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The Journal of Immunology, 2002, 168: 3329-3340.
Copyright © 2002 by The American Association of Immunologists

The First Cytokine Sequence Within Cartilaginous Fish: IL-1{beta} in the Small Spotted Catshark (Scyliorhinus canicula)1

Steve Bird*, Tiehui Wang*, Jun Zou*, Charlie Cunningham{dagger} and Chris J. Secombes2,*

* Department of Zoology, University of Aberdeen, Aberdeen, United Kingdom; and {dagger} Sars International Center for Marine Molecular Biology, Bergen, Norway


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cartilaginous fish are considered the most primitive living jawed vertebrates with a complex immune system typical of all jawed vertebrates. Cytokine homologs are found within jawless and bony fish, although no cytokine or cytokine receptor genes have been sequenced in cartilaginous fish. In this study the complete coding sequence of the small spotted catshark (Scyliorhinus canicula) IL-1{beta} gene is presented that contains a short 5' untranslated region (54 bp), a 903-bp open reading frame, a 379-bp 3' untranslated region, a polyadenylation signal, and eight mRNA instability motifs. The predicted translation (301 amino acids) has highest identity to trout IL-1{beta} (31.7%), with greatest homology within the putative 12 {beta}-sheets. The IL-1 family signature is also present, but there is no apparent signal peptide. As with other nonmammalian IL-1{beta} sequences, the IL-1-converting enzyme cut site is absent. Expression of the IL-1{beta} transcript is detectable by RT-PCR in the spleen and testes, induced in vivo with LPS. Furthermore, a 7-fold increase of transcript levels in splenocytes incubated for 5 h with LPS was seen. The genomic organization comprises six exons and five introns with highest homology seen in exons encoding the largest amount of secondary structure per amino acid. Southern blot analysis suggests at least two copies of the IL-1{beta} gene or genes related to the 3' end of the IL-1{beta} sequence are present in the catshark. The cloning of IL-1{beta} in S. canicula, the first cytokine sequenced within cartilaginous fish, verifies previous bioactivity evidence for the presence of inflammatory cytokines.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cartilaginous fish (sharks and rays) are generally considered the most primitive living jawed vertebrates (gnathostomes) and possess a skeleton made from calcified cartilage. They made their first appearance during the Ordovician period and most major lineages arose in the subsequent Carboniferous (362–290 million year (Myr))3 period. Their diversity was seen to decline during the Permian (290–250 Myr) and Triassic (250–208 Myr) periods. Fossil evidence reveals that ~45 families of sharks and their relatives were known to have lived during the late Palaeozoic and Mesozoic eras. Members of the class Chondrichthyes still in existence have been shown to be derived from Palaeozoic ancestors and can be divided into two subclasses: the elasmobranchs (sharks and rays) and the holocephalans (chimaeras). Modern elasmobranchs (the Neoselachii) radiated particularly during the Jurassic and Cretaceous periods to give ~760 species present today. It has been suggested that this radiation was in response to the appearance of abundant sources of food, which was brought about by the radiation of the actinopterygian bony fish (1).

Present-day elasmobranchs possess a complex immune system typical of all jawed vertebrates (2), and the continued study of this group will give a valuable insight into the origins of specific immunity. Elasmobranchs also possess a variety of nonspecific mechanisms, which act as a first line of defense. The first antimicrobial agent to be identified was squalamine (3), a steroid with broad-spectrum antibiotic activity. It may serve as a systemic antimicrobial agent in elasmobranchs and has been shown to act against bacteria such as Escherichia coli, strains of Staphylococcus and Streptococcus, and fungi (4). Lysozyme activity has also been detected in shark leukocyte lysates (5).

The initiation and regulation of these elasmobranch immune responses is likely to require intercellular signaling molecules, cytokines, as seen in mammals. Recently a cytokine was sequenced in Agnathans and identified as a member of the chemokine family, having significant homology with IL-8 (6). Many cytokine genes have also been isolated from different species of bony fish over the last few years. For example, in rainbow trout, two IL-1{beta} genes (7, 8), three isoforms of TGF-{beta} (9, 10, 11), two TNF-{alpha} genes (12), and several chemokines and their receptors (13, 14) have been cloned and sequenced. In cartilaginous fish no cytokine or cytokine receptor genes have been cloned or sequenced to date. The discovery of cytokine homologs within both jawless fish and bony fish suggests the existence of these molecules in cartilaginous fish.

IL-1{beta} is a primary regulator of inflammatory and immune responses in mammals (15). Until recently this important cytokine had only been sequenced in mammals. Over the last few years bony fish and amphibian IL-1{beta} has been extensively studied, and a number of regions conserved throughout vertebrates were identified (16, 17). Previous work has provided evidence for the presence of inflammatory cytokines in elasmobranchs (18, 19). In this study, primers designed to these conserved regions of IL-1{beta} were used for the homology cloning of the small spotted catshark (Scyliorhinus canicula) IL-1{beta} gene, the first cytokine gene sequenced in a cartilaginous fish.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Catshark

Sexually mature male S. canicula weighing 900-1000 g were obtained from the North Sea around Aberdeen, U.K. Adults were maintained in recirculating artificial saltwater (Peacock, Glasgow, U.K.) at 9–10°C and were fed squid three times weekly.

cDNA production

Splenocytes were isolated from individual animals and suspended in L-15 medium (Life Technologies, Paisley, U.K.) adjusted to cartilaginous fish osmolarity (20, 21), containing 0.2 M NaCl (Sigma-Aldrich, St. Louis, MO), 0.35 M urea (Sigma-Aldrich), 10% FCS (Life Technologies), 100 µg/ml streptomycin (Life Technologies) and 100 U/ml penicillin (Life Technologies). The cells were incubated at 25°C and used unstimulated or after stimulation for 4 h with LPS from E. coli, serotype 0127:B8 (Sigma-Aldrich) at 10 µg/ml, a concentration known to be stimulatory for IL-1{beta} expression in trout leukocytes (7). Total RNA was then isolated using RNAzol (Biogenesis, Poole, U.K.) following the manufacturer’s instructions and poly(A) mRNA was purified using an Oligotex mRNA kit (Qiagen, Valencia, CA). cDNA was synthesized from 2 µg of mRNA by Moloney murine leukemia virus reverse transcriptase (Life Technologies) at 42°C for 50 min with oligo(dT) primer (Life Technologies) and used as a template for PCR.

