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Interleukin-1 in fish
Fish & Shellfish Immunology (1999) 9, 335–343 Article ID: fsim.1998·0193 Available online at http://www.idealibrary.com on
Interleukin-1 in fish CHRISTOPHER J. SECOMBES1*, STEVE BIRD1, CHARLES CUNNINGHAM2
AND
JUN ZOU1
1
Department of Zoology, University of Aberdeen, Aberdeen, AB24 2TZ, Scotland, U.K. and 2SARS International Center for Marine Molecular Biology, 5008 Bergen, Norway (Received 21 December 1998, accepted 29 December 1998) It has been known for over a decade that IL-1 bioactivity exists in fish. Recently IL-1 has been cloned in several teleost species using two di#erent approaches; homology cloning or a suppression subtractive PCR technique. One of the most important di#erences of the fish sequences compared with mammalian IL-1 is the lack of a clear ICE cut site, and it remains to be determined whether the fish molecule is processed to a mature peptide. The IL-1 gene organisation has also been determined for rainbow trout, and Southern blot analysis suggests at least one related gene is present in this species. Expression of the IL-1 transcript can be induced in vitro by stimulation of head kidney leucocytes with LPS or PHA, or in vivo by challenge with Gram-negative bacteria. A number of incompletely spliced transcripts have also been detected in tissues from challenged fish. 1999 Academic Press
Key words:
Interleukin-1, cytokines, fish, gene.
I. The IL-1 gene family The interleukin-1 (IL-1) family of cytokines currently has four members: IL-1á, IL-1â, IL-1 receptor antagonist (IL-1ra) and IL-18 (also known as interferon-ãinducing factor) (Dinarello, 1997). In common with the fibroblast growth factor family of cytokines, members of the IL-1 family have a â-trefoil structure, composed of 12 â-sheets (Nicola, 1994). IL-1á and IL-1â share some 23% amino acid homology, and both are produced as precursor molecules (31 kDa) without a signal peptide. In the case of IL-1á, the precursor molecule is biologically active, and remains predominantly intracellular or associated with the cell surface. It can be released when cells die, and is then susceptible to cleavage by a number of proteases that generate a 17 kDa mature peptide. In contrast, IL-1â is inactive as a precursor, and must be cleaved to release the biologically active form, which is secreted from cells. The cleavage is achieved by the action of IL-1â converting enzyme (ICE), which has a specificity for aspartic acid (a caspase) and cleaves at Asp116. This process is part of the secretory process, in the absence of a signal peptide, and ICE deficient mice cannot release mature IL-1â. Recent crystal structure analysis has confirmed *Corresponding author. Email: [email protected] 1050–4648/99/040335+09 $30.00/0
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Fig. 1. Possible evolutionary events within the â-trefoil cytokine family.
site directed mutagenesis studies, showing that IL-1á and IL-1â bind to the IL-1 receptor (IL-1RI) at two sites, contacting the first two domains of the receptor (site A) or the third domain (site B)(Vigers et al., 1997). The IL-1ra is a specific antagonist of IL-1. It is produced as a secreted (sIL-1ra) or intracellular form (icIL-1ra), through alternative splicing of the transcript (Gabay et al., 1997), and is equivalent to the mature peptide of IL-1á and IL-1â with a signal peptide. The IL-1ra has highest amino acid homology (26%) to IL-1â, and this together with homology between the signal peptide of IL-1ra and the first exon of IL-1â (which is not translated) suggests that IL-1ra evolved from a duplicated IL-1â gene that arose some 350 million years ago (Fig. 1) (Hughes, 1994). Whilst IL-1ra binds to site A of the IL-1R in a similar manner to IL-1á and IL-1â, it makes a quite di#erent contact with domain 3 and does not interact with the proposed ‘receptor trigger site’ required for signal transduction (Schreuder et al., 1997). IL-18 is the newest member of the IL-1 cytokine family, based upon its structural homology (rather than sequence homology) to the other members (Bazan et al., 1996; Gillespie & Horwood, 1998). Like IL-1â, IL-18 is produced as a precursor without a signal peptide, and is cleaved by ICE to give the biologically active form (18 kDa) (Ghayur et al., 1997). It shares features of the IL-1 family signature but, significantly, is di#erent in the regions that bind to the IL-1R. Not surprisingly, the IL-18R is indeed a di#erent receptor to that used by IL-1á and IL-1â, and is a molecule previously known as the IL-1R related protein (IL-1Rrp). II. IL-1 bioactivity in fish It has been known since the pioneering work of Clem et al. (1985) that fish monocytes/macrophages are able to produce factors with IL-1 bioactivity. In this study, supernatants from LPS-stimulated monocytes of channel catfish
