures The authors have no financial conflict of interest.

References 1. Lodolce, J. P., P. R. Burkett, R. M. Koka, D. L. Boone, and A. Ma. 2002. Regulation of lymphoid homeostasis by interleukin-15. Cytokine Growth Factor Rev. 13: 429 – 439. 2. Grabstein, K. H., J. Eisenman, K. Shanebeck, C. Rauch, S. Srinivasan, V. Fung, C. Beers, J. Richardson, M. A. Schoenborn, M. Ahdieh, et al. 1994. Cloning of a T cell growth factor that interacts with the ␤ chain of the interleukin-2 receptor. Science 264: 965–968. 3. Giri, J. G., M. Ahdieh, J. Eisenman, K. Shanebeck, K. Grabstein, S. Kumaki, A. Namen, L. S. Park, D. Cosman, and D. Anderson. 1994. Utilization of the ␤ and ␥ chains of the IL-2 receptor by the novel cytokine IL-15. EMBO J. 13: 2822–2830. 4. Giri, J. G., D. M. Anderson, S. Kumaki, L. S. Park, K. H. Grabstein, and D. Cosman. 1995. IL-15, a novel T cell growth factor that shares activities and receptor components with IL-2. J. Leukocyte Biol. 57: 763–766. 5. Girard, D., M. E. Paquet, R. Paquin, and A. D. Beaulieu. 1996. Differential effects of interleukin-15 (IL-15) and IL-2 on human neutrophils: modulation of phagocytosis, cytoskeleton rearrangement, gene expression, and apoptosis by IL-15. Blood 88: 3176 –3184. 6. Carson, W. E., M. E. Ross, R. A. Baiocchi, M. J. Marien, N. Boiani, K. Grabstein, and M. A. Caligiuri. 1995. Endogenous production of interleukin 15 by activated human monocytes is critical for optimal production of interferon-␥ by natural killer cells in vitro. J. Clin. Invest. 96: 2578 –2582. 7. Musso, T., L. Calosso, M. Zucca, M. Millesimo, D. Ravarino, M. Giovarelli, F. Malavasi, A. N. Ponzi, R. Paus, and S. Bulfone-Paus. 1999. Human monocytes constitutively express membrane-bound, biologically active, and interferon-␥upregulated interleukin-15. Blood 93: 3531–3539.

The Journal of Immunology 8. Blauvelt, A., H. Asada, V. Klaus-Kovtun, D. J. Altman, D. R. Lucey, and S. I. Katz. 1996. Interleukin-15 mRNA is expressed by human keratinocytes Langerhans cells, and blood-derived dendritic cells and is downregulated by ultraviolet B radiation. J. Invest. Dermatol. 106: 1047–1052. 9. Lee, Y. B., J. Satoh, D. G. Walker, and S. U. Kim. 1996. Interleukin-15 gene expression in human astrocytes and microglia in culture. Neuroreport 7: 1062–1066. 10. Shurin, M. R., I. L. Tourkova, H. Hackstein, and G. V. Shurin. 2003. Interleukin-15 and 21, Chapter 18. Elsevier Science, London. 11. Meazza, R., A. Gaggero, F. Neglia, S. Basso, S. Sforzini, R. Pereno, B. Azzarone, and S. Ferrini. 1997. Expression of two interleukin-15 mRNA isoforms in human tumors does not correlate with secretion: role of different signal peptides. Eur. J. Immunol. 27: 1049 –1054. 12. Doherty, T. M., R. A. Seder, and A. Sher. 1996. Induction and regulation of IL-15 expression in murine macrophages. J. Immunol. 156: 735–741. 13. Bamford, R. N., A. P. Battiata, and T. A. Waldmann. 1996. IL-15: the role of translational regulation in their expression. J. Leukocyte Biol. 59: 476 – 480. 14. Bamford, R. N., A. P. DeFilippis, N. Azimi, G. Kurys, and T. A. Waldmann. 1998. The 5⬘ untranslated region, signal peptide, and the coding sequence of the carboxyl terminus of IL-15 participate in its multifaceted translational control. J. Immunol. 160: 4418 – 4426. 15. Kurys, G., Y. Tagaya, R. Bamford, J. A. Hanover, and T. A. Waldmann. 2000. The long signal peptide isoform and its alternative processing direct the intracellular trafficking of interleukin-15. J. Biol. Chem. 275: 30653–30659. 16. Tagaya, Y., G. Kurys, T. A. Thies, J. M. Losi, N. Azimi, J. A. Hanover, R. N. Bamford, and T. A. Waldmann. 1997. Generation of secretable and nonsecretable interleukin 15 isoforms through alternate usage of signal peptides. Proc. Natl. Acad. Sci. USA 94: 14444 –14449. 