Cloning and sequencing

Initially, PCR was performed using the cDNA prepared above, with primers universal-F17 and -R (Table IGo; Fig. 1Go) that were designed to conserved regions of all known IL-1{beta} sequences, to allow isolation of the S. canicula IL-1{beta} gene. Obtained PCR products were ligated into the pGEM-T Easy vector (Promega, Madison, WI). Following transfection into competent E. coli cells, recombinants were identified through red-white color selection when grown on MacConkey agar (Sigma-Aldrich). Plasmid DNA from at least three independent clones was recovered using an alkaline lysis-based method (22) and sequenced using an ABI 377 Automated Sequencer (Applied Biosystems, Foster City, CA). Sequences generated were analyzed for similarity with other known sequences using the FASTA (23) and basic local alignment search tool (24) suite of programs. Direct comparison between cDNA sequences was performed using the GAP program (25) within the Wisconsin Genetics Computer Group Sequence Analysis Software Package (version 10.0) and multiple sequence alignments generated using CLUSTAL W (version 1.74) (26).


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  		Table I. Oligonucleotide primers used to amplify the S. canicula IL-1{beta} cDNA and IL-1{beta} gene

 


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  		FIGURE 1. Position of primers used to obtain the S. canicula IL-1{beta} cDNA and the products obtained (P1–P4).

 
Having isolated a partial S. canicula IL-1{beta} sequence the 5' and 3' ends were obtained by RACE-PCR, using gene-specific primers (Table IGo). In 3' RACE-PCR, cDNA was transcribed from poly(A) mRNA using an oligo(dT) adapter primer. PCR was performed with a S. canicula IL-1{beta}-specific forward primer (dogfish-F1) and the adapter primer and further seminested with a second S. canicula IL-1{beta}-specific forward primer (dogfish-F2) and the adapter primer (Fig. 1Go). In 5' RACE-PCR, cDNA was transcribed from poly(A) mRNA using an oligo-dT primer (Life Technologies), treated with E. coli RNase H (Promega), purified using a PCR Purification kit (Qiagen), and tailed with poly(C) at the 5' end with terminal deoxynucleotidyl transferase (TdT; Promega). PCR was performed initially with a S. canicula IL-1{beta}-specific reverse primer (dogfish-R1) and the oligo(dG) primer and further seminested with a second S. canicula IL-1{beta}-specific reverse primer (dogfish-R2) and the oligo(dG) (Fig. 1Go). S. canicula IL-1{beta}-specific reverse primers (dogfish-R3 and -R4) were used to fully sequence the amplified product.

Finally, phylogenetic analysis was performed on the full-length amino acid sequences of the known IL-1{beta} molecules using the neighbor-joining method (27) and was drawn using the TreeView program v1.6.1 (28), and confidence limits were added (29). Predicted amino acid sequence was analyzed by SignalP v1.1 (30) and the hydrophobicity profile was determined using Kyte and Doolittle plots (31).

Expression studies

RT-PCR with primers IL-1{beta}SC-F and IL-1{beta}SC-R (Table IGo) was performed with cDNA from a range of tissues (peripheral blood leukocytes, brain, gill, heart, kidney, Leydig organ, liver, muscle, spleen, testes) isolated 24 h following peritoneal injection of S. canicula with 200 µg of LPS. cDNA was also produced from tissues taken from noninjected S. canicula and used as a control. After isolation of total RNA (as described above), 5 µg was reverse-transcribed to cDNA. Primers for S. canicula {beta}-actin (GenBank accession no. AJ312004) were used as a positive control for RT-PCR (Table IGo), because the gene is expressed constitutively in the tissues examined. Tissue expression was studied in three noninjected and injected S. canicula.

Northern blot analysis was also performed on total RNA from splenocytes stimulated in vitro at 25°C for 0, 1, 2, 3, 4, and 5 h with 10 µg/ml LPS (as described above). Unstimulated cells were also cultured for a variety of the above time periods. A probe was prepared from the PCR product using primers IL-1{beta}SC-F and IL-1{beta}SC-R. The probe was then labeled with 32P and used for hybridization to RNA (20 µg per lane) at 65°C for 4 h. A 32P-labeled S. canicula {beta}-actin probe was used as a control to ensure that any increases seen were not a result of a general increase in mRNA sample loading. Following stringent washing, the membrane was put into an x-ray cassette and film (Kodak, Rochester, NY) was exposed for 4 h. The relative levels of mRNA were quantified by densitometric scanning of exposed film, using a UVP imaging system (Ultraviolet Products, Cambridge, U.K.) and UVP Gelworks ID advanced software v3.01 (Ultraviolet Products), and expressed relative to the transcript in unstimulated cells. Northern blotting was performed from three S. canicula.