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(Ictalurus punctatus) could substitute for intact monocytes, allowing macrophage-depleted lymphocytes to undergo mitogen-induced proliferation and antigen-induced antibody production. Similarly, an IL-1-like factor is secreted by carp (Cyprinus carpio) macrophages and neutrophils (Verburg van Kemenade et al., 1995), and by carp epithelial and macrophage cell lines (Sigel et al., 1986; Weyts et al., 1997). The fish IL-1-like factor cross-reacts with mammalian cells, which respond in a manner typical of IL-1 stimulation (e.g. proliferation and IL-2 secretion by T cell lines). In addition, the activity can be inhibited by antisera to human IL-1á and IL-1â, and cDNA probes to mammalian IL-1á hybridise with mRNA from catfish monocytes. Such data suggest that IL-1 is highly conserved. Fractionation of catfish supernatants containing the IL-1-like factor found that a 70 kDa peak was active for catfish leucocytes (Clem et al., 1991; Ellsaesser & Clem, 1994). However, a peak of 15 kDa was active for mouse T cells. Western blot analysis of the catfish material showed that both fractions reacted with anti-human IL-1á and anti-human IL-1â. In carp a 15 kDa and 22 kDa band reacted with the anti-IL-1 sera in Western blot analysis, although immunoprecipitation revealed that the predominant newly synthesised protein was the 15 kDa species (Verburg van Kemenade et al., 1995). III. Approaches taken to sequence IL1 in fish In view of the biological evidence for IL-1 in fish, and the cross-reactivity of mammalian probes, several groups have attempted to isolate fish IL-1 using molecular approaches. The commonest approach taken has been PCR based homology cloning, where known mammalian sequences are used to reveal sites of conservation for primer design. For example, multiple alignment of the known IL-1â sequences reveals several areas of good homology (Fig. 2). Following PCR with degenerate primers based on these regions, products of the predicted size are cloned, sequenced and analysed for homology. However, the cDNA used as template is also an important consideration, and must be from cells likely to express IL-1â (e.g. macrophages) that have been stimulated, since IL-1â is not expected to be expressed constitutively. Stimulation can be simply achieved by bacterial challenge in vivo or incubation with LPS in vitro. Avirulent or attenuated bacterial strains are particularly useful for the former. To date, this approach has been successful for the cloning of rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar) and plaice (Pleuronectes platessa) IL-1â (Bird et al., 1998; Zou et al., 1999, in press). A related approach is to probe for IL-1â after expression cloning, using antisera against mammalian IL-1. Whilst this approach has been attempted with some fish species (e.g. carp), to date the molecules detected have not been IL-1. Nevertheless this approach is certainly feasible, and it has now been established that carp IL-1 is indeed recognised by a mammalian polyclonal antiserum (Verburg van Kemenade, personal communication). An alternative approach is to enrich for genes of interest and then generate a subtractive cDNA library that is used for random sequencing of individual clones. This approach has been used successfully for the isolation of carp IL-1â (Fujiki et al., 1998). Following in vivo stimulation (with sodium alginate),
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Fig. 2. Multiple alignment of mammalian IL-1â amino acid sequences, revealing regions of good homology (in bold) that were used for the design of primers for homology cloning. Identical (*) and similar (.or :) residues are indicated below the alignment.
peritoneal cells were collected and used in a suppression subtractive PCR technique to generate a cDNA library for sequencing. IV. IL-1â sequences The trout IL-1â cDNA obtained by homology cloning contains a 780 bp open reading frame (ORF), with 43–49% nucleotide identity to mammalian IL-1â sequences. The predicted translation (260 amino acids) has 49–56% amino acid similarity to mammalian IL-1â sequences and 42–45% amino acid similarity to
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Fig. 3. Multiple polyadenylation sites (AATAAA) in the 3 UTR of the trout IL-1â transcript. The first polyadenylation site was identified in the sequence reported by Zou et al. (in press) but some clones have now been found with a second polyadenylation site, 16 bp upstream from a 20 bp poly A tail. The extra sequence is underlined.
Fig. 4. Multiple alignment of trout, plaice and carp IL-1â, to show identical (*) and similar (. or :) residues in a 79–82 amino acid overlapping sequence representing the 3 end of the ORF (the 5 end of the plaice IL-1â cDNA has not been determined and the first six residues of the plaice sequence were used for priming).