17. Anderson, D. M., S. Kumaki, M. Ahdieh, J. Bertles, M. Tometsko, A. Loomis, J. Giri, N. G. Copeland, D. J. Gilbert, N. A. Jenkins, et al. 1995. Functional characterization of the human interleukin-15 receptor ␣ chain and close linkage of IL15RA and IL2RA genes. J. Biol. Chem. 270: 29862–29869. 18. Dubois, S., J. Mariner, T. A. Waldmann, and Y. Tagaya. 2002. IL-15R␣ recycles and presents IL-15 in trans to neighboring cells. Immunity 17: 537–547. 19. Schluns, K. S., K. D. Klonowski, and L. Lefrancois. 2004. Transregulation of memory CD8 T-cell proliferation by IL-15R␣⫹ bone marrow-derived cells. Blood 103: 988 –994. 20. Giron-Michel, J., A. Caignard, M. Fogli, D. Brouty-Boye, D. Briard, M. van Dijk, R. Meazza, S. Ferrini, C. Lebousse-Kerdiles, D. Clay, et al. 2003. Differential STAT3, STAT5, and NF-␬B activation in human hematopoietic progenitors by endogenous interleukin-15: implications in the expression of functional molecules. Blood 102: 109 –117. 21. McDonald, P. P., M. P. Russo, S. Ferrini, and M. A. Cassatella. 1998. Interleukin-15 (IL-15) induces NF-␬B activation and IL-8 production in human neutrophils. Blood 92: 4828 – 4835. 22. DiSanto, J. P. 1997. Cytokines: shared receptors, distinct functions. Curr. Biol. 7: R424 –R426. 23. Gosselin, J., A. TomoIu, R. C. Gallo, and L. Flamand. 1999. Interleukin-15 as an activator of natural killer cell-mediated antiviral response. Blood 94: 4210 – 4219. 24. Cooper, M. A., J. E. Bush, T. A. Fehniger, J. B. VanDeusen, R. E. Waite, Y. Liu, H. L. Aguila, and M. A. Caligiuri. 2002. In vivo evidence for a dependence on interleukin 15 for survival of natural killer cells. Blood 100: 3633–3638. 25. Mastroianni, C. M., G. d’Ettorre, G. Forcina, and V. Vullo. 2004. Teaching tired T cells to fight HIV: time to test IL-15 for immunotherapy? Trends Immunol. 25: 121–125. 26. Armitage, R. J., B. M. Macduff, J. Eisenman, R. Paxton, and K. H. Grabstein. 1995. IL-15 has stimulatory activity for the induction of B cell proliferation and differentiation. J. Immunol. 154: 483– 490. 27. Masuda, A., T. Matsuguchi, K. Yamaki, T. Hayakawa, M. Kubo, W. J. LaRochelle, and Y. Yoshikai. 2000. Interleukin-15 induces rapid tyrosine phosphorylation of STAT6 and the expression of interleukin-4 in mouse mast cells. J. Biol. Chem. 275: 29331–29337. 28. Badolato, R., A. N. Ponzi, M. Millesimo, L. D. Notarangelo, and T. Musso. 1997. Interleukin-15 (IL-15) induces IL-8 and monocyte chemotactic protein 1 production in human monocytes. Blood 90: 2804 –2809. 29. Mohamadzadeh, M., F. Berard, G. Essert, C. Chalouni, B. Pulendran, J. Davoust, G. Bridges, A. K. Palucka, and J. Banchereau. 2001. Interleukin 15 skews monocyte differentiation into dendritic cells with features of Langerhans cells. J. Exp. Med. 194: 1013–1020. 30. Lillehoj, H. S., W. Min, K. D. Choi, U. S. Babu, J. Burnside, T. Miyamoto, B. M. Rosenthal, and E. P. Lillehoj. 2001. Molecular, cellular, and functional characterization of chicken cytokines homologous to mammalian IL-15 and IL-2. Vet. Immunol. Immunopathol. 82: 229 –244. 31. Wang, T., and C. J. Secombes. 2003. Complete sequencing and expression of three complement components, C1r, C4 and C1 inhibitor, of the classical activation pathway of the complement system in rainbow trout Oncorhynchus mykiss. Immunogenetics 55: 615– 628. 32. Bei, J. X., H. Suetake, K. Araki, K. Kikuchi, Y. Yoshiura, H. R. Lin, and Y. Suzuki. 2006. Two interleukin (IL)-15 homologues in fish from two distinct origins. Mol. Immunol. 43: 860 – 869. 33. Gunimaladevi, I., R. Savan, K. Sato, R. Yamaguchi, and M. Sakai. 2007. Characterization of an interleukin-15 like (IL-15L) gene from zebrafish (Danio rerio). Fish Shellfish Immunol. 22: 351–362. 34. Fang, W., L. X. Xiang, J. Z. Shao, Y. Wen, and S. Y. Chen. 2006. Identification and characterization of an interleukin-15 homologue from Tetraodon nigroviridis. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 143: 335–343.