Genomic DNA extraction

Liver tissue was removed from an individual catshark and left to dry for 10 min in a vacuum concentrator. After drying, an extraction mix was added to each tube, consisting of 500 µl 10x TNE buffer (0.01 M Trizan base-HCl (pH 8; Sigma Aldrich), 0.1 M NaCl, 0.5 M EDTA (pH 8; Sigma Aldrich)), 50 µl of 1 M Trizan base-HCl (pH 8; Sigma Aldrich) and 20 µl 25% SDS (Sigma Aldrich), followed by 20 µl of proteinase K (20 mg/ml; Promega). The tubes were then incubated at 55°C for 3 h, with vortexing every 30 min. A layer of oil formed at the surface (due to the presence of squalene in the liver), which was removed by careful pipetting. Next, 5 µl of RNase A (10 mg/ml; Promega) was added to each tube and was incubated at 37°C for 1 h to digest the RNA. To separate the DNA, 300 µl phenol (Sigma Aldrich) and 300 µl chloroform (Sigma Aldrich) were added. The tubes were rotated for 10 min to mix well and centrifuged at 10,000 x g for 15 min, and the DNA in the aqueous layer was carefully aspirated. To this aqueous phase 600 µl chloroform/isoamyl alcohol (Sigma Aldrich) was added and the tubes were again rotated and centrifuged. The aqueous layer was collected, the DNA was precipitated with 50 µl 3 M sodium acetate (pH 8; Sigma-Aldrich) and 1 ml (2 volumes) of cold 100% ethanol (BDH Chemicals, Poole, U.K.), and the precipitate was pelleted by centrifugation. The pellet was washed in cold 70% ethanol and centrifuged at 10,000 x g for 5 min, and the ethanol was removed. Finally, the pellet was resuspended in 30–100 µl of autoclaved ddH20 and incubated at 4°C overnight before determining the 260:280 ratio. The quality of the genomic DNA was checked by running 1 µl of DNA (0.25 µg/µl) on an ethidium bromide (0.1 µg/ml)-stained agarose gel (0.5%). The DNA was placed at -20°C for long-term storage. Genomic DNA was also digested with several restriction enzymes (EcoRV, ScaI, DraI; Promega) and ligated into the GenomeWalker adapter (Clontech Laboratories, Palo Alto, CA) following the manufacturer’s instructions.

Genomic PCR

Based on the S. canicula IL-1{beta} cDNA sequence and genomic organization of IL-1{beta} from mammals, several primers were made to characterize the genomic structure of S. canicula IL-1{beta} (see Fig. 7Go and Table IGo). Where possible, gene-specific primers were designed in the same exon to avoid intron polymorphism. Gene walking in the 5' direction was performed using a nested approach with primers DB-R1, DB-R2, AP-1, and AP-2. The first-round PCR used the outer adapter primer (AP-1) and the outer gene-specific primer (DB-R2), and the second round used the nested adapter primer (AP-2) and the inner gene-specific primer (DB-R1). Gene walking in the 3' direction was performed using a nested approach with primers dogfish-F1, dogfish-F2, AP-1, and AP-2. The first-round PCR used the outer adapter primer (AP-2) and the outer gene-specific primer (dogfish-F1), and the second round used the nested adapter primer (AP-1) and the inner gene-specific primer (dogfish-F2). Wax-mediated hot start and touchdown PCR were performed using an enzyme mixture of Taq DNA polymerase (Bioline, London, U.K.) and PFU DNA polymerase (Promega) in a 50:1 U ratio. First- and second-round PCR amplification was performed using one cycle of 4 min (2 min for second round) at 94°C, 10 cycles of 1 min at 94°C, 1 min at 68°C (62°C for second round), and 3 min at 68°C, 10–20 cycles of 30 s at 94°C, 30 s at 62°C, and 3 min and 4 s per cycle at 68°C, with a final extension step of 5 min at 68°C. Specific primers DB-F7, -F8, -F9, -R5, -R6, and -R7 were used to fully sequence the amplified 5' product. A product that overlapped the two gene walking products was also amplified with gene-specific PCR using DB-F1, DB-F2, dogfish-R1, and dogfish-R2. Specific primers DB-F3, -F4, -F5, -F6, -R3, -R4, -R8, and -R9 were used to fully sequence the amplified product.



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  		FIGURE 7. Diagram indicating the position of the primers used to amplify the three products encoding the S. canicula IL-1{beta} gene.

 
Southern blot analysis

S. canicula genomic DNA extracted from spleen (as above) was digested completely with BamHI (Promega) and EcoRI (Promega). The digested DNA samples were electrophoresed (15 µg per lane) on a 1% agarose gel, transferred to a nylon membrane, and hybridized at 65°C for 4 h with the same probe used for the Northern blot analysis. After stringency washing the membrane was treated as for the Northern blot, except that it was exposed to film for 2 days. Southern blotting was performed from two S. canicula.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cloning and sequencing

Three overlapping products were obtained using PCR and universal and specific primers (Fig. 1Go) that contained the full-length S. canicula IL-1{beta} cDNA (Fig. 2Go). The transcript consisted of 1351 nt that translated in a single reading frame to give a predicted 301-aa IL-1{beta} molecule. The gene had a 54-bp 5' untranslated region (UTR) and a 379-bp 3' UTR. The 3' UTR contains eight RNA instability motifs (attta) and a polyadenylation signal 14 bp upstream from the poly(A) tail. The translated molecule contained two potential glycosylation sites and a readily identifiable IL-1 family signature(L e S A m y R g w Y V S T s r r n r q p I).