IL-1á sequences (Zou et al., in press). In addition, the transcript contains a 97 bp 5 untranslated region (UTR) and a 466 bp 3 UTR, with the latter containing multiple repeats of a known mRNA instability motif (attta) typical of inflammatory cytokines and two polyadenylation sites (Fig. 3). There is no apparent signal peptide, in agreement with mammalian sequences, and the predicted molecular weight of the precursor molecule is 29 kDa. One of the most important di#erences when compared to mammalian IL-1â is the lack of a clear ICE cut site. Multiple alignment reveals no aspartic acid in the region where IL-1á or IL-1â are known to be cleaved to release the mature IL-1 peptides (Secombes et al., 1998). So whether the precursor is biologically active or must be cleaved to release a biologically active mature peptide remains to be determined experimentally. Both the salmon and plaice IL-1â sequences show good homology to trout IL-1â, with 97% nucleotide identity (97% amino acid similarity) for the full length salmon molecule and 70% nucleotide identity (71% amino acid similarity) for a 237 bp partial plaice sequence (Fig. 4). However, the carp sequence is quite di#erent, and has only 49% and 42% nucleotide identity (57% and 45% amino acid similarity) to the trout and plaice sequences respectively. Nevertheless, the degree of homology of the carp sequences to mammalian IL-1â sequences is similar to that for trout: 48–52% amino acid similarity. The carp ORF is a little longer than in the trout sequence (810 bp), with the insertion at the 5 end of the molecule. However, in agreement with the trout sequence it also has no signal peptide, no clear ICE cut site and contains attta motifs in the 347 bp 3 UTR. Overall, there is far lower homology between IL-1â genes within fish relative to the homology within mammals, as apparent by comparison of Figs 2 and 4. The gene organisation of the trout IL-1â sequence has also been determined and shown to contain 5 introns, in contrast to mammals which have 6 introns
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(Zou et al., 1999), with exon 1 being entirely untranslated. The missing exon appears to be at the 5 end of the gene but may not be a universal feature in fish since this region shows di#erences between the trout and carp cDNA sequences. One less intron and smaller introns compared to mammalian IL-1â genes, makes the trout gene relatively small, approx. 3·1 kb. Southern blot analysis suggests there are two IL-1â genes in trout, or an extra gene with homology to IL-1â (Zou et al., 1999). Whilst it is not unusual for tetraploid fish to have two loci for many genes, a precedent for two IL-1â genes does exist in mammals where pigs have two genes that code for molecules with 86% amino acid similarity (Vandenbroeck & Billiau, 1997). V. IL-1â expression Whilst waiting for the production of antisera to detect fish IL-1â it is possible to look at the induction of IL-1â messenger RNA by Northern blot analysis or RT-PCR. Both techniques have confirmed that in trout there is no constitutive expression of IL-1â in lymphoid tissues (Zou et al., 1999, in press). However, isolation and culture of head kidney leucocytes for 4 h is su$cient to induce a low level expression of the IL-1â transcript, similar to the situation with mammalian monocytes following adherance to culture vessels (Dinarello, 1997). In mammals, no significant translation occurs under these circumstances and it is clear that post-transcriptional events tightly regulate IL-1â expression. Stimulation of trout head kidney leucocytes or macrophage cultures with LPS (Zou et al., in press) or PHA (Fig. 5) results in a large increase in IL-1â expression. Similarly, challenge of fish with Gram negative bacteria (injection with aroA Aeromonas salmonicida, Marsden et al., 1996) results in IL-1â expression in lymphoid tissues such as the spleen, kidney, gill and blood (Zou et al., 1999). Whilst it remains to be proven whether translation occurs in these circumstances, in mammals LPS does have this e#ect (Dinarello, 1997). RT-PCR analysis of tissues from challenged fish has also revealed the presence of a number of incompletely spliced transcripts of IL-1â, that are not detected by Northern blot analysis (Zou et al., 1999). These transcripts contain either intron 5 or introns 4 and 5. Precedents for this also exist in the mammalian IL-1â literature, where there appears to be a mechanism to prevent the processing of precursor mRNA into mature mRNA, and some stimuli (e.g. retinoic acid) cannot overcome this block resulting in the accumulation of the precursor transcripts (Jarrous & Kaempfer, 1994). Kinetic studies looking at expression levels of the three trout transcripts following stimulation are on-going. VI. Conclusions Sequence evidence for the existence of IL-1â in fish now exists. The known IL-1-like bioactivity in fish, together with the induction of the IL-1â transcript in leucocytes by stimulation with LPS in vitro or challenge with bacteria in vivo strongly suggest that IL-1â is a major player in immune responses of fish as in mammals. However, unequivocal evidence that fish IL-1â is biologically
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Fig. 5. (A) Northern blot analysis of IL-1â expression in trout head kidney leucocytes after culture for 4 h (Con) or after stimulation for 4 h with 5 ìg ml 1 phytohaemagglutinin (PHA). (B) The relative levels of mRNA were quantified by densitometric scanning of exposed film and expressed as a ratio relative to the values obtained for â-actin expression with the same samples. The value above the PHA bar is the increase in ratio seen compared with the control ratio.