1487 35. Zhou, H., I. Wertz, K. O’Rourke, M. Ultsch, S. Seshagiri, M. Eby, W. Xiao, and V. M. Dixit. 2004. Bcl10 activates the NF-␬B pathway through ubiquitination of NEMO. Nature 427: 167–171. 36. Norris, F. A., R. C. Atkins, and P. W. Majerus. 1997. The cDNA cloning and characterization of inositol polyphosphate 4-phosphatase type. II. Evidence for conserved alternative splicing in the 4-phosphatase family. J. Biol. Chem. 272: 23859 –23864. 37. Ganassin, R. C., and N. C. Bols. 1998. Development of a monocyte/macrophagelike cell line, RTS11, from rainbow trout spleen. Fish Shellfish Immunol. 8: 457– 476. 38. Wolf, K., and M. C. Quimby. 1962. Established eurythermic lines of fish cells in vitro. Science 135: 1065–1066. 39. Lee, L. E., J. H. Clemons, D. G. Bechtel, S. J. Caldwell, K. B. Han, M. Pasitschniak-Arts, D. D. Mosser, and N. C. Bols. 1993. Development and characterization of a rainbow trout liver cell line expressing cytochrome P450dependent monooxygenase activity. Cell. Biol. Toxicol. 9: 279 –294. 40. Barnes, A. C., M. T. Horne, and A. E. Ellis. 1996. Effect of iron on expression of superoxide dismutase by Aeromonas salmonicida and associated resistance to superoxide anion. FEMS Microb. Lett. 142: 19 –26. 41. Pearson, W. R., and D. I. Lipman. 1988. Improved tools for biological sequence comparison. Proc. Natl. Acad. Sci. USA 85: 2444 –2448. 42. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215: 403– 410. 43. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389 –3402. 44. Needleman, S. B., and C. D. Wunsch. 1970. A general method applicable to the search for similarities in the amino acid sequence of two proteins. J. Mol. Biol. 48: 443– 453. 45. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: 4673– 4680. 46. Saitou, N., and M. Nei. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406 – 425. 47. Wang, T., J. Zou, C. Cunningham, and C. J. Secombes. 2002. Cloning and functional characterisation of the interleukin-1␤1 promoter of rainbow trout (Oncorhynchus mykiss). Biochim. Biophys. Acta 1575: 108 –116. 48. Wang, T., N. Johnson, J. Zou, N. Bols, and C. J. Secombes. 2004. Sequencing and expression of the second allele of the interleukin-1␤1 gene in rainbow trout (Oncorhynchus mykiss): identification of a novel SINE in the third intron. Fish Shellfish Immunol. 16: 335–358. 49. Prestridge, D. S. 1991. SIGNAL SCAN: a computer program that scans DNA sequences for eukaryotic transcriptional elements. Comput. Appl. Biosci. 7: 203–206. 50. Wang, T., M. Ward, P. Grabowski, and C. J. Secombes. 2001. Molecular cloning, gene organization and expression of rainbow trout (Oncorhynchus mykiss) inducible nitric oxide synthase (iNOS) gene. Biochem. J. 358: 747–755. 51. Zou, J., A. Carrington, B. Collet, J. M. Dijkstra, Y. Yoshiura, N. Bols, and C. Secombes. 2005. Identification and bioactivities of IFN-␥ in rainbow trout Oncorhynchus mykiss: the first Th1-type cytokine characterized functionally in fish. J. Immunol. 175: 2484 –2494. 52. Hong, S., J. Zou, M. Crampe, S. Peddie, G. Scapigliati, N. Bols, C. Cunningham, and C. J. Secombes. 2001. The production and bioactivity of rainbow trout (Oncorhynchus mykiss) recombinant IL-1␤. Vet. Immunol. Immunopathol. 81: 1–14. 53. Wang, T., J. W. Holland, N. Bols, and C. J. Secombes. 2005. Cloning and expression of the first nonmammalian interleukin-11 gene in rainbow trout Oncorhynchus mykiss. FEBS J. 272: 1136 –1147. 