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  		FIGURE 2. Compiled full-length S. canicula IL-1{beta} cDNA sequence. Features highlighted include the start and stop codon and potential glycosylation sites. In the 3' UTR the RNA instability motifs (ATTTA) are in bold and the polyadenylation signal (AATAAA) is underlined. The IL-1 family signature is boxed.

 
Multiple alignment with other known IL-1{beta} amino acidsequences (Fig. 3Go) revealed many areas of strong amino acid conservation throughout the vertebrates. Good conservation is seen especially in the regions that would be expected to represent the 12 {beta}-sheets found in the {beta}-trefoil family members (32). As with other nonmammalian IL-1{beta} genes sequenced to date, the S. canicula IL-1{beta} gene lacks an aspartic acid found at the cut site region of mammalian IL-1{beta}s. This site is required for cleavage by IL-1-converting enzyme (ICE), a member of the caspase family.



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  		FIGURE 3. Multiple alignment of the predicted S. canicula IL-1{beta} translation with known IL-1{beta}s. Identical (*) and similar (: or .) residues identified by the CLUSTAL W program are indicated. The ICE cut site (aspartic acid) seen in all the mammalian sequences is highlighted.

 
The S. canicula IL-1{beta} sequence had highest nucleotide identity (45.3%) and amino acid similarity (43%) and identity (31.7%) with trout IL-1{beta} (Table IIGo), with chicken amino acid identities next highest, followed by Xenopus. All amino acid identities were <32%, indicating low homology with other known IL-1{beta} genes. The closer relationship between the trout and chicken IL-1{beta} with S. canicula IL-1{beta} was also apparent in the phylogenetic tree obtained using amino acid data (Fig. 4Go). In this study the S. canicula sequence branches with the fish and chicken sequences away from the mammalian IL-1{beta}s, although the genetic distance of S. canicula from the trout and chicken sequence is high.


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  		Table II. Amino acid and nucleotide homology of S. canicula IL-1{beta} with other known IL-1{beta} sequences1

 


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  		FIGURE 4. Unrooted phylogenetic tree showing the relationship between the S. canicula IL-1{beta} amino acid sequence for the full-length molecule with other known IL-1{beta} sequences. This tree was constructed by the neighbor-joining method using the CLUSTAL W and TreeView packages and was bootstrapped 10,000 times. Bootstrap values <75 are shown within the circles. The accession numbers of the nonmammalian IL-1{beta} sequences are as follows: chicken, Y15006; Xenopus, AJ010497; seabass, AJ269472; trout 1, AJ223954; trout 2, AJ245925; carp 1, AB010701; carp 2-1, AJ401030; carp 2-2, AJ401031; catshark, AJ251201.

 
Analysis of the hydrophobicity of the amino acid sequence revealed some similarity to the other known sequences. It was apparent from a Kyte and Doolittle (31) plot that although the first 10 or so S. canicula IL-1{beta} amino acids were hydrophobic there was no suggestion that a signal peptide was present, in common with other known IL-1{beta} sequences. This was also confirmed using SignalP v1.1.

Expression studies

Following injection of 200 µg LPS 24 h earlier, up-regulation of IL-1{beta} transcripts could be detected by RT-PCR in only the spleen and testes (Fig. 5Go). No expression was seen in control S. canicula gill, heart, kidney, and muscle tissue. Slight background expression of IL-1{beta} was found in the blood, brain, Leydig organ, and liver, but no increase in expression was detectable in response to LPS. Northern blot analysis (Fig. 6Go) of samples from splenocytes stimulated in vitro with LPS confirmed that IL-1{beta} expression could be up-regulated in these cells over a 5-h period. A transcript of the correct size (1.35 kb) was detectable, with optimum expression seen at 5 h, where a 7-fold increase of expression was detected relative to RNA from control 0-h splenocytes. A second, smaller transcript of ~0.9 kb was also detected with the probe, which followed a similar expression pattern as the 1.35-kb transcript, but it remains to be determined whether this molecule is related to IL-1{beta}.



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  		FIGURE 5. Analysis of the tissue expression of S. canicula IL-1{beta}. RT-PCR was performed using primers specific for S. canicula {beta}-actin and IL-1{beta} with cDNA from a variety of tissues. S. canicula were either injected (S) with 200 µg LPS and left for 24 h after injection or noninjected (C). Data shown are for a representative fish of three analyzed.

 


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  		FIGURE 6. Northern blot analysis of S. canicula splenocyte RNA obtained 0–5 h after cell stimulation in vitro with 10 µg/ml LPS. In all cases, 20 µg RNA was loaded per lane and the blot was hybridized with a 32P-labeled S. canicula {beta}-actin (A) and IL-1{beta} (B) probe. C, The level of expression of IL-1{beta} relative to {beta}-actin. Data shown are for a representative fish of three analyzed.

 
IL-1{beta} gene organization

Three overlapping products were obtained using PCR and a gene walking approach with gene-specific primers (Fig. 7Go). PCR with primers DB-F2 and dogfish-R1 gave a 3.3-kb product. Gene walking in the 5' direction with primers DB-R2 and DB-R1 using nested PCR gave a 3.1-kb product. Gene walking in the 3' direction with primers dogfish-F1 and dogfish-F2 using nested PCR gave a 0.7-kb product. The full gene sequence obtained by PCR is shown in Fig. 8Go, with the genomic organization of S. canicula IL-1{beta} consisting of five introns and six exons (Table IIIGo). S. canicula has the same genomic organization as chicken when compared with other known IL-1{beta} genomic organizations (Fig. 9Go), which, relative to the human gene, is missing an intron within the 5' UTR. The IL-1{beta} introns in S. canicula were similar to mammalian introns in size relative to other known IL-1{beta} introns. Multiple alignment of known IL-1{beta} amino acid sequences by exons (Fig. 10Go) showed that the junction amino acids between exons are quite conserved within exon 5 (exon 4 in S. canicula) only. Table IVGo shows the amino acid and nucleotide homologies of the coding regions of known IL-1{beta} exons using the GAP program in theGenetics Computer Group package. The highest amino acid homology was between chicken and S. canicula exon 3 (exon 4 in mammals), with the next highest between Xenopus exon 6 and S. canicula exon 5 (exon 6 in mammals). The highest nucleotide homology was between mouse exon 6 and S. canicula exon 5 (exon 6 in mammals). Generally, on comparison of each individual exon the highest homologies for S. canicula amino acid and nucleotides was with nonmammalian vertebrates.