active awaits the production of the recombinant proteins. This is hindered by the lack of an indentifiable ICE cut site. Indeed, it is not impossible that the precursor molecule could be active in fish, as seen with precursor IL-1á. Conclusive data on whether fish IL-1â must be cleaved for biological activity may require sequencing of the N-terminus of the native secreted molecule, possibly aided by the production of antisera to the recombinant protein to allow rapid purification. Whether fish also possess IL-1á and the IL-1ra will be interesting to determine. The second gene detectable in trout could be a related gene rather than a second IL-1â. It has been estimated that IL-1ra diverged from IL-1â approximately 350 million years ago (Eisenberg et al., 1991), and thus could be absent in fish. It has also been suggested that the common ancestor of IL-1â and the IL-1ra may have undergone alternative splicing to produce the two di#erent molecules (Hughes, 1994). There is as yet no evidence of this in fish. Hughes also suggests that the mechanism of secretion of IL-1â without a signal peptide may be a recent event. Whilst the use of ICE to cleave the IL-1â precursor may indeed be a recent event, the fish IL-1â sequences lack a hydrophobic region at the N-terminus, indicative of a signal peptide; assuming the fish molecule is secreted, a more ancient mechanism must therefore exist.
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Undoubtedly we have a lot to learn about IL-1 in fish. Nevertheless, we are now in a position to make real progress into its role and regulation in fish, and the evolution of this important cytokine gene family. This research was supported by a grant from the EC (FAIR CT95-666).
References Bazan, J. F., Timans, J. C. & Kastelein, R. A. (1996). A newly defined interleukin-1? Nature 379, 591. Bird, S., Zou, J. & Secombes, C. J. (1998). Evolution of interleukin-1 â gene in vertebrates. EMBL accession No. AJ010640. Clem, L. W., Sizemore, R. C., Ellsaesser, C. F., Miller, N. W. (1985). Monocytes as accessory cells in fish immune responses. Developmental and Comparative Immunology 9, 803–809. Clem, L. W., Miller, M. W. & Bly, J. E. (1991). Evolution of lymphocyte subpopulations, their interactions and temperature sensitivities. In Phylogenesis of Immune Functions (G. W. Warr & N. Cohen, eds). pp. 191–213. Boca Raton, Florida: CRC Press. Dinarello, C. A. (1997). Interleukin-1. Cytokine & Growth Factor Reviews 8, 253–265. Eisenberg, S. P., Brewer, M. T., Verderber, E. Heimdal, P., Brandhuber, B. J. & Thompson, R. C. (1991). Interleukin 1 receptor antagonist is a member of the interleukin 1 gene family: Evolution of a cytokine control mechanism. Proceedings of the National Academy of Sciences USA 88, 5232–5236. Ellsaesser, C. F. & Clem, L. W. (1994). Functionally distinct high and low molecular weight species of channel catfish and mouse IL-1. Cytokine 6, 10–20. Fujiki, K., Nakao, M., Shin, D. & Yano, T. (1998). cDNA cloning of a carp homologue of mammalian interleukin-1 beta. EMBL accession No. AB010701. Gabay, C., Porter, B., Fantuzzi, G. & Arend, W. P. (1997). Mouse IL-1 receptor antagonist isoforms: complementary DNA cloning and protein expression of intracellular isoform and tissue distribution of secreted and intracellular IL-1 receptor antagonist in vivo. Journal of Immunology 159, 5905–5913. Ghayur, T., Banerjee, S., Hugunin, M., Butler, D., Herzog, L., Carter, A., Quintal, L., Sekut, L., Talanian, R., Paskind, M., Wong, W., Kamen, R., Tracey, D. & Allen, H. (1997). Caspase-1 processes IFN-ã-inducing factor and regulates LPS-induced IFN-ã production. Nature 386, 619–623. Gillespie, M. T. & Horwood, N. J. (1998). Interleukin-18: Perspectives on the newest interleukin. Cytokine & Growth Factor Reviews 9, 109–116. Hughes, A. L. (1994). Evolution of the interleukin-1 gene family in mammals. Journal of Molecular Evolution 39, 6–12. Jarrous, N. & Kaempfer, R. (1994). Induction of human interleukin-1 gene expression by retinoic acid and its regulation at processing of the precursor transcripts. Journal of Biological Chemistry 269, 23141–23149. Marsden, M. J., Vaughan, L. M., Foster, T. J. & Secombes, C. J. (1996). A live (ÄaroA) Aeromonas salmonicida vaccine for furunculosis preferentially stimulates enhanced T cell responses relative to B cell responses in rainbow trout (Oncorhynchus mykiss). Infection and Immunity 64, 3863–3869. Nicola, N. A. (1994). Guidebook to cytokines and their receptors. Oxford: Sambrook and Tooze. Schreuder, H., Tardif, C., Trump-Kallmeyer, S., So$entini, A., Sarubbi, E., Akeson, A., Bowlin, T., Yanofsky, S. & Barrett, R. W. (1997). A new cytokine-receptor binding mode revealed by the crystal structure of the IL-1 receptor with an antagonist. Nature 386, 194–200. Secombes, C. J., Zou, J., Daniels, G., Cunningham, C., Koussounadis, A. & Kemp, G. (1998). Rainbow trout cytokine and cytokine receptor genes. Immunological Reviews 166, 333–340.