54. Zou, J., J. Holland, O. Pleguezuelos, C. Cunningham, and C. J. Secombes. 2000. Factors influencing the expression of interleukin-1␤ in cultured rainbow trout (Oncorhynchus mykiss) leucocytes. Dev. Comp. Immunol. 24: 575–582. 55. Zhu, X. Q., S. X. Li, H. J. He, and Q. S. Yuan. 2005. On-column refolding of an insoluble His6-tagged recombinant EC-SOD over expressed in Escherichia coli. Acta Biochim. Biophys. Sin. 37: 265–269. 56. Nielsen, H., J. Engelbrecht, S. Brunak, and G. von Heijne. 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10: 1– 6. 57. Bernard, J., C. Harb, E. Mortier, A. Quemener, R. H. Meloen, C. VermotDesroches, J. Wijdeness, P. van Dijken, J. Grotzinger, J. W. Slootstra, et al. 2004. Identification of an interleukin-15␣ receptor-binding site on human interleukin-15. J. Biol. Chem. 279: 24313–24322. 58. Nakai, K., and M. Kanehisa. 1992. A knowledge base for predicting protein localization sites in eukaryotic cells. Genomics 14: 897–911. 59. Horton, P., and K. Nakai. 1997. Better prediction of protein cellular localization sites with the k nearest neighbors classifier. Proc. Int. Conf. Intell. Syst. Mol. Biol. 5: 147–152. 60. Hopp, T. P., and K. R. Woods. 1981. Prediction of protein antigenic determinants from amino-acid-sequences. Proc. Natl. Acad. Sci. USA 78: 3824 –3828. 61. Roberts, B. L., W. D. Richardson, and A. E. Smith. 1987. The effect of protein context on nuclear location signal function. Cell 50: 465– 475. 62. Willis, T. G., D. M. Jadayel, M. Q. Du, H. Peng, A. R. Perry, M. Abdul-Rauf, H. Price, L. Karran, O. Majekodunmi, I. Wlodarska, et al. 1999. Bcl10 is involved in t(1;14)(p22;q32) of MALT B cell lymphoma and mutated in multiple tumor types. Cell 96: 35– 45. 63. Fujita, T., S. Ohno, H. Yasumitsu, and T. Taniguchi. 1985. Delimitation and properties of DNA sequences required for the regulated expression of human interferon-␤ gene. Cell 41: 489 – 496.

1488 64. Yang, Z., M. Sugawara, P. D. Ponath, L. Wessendorf, J. Banerji, Y. Li, and J. L. Strominger. 1990. Interferon ␥ response region in the promoter of the human DPA gene. Proc. Natl. Acad. Sci. USA 87: 9226 –9230. 65. Nimer, S., J. Fraser, J. Richards, M. Lynch, and J. Gasson. 1990. The repeated sequence CATT(A/T) is required for granulocyte-macrophage colony-stimulating factor promoter activity. Mol. Cell Biol. 10: 6084 – 6088. 66. Beguinot, L., H. Yamazaki, I. Pastan, and A. C. Johnson. 1995. Biochemical characterization of human GCF transcription factor in tumor cells. Cell Growth Differ. 6: 699 –706. 67. De Bosscher, K., Berghe, W. V., and Haegeman, G. 2003. The interplay between the glucocorticoid receptor and nuclear factor-␬B or activator protein-1: molecular mechanisms for gene repression. Endocr. Rev. 24: 488 –522. 68. Howe, K. M., C. F. Reakes, and R. J. Watson. 1990. Characterization of the sequence-specific interaction of mouse c-myb protein with DNA. EMBO J. 9: 161–169. 69. Azimi, N., K. Brown, R. N. Bamford, Y. Tagaya, U. Siebenlist, and T. A. Waldmann. 1998. Human T cell lymphotropic virus type I Tax protein transactivates interleukin 15 gene transcription through an NF-␬B site. Proc. Natl. Acad. Sci. USA 95: 2452–2457. 70. Azimi, N., K. M. Shiramizu, Y. Tagaya, J. Mariner, and T. A. Waldmann. 2000. Viral activation of interleukin-15 (IL-15): characterization of a virus-inducible element in the IL-15 promoter region. J. Virol. 