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  		FIGURE 8. Compiled genomic organization of S. canicula IL-1{beta} gene. Exons are in uppercase and introns are in lowercase. The translation of the exon-coding regions is also given.

 

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  		Table III. Number of base pairs in the introns and exons of human and S. canicula IL-1{beta}

 


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  		FIGURE 9. Comparison of the gene organization and intron/exon sizes between known IL-1{beta} genes and S. canicula IL-1{beta}. The accession numbers of the IL-1{beta} genes are as follows: human, M15840; chicken, AJ245728; carp, AJ245635; trout, AJ004821; catshark, AJ295947.

 


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  		FIGURE 10. Multiple alignment of the predicted translation of S. canicula IL-1{beta} exons with exons from other known genes. Mammalian exon numbering is used.

 

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  		Table IV. Homology (%) of exons of S. caniculaIL-1{beta} with other known IL-1{beta} sequences.1

 
Southern blot analysis

To determine the number of IL-1{beta} gene copies in the S. canicula genome, genomic DNA was digested with BamHI and EcoRI and hybridized with the IL-1{beta} cDNA probes (Fig. 11Go). Using these enzymes two bands were expected, one of 1.8 kb and one >2.7 kb, because the cDNA probe would potentially bind to exon 4 before the EcoRI cut site and to exons 5 and 6 after the EcoRI cut site. Hybridization revealed two clear bands; one was of the expected size (~3.6 kb) for hybridization to exons 5 and 6 the other was and smaller (~0.9 kb) than the expected (1.8 kb) for the region containing exon 4. The 1.8-kb band was probably too weak to be detected, due to the exon region that the probe binds to being very short (40 bp).



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  		FIGURE 11. Southern blot analysis of S. canicula spleen DNA completely digested with BamHI and EcoRI before electrophoresis. After blotting, the membrane was hybridized with a 32P-labeled S. canicula IL-1{beta} probe. The exon position of the probe is shown on the gene organization in black. Data shown are for a representative fish of two analyzed.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This paper reports the isolation and sequencing of the IL-1{beta} gene in S. canicula, the first cytokine gene to be cloned within cartilaginous fish. S. canicula is found within the group Galeomorphs and belongs to the order Carcharhiniformes, family Scyliorhinidae. They are not true dogfish and it is more accurate to call them catsharks due to a number of different morphological characteristics (33). They were used in this investigation as they were an abundant representative from the cartilaginous group of fishes, available locally and easily kept in an aquarium.

The S. canicula IL-1{beta} gene cDNA had highest nucleotide (45.3%) and amino acid (31.7%) identity to trout IL-1{beta} (7, 34). Chicken (35) and Xenopus (17) IL-1{beta} had the next highest amino acid identities. A closer relationship with the trout and chicken IL-1{beta} was also apparent in the phylogenetic tree, where S. canicula branched with them away from the mammals, although in each case the genetic distance between the sequences was still quite high. Amino acid homology was highest in regions containing the secondary structure of the molecule, the 12 {beta}-sheets characteristic of the {beta}-trefoil cytokines (32). The predicted mature peptide also contains the IL-1 family signature, which spans {beta}-sheets 9–11. The IL-1 family signature pattern or motif has been taken from a selected conserved region in the C-terminal section, which has the following consensus pattern: [FC]-x-S-[ASLV]-x(2)-P-x(2)-[FYLIV]-[LI]-[SCA]-T-x(7)-[LIVM] and can be found in the PROSITE database (36). All mammalian, chicken, and carp IL-1{beta} sequences contain this exact motif, although to allow the inclusion of the trout and S. canicula IL-1{beta} sequences slight changes are required in the consensus sequence. The consensus sequence would need to read [FCL]-x-S-[ASLV]-x(2)-[PR]-x(2)-[FYLIV]-[LIV]-[SCA]-T-x(7)-[LIVMK]. The observation of homology within the predicted {beta}-sheets, along with the presence of the IL-1 family signature in the predicted mature protein, suggests that the S. canicula protein is also a {beta}-trefoil cytokine.

When analyzed using SignalP v1.1 it was shown that no apparent signal peptide was present in S. canicula IL-1{beta}. This is in common with other IL-1{beta} sequences (37), indicating that the molecule is secreted through a nonclassical pathway that does not involve the Golgi/endoplasmic reticulum route (38). Also found in the 3' UTR of S. canicula IL-1{beta} are numerous copies of an mRNA instability motif (attta). The degradation of mRNA in eukaryotic cells is a regulated process and can determine the level of expression of the gene. The presence of the consensus sequence, attta, in the 3' UTR of mRNA can play a role in the degradation of mRNA (39). The presence of this motif is typical of genes coding for inflammatory mediators (40) and suggests this gene will be biologically functional.