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Sigel, M. M., Hamby, B. A. & Huggins, E. M. Jr. (1986). Phylogenetic studies on lymphokines. Fish lymphocytes respond to human IL-1 and epithelial cells produce an IL-1-like factor. Veterinary Immunology and Immunopathology 12, 47–58. Vandenbroeck, K. & Billiau, A. (1997). Recent progress in the molecular characterization of porcine cytokines. In Cytokines in Veterinary Medicine (V. E. C. J. Schijns & M. C. Horzinek, eds) pp. 139–153. Oxon: CAB International. Verburg van Kemenade, B. M. L., Weyts, F. A. A., Debets, R. & Flik, G. (1995). Carp macrophages and neutrophilic granulocytes secrete an interleukin-1-like factor. Developmental and Comparative Immunology 19, 59–70. Vigers, G. P. A., Anderson, L. J., Ca#es, P. & Brandhuber, B. J. (1997). Crystal structure of the type-1 interleukin-1 receptor complexed with interleukin-1 â. Nature 386, 190–194. Weyts, F. A. A.. Rombout, J. H. W. M., Flik, G. & Verburg van Kemenade, B. M. L. (1997). A common carp (Cyprinus carpio L.) leucocyte cell line shares morphological and functional characteristics with macrophages. Fish & Shellfish Immunology 7, 123–133. Zou, J., Grabowski, P. S., Cunningham, C. & Secombes, C. J. (in press). Molecular cloning of interleukin 1â from rainbow trout Oncorhynchus mykiss reveals no evidence of an ICE cut site. Cytokine. Zou, J., Cunningham, C. & Secombes, C. J. (1999). The rainbow trout Oncorhynchus mykiss interleukin 1â gene has a di#erent organisation to mammals and undergoes incomplete splicing. European Journal of Biochemistry 259, 901–908.
Interleukin-1 in fish CHRISTOPHER J. SECOMBES1*, STEVE BIRD1, CHARLES CUNNINGHAM2
AND
JUN ZOU1
1
Department of Zoology, University of Aberdeen, Aberdeen, AB24 2TZ, Scotland, U.K. and 2SARS International Center for Marine Molecular Biology, 5008 Bergen, Norway (Received 21 December 1998, accepted 29 December 1998) It has been known for over a decade that IL-1 bioactivity exists in fish. Recently IL-1 has been cloned in several teleost species using two di#erent approaches; homology cloning or a suppression subtractive PCR technique. One of the most important di#erences of the fish sequences compared with mammalian IL-1 is the lack of a clear ICE cut site, and it remains to be determined whether the fish molecule is processed to a mature peptide. The IL-1 gene organisation has also been determined for rainbow trout, and Southern blot analysis suggests at least one related gene is present in this species. Expression of the IL-1 transcript can be induced in vitro by stimulation of head kidney leucocytes with LPS or PHA, or in vivo by challenge with Gram-negative bacteria. A number of incompletely spliced transcripts have also been detected in tissues from challenged fish. 1999 Academic Press
Key words:
Interleukin-1, cytokines, fish, gene.