74: 7338 –7348. 71. Ahmed, C. M., M. A. Burkhart, M. G. Mujtaba, P. S. Subramaniam, and H. M. Johnson. 2003. The role of IFN␥ nuclear localization sequence in intracellular function. J. Cell. Sci. 116: 3089 –3098. 72. Jans, D. A., L. J. Briggs, S. E. Gustin, P. Jans, S. Ford, and I. G. Young. 1997. A functional bipartite nuclear localisation signal in the cytokine interleukin-5. FEBS Lett. 406: 315–320. 73. Johnson, H. M., B. A. Torres, M. M. Green, B. E. Szente, K. I. Siler, J. Larkin, 3rd, and P. S. Subramaniam. 1998. Cytokine-receptor complexes as chaperones for nuclear translocation of signal transducers. Biochem. Biophys. Res. Commun. 244: 607– 614. 74. Bulanova, E., V. Budagian, Z. Orinska, H. Krause, R. Paus, and S. Bulfone-Paus. 2003. Mast cells express novel functional IL-15 receptor ␣ isoforms. J. Immunol. 170: 5045–5055.

RAINBOW TROUT IL-15 75. Nishimura, H., J. Washizu, N. Nakamura, A. Enomoto, and Y. Yoshikai. 1998. Translational efficiency is up-regulated by alternative exon in murine IL-15 mRNA. J. Immunol. 160: 936 –942. 76. Gaggero, A., B. Azzarone, C. Andrei, Z. Mishal, R. Meazza, E. Zappia, A. Rubartelli, and S. Ferrini. 1999. Differential intracellular trafficking, secretion and endosomal localization of two IL-15 isoforms. Eur. J. Immunol. 29: 1265–1274. 77. Volloch, V., B. Schweitzer, and S. Rits. 1994. Ligation-mediated amplification of RNA from murine erythroid cells reveals a novel class of ␤ globin mRNA with an extended 5⬘-untranslated region. Nucleic Acids Res. 22: 2507–2511. 78. Curnock, A. P., M. K. Logan, and S. G. Ward. 2002. Chemokine signalling: pivoting around multiple phosphoinositide 3-kinases. Immunology 105: 125–136. 79. Yano, S., M. Komine, M. Fujimoto, H. Okochi, and K. Tamaki. 2003. Interleukin 15 induces the signals of epidermal proliferation through ERK and PI 3-kinase in a human epidermal keratinocyte cell line, HaCaT. Biochem. Biophys. Res. Commun. 301: 841– 847. 80. Vyas, P., F. A. Norris, R. Joseph, P. W. Majerus, and S. H. Orkin. 2000. Inositol polyphosphate 4-phosphatase type I regulates cell growth downstream of transcription factor GATA-1. Proc. Natl. Acad. Sci. USA 97: 13696 –13701. 81. Maeurer, M., B. Seliger, P. Trinder, J. Gerdes, and U. Seitzer. 1999. Interleukin-15 in mycobacterial infection of antigen-presenting cells. Scand. J. Immunol. 50: 280 –288. 82. Mattei, F., G. Schiavoni, F. Belardelli, and D. F. Tough. 2001. IL-15 is expressed by dendritic cells in response to type I IFN, double-stranded RNA, or lipopolysaccharide and promotes dendritic cell activation. J. Immunol. 167: 1179 –1187. 83. Gil, S., M. Spagnuolo-Weaver, A. Canals, N. Sepulveda, J. Oliveira, A. Aleixo, G. Allan, A. Leitao, and C. L. Martins. 2003. Expression at mRNA level of cytokines and A238L gene in porcine blood-derived macrophages infected in vitro with African swine fever virus (ASFV) isolates of different virulence. Arch. Virol. 148: 2077–2097. 84. Neely, G. G., S. M. Robbins, E. K. Amankwah, S. Epelman, H. Wong, J. C. Spurrell, K. K. Jandu, W. Zhu, D. K. Fogg, C. B. Brown, and C. H. Mody. 2001. Lipopolysaccharide-stimulated or granulocyte-macrophage colony-stimulating factor-stimulated monocytes rapidly express biologically active IL-15 on their cell surface independent of new protein synthesis. J. Immunol. 167: 5011–5017.