Until the cloning of trout IL-1{beta} (7), the processing of the precursor IL-1{beta} molecule was not in question. In all mammalian IL-1{beta} homologs ICE or caspase-1 cuts the precursor at an aspartic acid, Asp116 (41). Mammal pro-IL-1{beta} is biologically inactive until ICE cleaves it (42). Like trout and carp IL-1{beta}, S. canicula IL-1{beta} has no identifiable sequence that corresponds to an ICE cut site in the region where mammalian IL-1{beta} is cleaved to release the functional, mature peptide. Ala145 aligned with the first residue of the mammalian peptides, but it remains to be determined whether the S. canicula precursor molecule is cleaved naturally at this residue and whether this predicted "mature peptide" is active biologically.

Localization of IL-1{beta} expression in S. canicula was also investigated and indicates that the IL-1{beta} molecule is biologically relevant to cartilaginous fish immune responses to Gram-negative bacteria. Expression of the S. canicula IL-1{beta} transcript was clearly detectable by RT-PCR in the spleen and testes, tissues known to contain a variety of leukocytes (43), and was shown to be inducible in vivo following injection with LPS 24 h earlier. Stimulation of splenocytes in vitro with LPS for 0–5 h also resulted in a significant increase in transcript expression, with maximal expression seen at 5 h (the last time point examined). Northern blot analysis showed that there was a 7-fold increase in the transcript level after a 5-h incubation with LPS. Similar patterns of expression have also been seen in tissues of bony fish (34, 44). In carp, administration of PMA and LPS to phagocytes isolated from the head kidney induced expression of IL-1{beta} in these cells after 2 h of stimulation (44). In trout, head kidney leukocytes and isolated macrophages express IL-1{beta} after stimulation with LPS (34). A total of 5 µg/ml LPS was shown to produce maximal expression, which was first detectable 1–2 h poststimulation, with maximal induction at 4 h. While the kidney is a major source of phagocytes in bony fish, in cartilaginous fish it has been shown to contain lymphohemopoietic tissue only during embryonic development (45). In elasmobranchs, tissues such as the Leydig organ and testis (epigonal tissue) are found to contain lymphohemopoietic tissue. The lack of expression of IL-1{beta} in the Leydig organ in this study may reflect a redundancy between this tissue and the testis, as noted for B cell development in the clearnose skate (46). Temperature has also been shown to have a marked effect on trout IL-1{beta} expression (47) because fish are poikilothermic. While this is likely to be true also for cartilaginous fish, the temperatures used reflect those encountered naturally by this species.

Similar to the human IL-1{beta} gene (48), the carp IL-1{beta} gene has seven exons and six introns (44), whereas S. canicula, chicken, trout IL-1{beta}1 (34), and trout IL-1{beta}2 (8) have six exons and five introns. In S. canicula and chicken it is clear that the first intron is missing in the 5' UTR, whereas in trout the equivalent of an exon and an intron appear to be missing at the 5' end (34). In S. canicula and chicken the first exon is translated, unlike all other known IL-1{beta} genes where it is part of the 5' UTR. The sizes of the introns in S. canicula are more similar to those in humans, although introns 2, 3, and 4 (3, 4, and 5 in mammals) are smaller and 1 and 5 (2 and 6 in mammals) are larger than their human counterparts. All the S. canicula intron and exon boundaries follow the GT-AG consensus (49). The sizes of the coding exons 4, 5, and 6 (5, 6, and 7 in mammals) are similar to the exon sizes of mammalian IL-1{beta}, whereas exons 2 and 3 (3 and 4 in mammals) are larger. Exon 1 (2 in mammals) is also larger, but this is due to the missing intron. Generally, the highest homology seen between the different exons using both nucleotide and amino acid data was with S. canicula exon 5 (6 in mammals), chicken and trout exon 5, and mammalian exon 6. This exon is also known to have highest homology within mammals, and it contains the largest amount of secondary structure (four {beta}-sheets) per amino acid coded, even relative to the coding region of exon 7, which contains the IL-1 motif region. This adds further evidence to the statement that the most conserved region of the IL-1{beta} molecule is found within the 12 {beta}-sheets that form the secondary structure. S. canicula amino acid homology for exons 1 and 2 (exons 2 and 3 in mammals) showed very little homology to other species.

Southern blot analysis suggests there is a second S. canicula IL-1{beta} gene, or at least a related gene with homology to the 3' end of S. canicula IL-1{beta}. The presence of two IL-1{beta} genes has beenknown in mammals for some time (50), and in bony fish it is now clear that more than one gene can exist for IL-1{beta} (Ref. 8 ; GenBank accession no. AJ401030). Until it is sequenced this gene could also be a second member of the IL-1 gene family present in S. canicula, such as the IL-1R antagonist or IL-1{alpha}, which would potentially show homology with the 3' end of the gene. Whatever the outcome, it is clear that at least one member of the IL-1 gene family exists in cartilaginous fish.

In conclusion, previous bioactivity evidence for the presence of inflammatory cytokines in elasmobranchs has now be verified with the cloning of IL-1{beta} in S. canicula. The discovery of the S. canicula IL-1{beta} gene will greatly increase knowledge on the role of cytokines in the cartilaginous fish immune system. Future work will involve the production of the recombinant protein and the study of its immunological role directly. In addition it should be feasible to use the S. canicula IL-1{beta} gene to probe for other IL-1 family members to determine whether they had evolved before the divergence of bony and cartilaginous fish.