I. The IL-1 gene family The interleukin-1 (IL-1) family of cytokines currently has four members: IL-1á, IL-1â, IL-1 receptor antagonist (IL-1ra) and IL-18 (also known as interferon-ãinducing factor) (Dinarello, 1997). In common with the fibroblast growth factor family of cytokines, members of the IL-1 family have a â-trefoil structure, composed of 12 â-sheets (Nicola, 1994). IL-1á and IL-1â share some 23% amino acid homology, and both are produced as precursor molecules (31 kDa) without a signal peptide. In the case of IL-1á, the precursor molecule is biologically active, and remains predominantly intracellular or associated with the cell surface. It can be released when cells die, and is then susceptible to cleavage by a number of proteases that generate a 17 kDa mature peptide. In contrast, IL-1â is inactive as a precursor, and must be cleaved to release the biologically active form, which is secreted from cells. The cleavage is achieved by the action of IL-1â converting enzyme (ICE), which has a specificity for aspartic acid (a caspase) and cleaves at Asp116. This process is part of the secretory process, in the absence of a signal peptide, and ICE deficient mice cannot release mature IL-1â. Recent crystal structure analysis has confirmed *Corresponding author. Email: [email protected] 1050–4648/99/040335+09 $30.00/0
335
1999 Academic Press
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C. J. SECOMBES ET AL.
Fig. 1. Possible evolutionary events within the â-trefoil cytokine family.
site directed mutagenesis studies, showing that IL-1á and IL-1â bind to the IL-1 receptor (IL-1RI) at two sites, contacting the first two domains of the receptor (site A) or the third domain (site B)(Vigers et al., 1997). The IL-1ra is a specific antagonist of IL-1. It is produced as a secreted (sIL-1ra) or intracellular form (icIL-1ra), through alternative splicing of the transcript (Gabay et al., 1997), and is equivalent to the mature peptide of IL-1á and IL-1â with a signal peptide. The IL-1ra has highest amino acid homology (26%) to IL-1â, and this together with homology between the signal peptide of IL-1ra and the first exon of IL-1â (which is not translated) suggests that IL-1ra evolved from a duplicated IL-1â gene that arose some 350 million years ago (Fig. 1) (Hughes, 1994). Whilst IL-1ra binds to site A of the IL-1R in a similar manner to IL-1á and IL-1â, it makes a quite di#erent contact with domain 3 and does not interact with the proposed ‘receptor trigger site’ required for signal transduction (Schreuder et al., 1997). IL-18 is the newest member of the IL-1 cytokine family, based upon its structural homology (rather than sequence homology) to the other members (Bazan et al., 1996; Gillespie & Horwood, 1998). Like IL-1â, IL-18 is produced as a precursor without a signal peptide, and is cleaved by ICE to give the biologically active form (18 kDa) (Ghayur et al., 1997). It shares features of the IL-1 family signature but, significantly, is di#erent in the regions that bind to the IL-1R. Not surprisingly, the IL-18R is indeed a di#erent receptor to that used by IL-1á and IL-1â, and is a molecule previously known as the IL-1R related protein (IL-1Rrp). II. IL-1 bioactivity in fish It has been known since the pioneering work of Clem et al. (1985) that fish monocytes/macrophages are able to produce factors with IL-1 bioactivity. In this study, supernatants from LPS-stimulated monocytes of channel catfish
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(Ictalurus punctatus) could substitute for intact monocytes, allowing macrophage-depleted lymphocytes to undergo mitogen-induced proliferation and antigen-induced antibody production. Similarly, an IL-1-like factor is secreted by carp (Cyprinus carpio) macrophages and neutrophils (Verburg van Kemenade et al., 1995), and by carp epithelial and macrophage cell lines (Sigel et al., 1986; Weyts et al., 1997). The fish IL-1-like factor cross-reacts with mammalian cells, which respond in a manner typical of IL-1 stimulation (e.g. proliferation and IL-2 secretion by T cell lines). In addition, the activity can be inhibited by antisera to human IL-1á and IL-1â, and cDNA probes to mammalian IL-1á hybridise with mRNA from catfish monocytes. Such data suggest that IL-1 is highly conserved. Fractionation of catfish supernatants containing the IL-1-like factor found that a 70 kDa peak was active for catfish leucocytes (Clem et al., 1991; Ellsaesser & Clem, 1994). However, a peak of 15 kDa was active for mouse T cells. Western blot analysis of the catfish material showed that both fractions reacted with anti-human IL-1á and anti-human IL-1â. In carp a 15 kDa and 22 kDa band reacted with the anti-IL-1 sera in Western blot analysis, although immunoprecipitation revealed that the predominant newly synthesised protein was the 15 kDa species (Verburg van Kemenade et al., 1995). III. Approaches taken to sequence IL1 in fish In view of the biological evidence for IL-1 in fish, and the cross-reactivity of mammalian probes, several groups have attempted to isolate fish IL-1 using molecular approaches. The commonest approach taken has been PCR based homology cloning, where known mammalian sequences are used to reveal sites of conservation for primer design. For example, multiple alignment of the known IL-1â sequences reveals several areas of good homology (Fig. 2). Following PCR with degenerate primers based on these regions, products of the predicted size are cloned, sequenced and analysed for homology. However, the cDNA used as template is also an important consideration, and must be from cells likely to express IL-1â (e.g. macrophages) that have been stimulated, since IL-1â is not expected to be expressed constitutively. Stimulation can be simply achieved by bacterial challenge in vivo or incubation with LPS in vitro. Avirulent or attenuated bacterial strains are particularly useful for the former. To date, this approach has been successful for the cloning of rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar) and plaice (Pleuronectes platessa) IL-1â (Bird et al., 1998; Zou et al., 1999, in press). A related approach is to probe for IL-1â after expression cloning, using antisera against mammalian IL-1. Whilst this approach has been attempted with some fish species (e.g. carp), to date the molecules detected have not been IL-1. Nevertheless this approach is certainly feasible, and it has now been established that carp IL-1 is indeed recognised by a mammalian polyclonal antiserum (Verburg van Kemenade, personal communication). An alternative approach is to enrich for genes of interest and then generate a subtractive cDNA library that is used for random sequencing of individual clones. This approach has been used successfully for the isolation of carp IL-1â (Fujiki et al., 1998). Following in vivo stimulation (with sodium alginate),