   Footnotes
 
1 This work was supported by a grant from the Biotechnology and Biological Sciences Research Council (1/S09641), and a studentship (to S.B.). Back

2 Address correspondence and reprint requests to Dr. Chris J. Secombes, Department of Zoology, University of Aberdeen, Tillydron Avenue, Aberdeen AB24 2TZ, U.K. E-mail address: c.secombes{at}abdn.ac.uk Back

3 Abbreviations used in this paper: Myr, million year; UTR, untranslated region; ICE, IL-1-converting enzyme. Back

Received for publication November 7, 2001. Accepted for publication January 18, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Thies, D., W. Reif. 1985. Phylogeny and evolutionary ecology of Mesozoic Neoselachii. Neues Jahrb. Geol. Palaontol. Abh. 169:333.
  2. Secombes, C. J., L. Pilstrom. 2000. Evolution of adaptive immunity. Biologist 47:44.[Medline]
  3. Senior, K.. 2000. New aminosterols from the dogfish shark. Drug Disc. Today 5:267.[Medline]
  4. Moore, K. S., S. Wehrli, H. Roder, M. Rogers, J. N. Forrest, D. McCrimmon, M. Zasloff. 1993. Squalamine: an aminosterol antibiotic from the shark. Proc. Natl. Acad. Sci. USA 90:1354.[Abstract/Free Full Text]
  5. Smith, S. L.. 2000. Cellular and humoral aspects of innate immunity in the shark. Fish Shellfish Immunol. 10:287. (Abstr.).
  6. Najakshin, A. M., L. V. Mechetina, B. Y. Alabyev, A. V. Taranin. 1999. Identification of an IL-8 homolog in lamprey (Lampetra fluviatilis): early evolutionary divergence of chemokines. Eur. J. Immunol. 29:375.[Medline]
  7. Zou, J., P. S. Gabowski, C. Cunningham, C. J. Secombes. 1999. Molecular cloning of interleukin-1{beta} from rainbow trout Oncorhynchus mykiss reveals no evidence of an ICE cut site. Cytokine 11:552.[Medline]
  8. Pleguezuelos, O., J. Zou, C. Cunningham, C. J. Secombes. 2000. Cloning, sequencing and analysis of expression of a second IL-1{beta} gene in rainbow trout (Oncorhynchus mykiss). Immunogenetics 51:1002.[Medline]
  9. Hardie, L. J., K. J. Laing, G. D. Daniels, P. S. Grabowski, C. Cunningham, C. J. Secombes. 1998. Isolation of the first piscine transforming growth factor {beta} gene: analysis reveals tissue specific expression and a potential regulatory sequence in rainbow trout (Oncorhynchus mykiss). Cytokine 10:555.[Medline]
  10. Laing, K. J., L. Pilstrom, C. Cunningham, C. J. Secombes. 1999. TGF-{beta}3 exists in bony fish. Vet. Immunol. Immunopathol. 72:45.[Medline]
  11. Laing, K. J., C. Cunningham, C. J. Secombes. 2000. Genes for three different isoforms of transforming growth factor-{beta} are present in plaice (Pleuronectes platessa) DNA. Fish Shellfish Immunol. 10:261.[Medline]
  12. Laing, K. J., T. H. Wang, J. Zou, J. Holland, S. H. Hong, N. Bols, I. Hirono, T. Aoki, C. J. Secombes. 2001. Cloning and expression analysis of rainbow trout Oncorhynchus mykiss tumour necrosis factor-{alpha}. Eur. J. Biochem. 268:1315.[Medline]
  13. Dixon, B., B. Shum, E. J. Adams, K. E. Magor, R. P. Hedrick, D. G. Muir, P. Parham. 1998. CK-1, a putative chemokine of rainbow trout (Oncorhynchus mykiss). Immunol. Rev. 166:341.[Medline]
  14. Daniels, G. D., J. Zou, J. Charlemagne, S. Partula, C. Cunningham, C. J. Secombes. 1999. Cloning of two chemokine receptor homologs (CXC-R4 and CC-R7) in rainbow trout Oncorhynchus mykiss. J. Leukocyte Biol. 65:684.[Abstract]
  15. Dinarello, C. A.. 1997. Interleukin-1. Cytokine Growth Factor Rev. 8:253.[Medline]
  16. Secombes, C. J., J. Zou, G. Daniels, C. Cunningham, A. Koussounadis, G. Kemp. 1998. Rainbow trout cytokine and cytokine receptor genes. Immunol. Rev. 166:333.[Medline]
  17. Zou, J., S. Bird, R. Minter, J. Horton, C. Cunningham, C. J. Secombes. 2000. Molecular cloning of the gene for interleukin-1{beta} from Xenopus laevis and analysis of expression in vivo and in vitro. Immunogenetics 49:332.
  18. Grogan, E. D., R. Lund. 1991. Reactivity of human white blood cells to factors of elasmobranch origin. Copeia 2:402.
  19. Luer, C. A., C. J. Walsh, A. B. Bodine, J. T. Wyffels, T. R. Scott. 1995. The elasmobranch thymus: anatomical, histological and preliminary functional characterization. J. Exp. Zool. 273:342.
  20. Obenauf, S., S. L. Hyder, S. L. Smith. 1992. Migratory response of nurse shark leucocytes to activated mammalian sera and porcine C5a. Fish Shellfish Immunol. 2:173.
  21. Walsh, C. J., C. A. Luer. 1998. Comparative phagocytic and pinocytic activities of leucocytes from peripheral blood and lymphomyeloid tissues of the nurse shark (Ginglymostoma cirratum Bonaterre) and the clearnose skate (Raja eglanteria Bosc). Fish Shellfish Immunol. 8:197.
  22. Birnboim, H. C.. 1983. A rapid alkaline extraction method for the isolation of plasmid DNA. Methods Enzymol. 100:243.[Medline]