338
C. J. SECOMBES ET AL.
Fig. 2. Multiple alignment of mammalian IL-1â amino acid sequences, revealing regions of good homology (in bold) that were used for the design of primers for homology cloning. Identical (*) and similar (.or :) residues are indicated below the alignment.
peritoneal cells were collected and used in a suppression subtractive PCR technique to generate a cDNA library for sequencing. IV. IL-1â sequences The trout IL-1â cDNA obtained by homology cloning contains a 780 bp open reading frame (ORF), with 43–49% nucleotide identity to mammalian IL-1â sequences. The predicted translation (260 amino acids) has 49–56% amino acid similarity to mammalian IL-1â sequences and 42–45% amino acid similarity to
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339
Fig. 3. Multiple polyadenylation sites (AATAAA) in the 3 UTR of the trout IL-1â transcript. The first polyadenylation site was identified in the sequence reported by Zou et al. (in press) but some clones have now been found with a second polyadenylation site, 16 bp upstream from a 20 bp poly A tail. The extra sequence is underlined.
Fig. 4. Multiple alignment of trout, plaice and carp IL-1â, to show identical (*) and similar (. or :) residues in a 79–82 amino acid overlapping sequence representing the 3 end of the ORF (the 5 end of the plaice IL-1â cDNA has not been determined and the first six residues of the plaice sequence were used for priming).
IL-1á sequences (Zou et al., in press). In addition, the transcript contains a 97 bp 5 untranslated region (UTR) and a 466 bp 3 UTR, with the latter containing multiple repeats of a known mRNA instability motif (attta) typical of inflammatory cytokines and two polyadenylation sites (Fig. 3). There is no apparent signal peptide, in agreement with mammalian sequences, and the predicted molecular weight of the precursor molecule is 29 kDa. One of the most important di#erences when compared to mammalian IL-1â is the lack of a clear ICE cut site. Multiple alignment reveals no aspartic acid in the region where IL-1á or IL-1â are known to be cleaved to release the mature IL-1 peptides (Secombes et al., 1998). So whether the precursor is biologically active or must be cleaved to release a biologically active mature peptide remains to be determined experimentally. Both the salmon and plaice IL-1â sequences show good homology to trout IL-1â, with 97% nucleotide identity (97% amino acid similarity) for the full length salmon molecule and 70% nucleotide identity (71% amino acid similarity) for a 237 bp partial plaice sequence (Fig. 4). However, the carp sequence is quite di#erent, and has only 49% and 42% nucleotide identity (57% and 45% amino acid similarity) to the trout and plaice sequences respectively. Nevertheless, the degree of homology of the carp sequences to mammalian IL-1â sequences is similar to that for trout: 48–52% amino acid similarity. The carp ORF is a little longer than in the trout sequence (810 bp), with the insertion at the 5 end of the molecule. However, in agreement with the trout sequence it also has no signal peptide, no clear ICE cut site and contains attta motifs in the 347 bp 3 UTR. Overall, there is far lower homology between IL-1â genes within fish relative to the homology within mammals, as apparent by comparison of Figs 2 and 4. The gene organisation of the trout IL-1â sequence has also been determined and shown to contain 5 introns, in contrast to mammals which have 6 introns
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(Zou et al., 1999), with exon 1 being entirely untranslated. The missing exon appears to be at the 5 end of the gene but may not be a universal feature in fish since this region shows di#erences between the trout and carp cDNA sequences. One less intron and smaller introns compared to mammalian IL-1â genes, makes the trout gene relatively small, approx. 