  23. Pearson, W. R., D. I. Lipman. 1988. Improved tools for biological sequence comparison. Proc. Natl. Acad. Sci. USA 85:2444.[Abstract/Free Full Text]
  24. Altschul, S. F., W. Gish, W. Miller, E. Myers, D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403.[Medline]
  25. Needleman, S. B., 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.[Medline]
  26. Thompson, J. D., D. G. Higgins, 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.[Abstract/Free Full Text]
  27. Saitou, N., M. Nei. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406.[Abstract]
  28. Page, R. D. M.. 1996. TreeView: an application to display phylogenetic trees on personal computers. Comp. Appl. Biol. Sci. 12:357.
  29. Felsenstein, J.. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783.
  30. Nielsen, H., J. Engelbrecht, S. Brunak, G. vonHeijne. 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10:1.[Abstract/Free Full Text]
  31. Kyte, J., R. F. Doolittle. 1982. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157:105.[Medline]
  32. Nicola, N.. 1994. Guidebook to Cytokines and Their Receptors Sambrook and Tooze, Oxford.
  33. Miller, P., M. Loates. 1997. Fish of Britain and Europe HarperCollins, London.
  34. Zou, J., C. Cunningham, C. J. Secombes. 1999. The rainbow trout Oncorhynchus mykiss interleukin-1{beta} gene has a different organisation to mammals and undergoes incomplete splicing. Eur. J. Biochem. 259:901.[Medline]
  35. Weining, K. C., C. Sick, B. Kaspers, P. Staeheli. 1998. A chicken homolog of mammalian interleukin-1{beta}: cDNA cloning and purification of active recombinant protein. Eur. J. Biochem. 258:994.[Medline]
  36. Hofmann, K., P. Bucher, L. Falquet, A. Bairoch. 1999. The PROSITE database: its status in 1999. Nucleic Acids Res. 27:215.[Abstract/Free Full Text]
  37. Secombes, C. J., S. Bird, C. Cunningham, J. Zou. 1999. Interleukin-1 in fish. Fish Shellfish Immunol. 9:335.
  38. Rubartelli, A., F. Cozzolino, M. Talio, R. Sitia. 1990. A novel secretory pathway for interleukin-1{beta}, a protein lacking a signal sequence. EMBO J. 9:1503.[Medline]
  39. Sachs, A. B.. 1993. Messenger RNA degradation in eukaryotes. Cell 74:413.[Medline]
  40. Caput, D., B. Beutler, K. Hartog, R. Thayer, S. Brown-Shimer, A. Cermi. 1986. Identification of a common nucleotide sequence in the 3'-untranslated region of mRNA molecules specifying inflammatory mediators. Proc. Natl. Acad. Sci. USA 83:1670.[Abstract/Free Full Text]
  41. Howard, A. D., M. J. Kostura, N. Thornberry, G. J. F. Ding, G. Limjuco, J. Weidner, J. P. Salley, K. A. Hogquist, D. D. Chaplin, R. A. Mumford, et al 1991. IL-1-converting enzyme requires aspartic acid residues for processing of the IL-1{beta} precursor at two distinct sites and does not cleave 31-kDa IL-1{alpha}. J. Immunol. 147:2964.[Abstract]
  42. Fantuzzi, G., C. A. Dinarello. 1999. Interleukin-18 and interleukin-1{beta}: two cytokine substrates for ICE (caspase-1). J. Clin. Immunol. 19:1.[Medline]
  43. Zapata, A. G., M. Torroba, R. Sacedon, A. Varas, A. Vicente. 1996. Structure of the lymphoid organs of elasmobranchs. J. Exp. Zool. 275:125.
  44. Engelsma, M. Y., R. J. M. Stet, H. Schipper, B. M. L. Verburg-van Kemenade. 2001. Regulation of interleukin 1{beta} RNA expression in the common carp Cyprinus carpio L. Dev. Comp. Immunol. 25:195.[Medline]
  45. Zapata, A. G., M. Torroba, A. Vicente, A. Varas, R. Sacedon, E. Jimenez. 1995. The relevance of cell microenvironments for the appearance of lympho-haemopoietic tissues in primitive vertebrates. Histol. Hisopathol. 10:761.
  46. Miracle, L., M. K. Anderson, R. T. Litman, C. J. Walsh, C. A. Luer, E. V. Rothenberg, G. W. Litman. 2001. Complex expression patterns of lymphocyte-specific genes during the development of cartilaginous fish implicate unique lymphoid tissues in generating an immune repertoire. Int. Immunol. 13:567.[Abstract/Free Full Text]
  47. Zou, J., J. Holland, O. Pleguezuelos, C. Cunningham, C. J. Secombes. 2000. Factors influencing the expression of interleukin-1{beta} in cultured rainbow trout (Oncorhynchus mykiss) leucocytes. Dev. Comp. Immunol. 24:575.[Medline]
  48. Bensi, G., G. Raugei, E. Palla, V. Carinci, D. T. Buonamassa, M. Melli. 1987. Human interleukin-1{beta} gene. Gene 52:95.[Medline]
  49. Breathnach, R., P. Chambon. 1981. Organization and expression of eukaryotic split genes coding for proteins. Annu. Rev. Biochem. 50:349.[Medline]
  50. Vandenbroeck, K., A. Billiau. 1997. Recent progress in the molecular characterization of porcine cytokines. V. E. C. J. Schijns, and M. C. Horzinek, eds. Cytokines in Veterinary Medicine 139. CAB International, Wallingford, U.K.



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