3·1 kb. Southern blot analysis suggests there are two IL-1â genes in trout, or an extra gene with homology to IL-1â (Zou et al., 1999). Whilst it is not unusual for tetraploid fish to have two loci for many genes, a precedent for two IL-1â genes does exist in mammals where pigs have two genes that code for molecules with 86% amino acid similarity (Vandenbroeck & Billiau, 1997). V. IL-1â expression Whilst waiting for the production of antisera to detect fish IL-1â it is possible to look at the induction of IL-1â messenger RNA by Northern blot analysis or RT-PCR. Both techniques have confirmed that in trout there is no constitutive expression of IL-1â in lymphoid tissues (Zou et al., 1999, in press). However, isolation and culture of head kidney leucocytes for 4 h is su$cient to induce a low level expression of the IL-1â transcript, similar to the situation with mammalian monocytes following adherance to culture vessels (Dinarello, 1997). In mammals, no significant translation occurs under these circumstances and it is clear that post-transcriptional events tightly regulate IL-1â expression. Stimulation of trout head kidney leucocytes or macrophage cultures with LPS (Zou et al., in press) or PHA (Fig. 5) results in a large increase in IL-1â expression. Similarly, challenge of fish with Gram negative bacteria (injection with aroA Aeromonas salmonicida, Marsden et al., 1996) results in IL-1â expression in lymphoid tissues such as the spleen, kidney, gill and blood (Zou et al., 1999). Whilst it remains to be proven whether translation occurs in these circumstances, in mammals LPS does have this e#ect (Dinarello, 1997). RT-PCR analysis of tissues from challenged fish has also revealed the presence of a number of incompletely spliced transcripts of IL-1â, that are not detected by Northern blot analysis (Zou et al., 1999). These transcripts contain either intron 5 or introns 4 and 5. Precedents for this also exist in the mammalian IL-1â literature, where there appears to be a mechanism to prevent the processing of precursor mRNA into mature mRNA, and some stimuli (e.g. retinoic acid) cannot overcome this block resulting in the accumulation of the precursor transcripts (Jarrous & Kaempfer, 1994). Kinetic studies looking at expression levels of the three trout transcripts following stimulation are on-going. VI. Conclusions Sequence evidence for the existence of IL-1â in fish now exists. The known IL-1-like bioactivity in fish, together with the induction of the IL-1â transcript in leucocytes by stimulation with LPS in vitro or challenge with bacteria in vivo strongly suggest that IL-1â is a major player in immune responses of fish as in mammals. However, unequivocal evidence that fish IL-1â is biologically
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Fig. 5. (A) Northern blot analysis of IL-1â expression in trout head kidney leucocytes after culture for 4 h (Con) or after stimulation for 4 h with 5 ìg ml 1 phytohaemagglutinin (PHA). (B) The relative levels of mRNA were quantified by densitometric scanning of exposed film and expressed as a ratio relative to the values obtained for â-actin expression with the same samples. The value above the PHA bar is the increase in ratio seen compared with the control ratio.
active awaits the production of the recombinant proteins. This is hindered by the lack of an indentifiable ICE cut site. Indeed, it is not impossible that the precursor molecule could be active in fish, as seen with precursor IL-1á. Conclusive data on whether fish IL-1â must be cleaved for biological activity may require sequencing of the N-terminus of the native secreted molecule, possibly aided by the production of antisera to the recombinant protein to allow rapid purification. Whether fish also possess IL-1á and the IL-1ra will be interesting to determine. The second gene detectable in trout could be a related gene rather than a second IL-1â. It has been estimated that IL-1ra diverged from IL-1â approximately 350 million years ago (Eisenberg et al., 1991), and thus could be absent in fish. It has also been suggested that the common ancestor of IL-1â and the IL-1ra may have undergone alternative splicing to produce the two di#erent molecules (Hughes, 1994). There is as yet no evidence of this in fish. Hughes also suggests that the mechanism of secretion of IL-1â without a signal peptide may be a recent event. Whilst the use of ICE to cleave the IL-1â precursor may indeed be a recent event, the fish IL-1â sequences lack a hydrophobic region at the N-terminus, indicative of a signal peptide; assuming the fish molecule is secreted, a more ancient mechanism must therefore exist.
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Undoubtedly we have a lot to learn about IL-1 in fish. Nevertheless, we are now in a position to make real progress into its role and regulation in fish, and the evolution of this important cytokine gene family. This research was supported by a grant from the EC (FAIR CT95-666).
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