Cytokine & Growth Factor Reviews 13 (2002) 483–502

Survey

Evolution of interleukin-1�

Steve Birda, Jun Zoua, Tiehui Wanga, Barry Mundayb,Charlie Cunninghamc, Chris J. Secombesa,∗

a Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, UKb School of Human Life Sciences, University of Tasmania, Locked Bag 1-320, Launceston, Tasmania 7250, Australia

c Sars International Center for Marine Molecular Biology, 5008 Bergen, Norway

Abstract

All jawed vertebrates possess a complex immune system, which is capable of anticipatory and innate immune responses. Jawlessvertebrates posses an equally complex immune system but with no evidence of an anticipatory immune response. From these findings it hasbeen speculated that the initiation and regulation of the immune system within vertebrates will be equally complex, although very little hasbeen done to look at the evolution of cytokine genes, despite well-known biological activities within vertebrates. In recent years, cytokines,which have been well characterised within mammals, have begun to be cloned and sequenced within non-mammalian vertebrates, with thenumber of cytokine sequences available from primitive vertebrates growing rapidly. The identification of cytokines, which are mammalianhomologues, will give a better insight into where immune system communicators arose and may also reveal molecules, which are uniqueto certain organisms. Work has focussed on interleukin-1 (IL-1), a major mediator of inflammation which initiates and/or increases a widevariety of non-structural, function associated genes that are characteristically expressed during inflammation. Other than mammalian IL-1�sequences there are now full cDNA sequences and genomic organisations available from bird, amphibian, bony fish and cartilaginous fish,with many of these genes having been obtained using an homology cloning approach. This review considers how the IL-1� gene haschanged through vertebrate evolution and whether its role and regulation are conserved within selected non-mammalian vertebrates.© 2002 Elsevier Science Ltd. All rights reserved.

Keywords:Interleukin-1; Comparative immunology; Vertebrate; Gene evolution; ICE cut site

Contents

1. Evolution of immune system communication. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4832. Interleukin-1 family. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4853. IL-1 in vertebrates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4874. Known vertebrate IL-1� sequences. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4885. Processing of IL-1� in vertebrates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4906. Vertebrate IL-1� gene organisation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4927. Vertebrate IL-1� expression and regulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4928. Vertebrate IL-1 receptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4949. Interleukin-18. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494

10. Cytokine and IL-1 evolution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49511. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 496Acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497

∗ Corresponding author. Tel.:+44-1224-272872;fax: +44-1224-272396.E-mail address:c.secombes@abdn.ac.uk (C.J. Secombes).

1. Evolution of immune system communication

The study of the immune system of non-mammalian ver-tebrates has become increasingly important in organismsthat have commercial value such as birds and fish. Due tothis work, the origin of immune system components havealso become the focus of investigation. It is now accepted,

1359-6101/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved.PII: S1359-6101(02)00028-X

484 S. Bird et al. / Cytokine & Growth Factor Reviews 13 (2002) 483–502

Table 1Cytokines cloned and sequenced from non-mammalian vertebrates (excluding IL-1�)

Organism Gene GenBank accession number Reference

BirdsGallus gallus(Chicken) IL-2 AF000631 [10]

IL-6 AJ309540 [11]IL-8 AJ009800 [12]IL-15 AF139097 [13]IL-18 AJ277865 [14]

IFN-� X92476 [15]IFN-� X92479 [15]IFN-� X99774 [16]

FGF-2 M95707 [17]FGF-3 Z47555 [18]FGF-4 U14654 [19]FGF-8 U55189 [20]FGF-10 D86333 [21]FGF-12 AF108754 [22]FGF-13 AF108755 [22]FGF-18 AB030229 [23]FGF-19 AF315355 [24]

TGF-�2 X58071 [25]TGF-�3 M31154 [26]TGF-�4 M31160 [27]

MIF M95776 [28]

K60 (CXC chemokine) AF277660 [29]K203 (CC chemokine) Y18692 [29]

Stem cell factor D13516 [30]

cMGF X14477 [31]

Meleagris gallopavo(Turkey) IL-2 AJ007463 [32]

Type I interferon U28140 [33]IFN-� AJ000725 [34]

Coturnix japonica(Japanese Quail) IFN-� AJ001678 [34]

Phasianus colchicus(Ring-Necked Pheasant) IFN-� AJ001289 [34]

AmphibiansXenopus laevis(African Clawed Frog) Embryonic FGF X62593 [35]

FGF-2 M18067 [36]FGF-3 Z25539 [37]FGF-8 Y10312 [38]FGF-9 U47622 [39]FGF-10 AB073747 [40]

TGF-�2 X51817 [41]TGF-�5 J05180 [42]

Bony fishOncorhynchus mykiss(Rainbow Trout) IL-8 AJ279069

TNF-� AJ277604 [43]

TGF-�1 X99303 [44]TGF-�2 AJ318935TGF-�3 AJ318928 [45]

FGF6 Y16850 [46]

CK-1 (CXC chemokine) AF093802 [47]Putative chemokine (CXC chemokine) AJ300835Putative chemokine (CC chemokine) AJ315149

Cyprinus carpio(Common Carp) Allograft inflammatory factor 1 AB012309 [48]

Chemokine-1 (CC chemokine) AB010469 [48]

S. Bird et al. / Cytokine & Growth Factor Reviews 13 (2002) 483–502 485

Table 1 (Continued)

Organism Gene GenBank accession number Reference

NK cell enhancing factor AB010959 [48]

Danio rerio (Zebrafish) FGF8 AF034264 [49]

Scyba (CXC chemokine) AF279919 [50]CCL1 (CC chemokine) AF201450

Paralichthys olivaceus(Bastard Halibut) IL-8 AF216646 [51]

TNF-� AB040448 [52]

Acipenser bari(Siberian Sturgeon) TGF-�3 AJ318926 [45]

Anguilla anguilla (European Eel) TGF-�1 AJ318934TGF-�3 AJ318927 [45]

Pleuronectes platessa(Plaice) TGF-�1 AJ318929 [53]TGF-�2 AJ318930 [53]TGF-�3 AJ318931 [53]

Cartilaginous fishTriakis scyllium(Banded Dogfish) IL-8 AB063299

Jawless fishLampetra fluviatilis(Lamprey) LFCA-1 (CXC chemokine) AJ231072 [54]

Publications cited where available.

with the sequencing of various immune genes, that all jawedvertebrates (Gnathostomes) possess a complex immune sys-tem, which is capable of anticipatory and innate immuneresponses[1,2]. Jawless vertebrates posses an equally com-plex immune system but no evidence has been presentedto prove the presence of an anticipatory immune response.However, innate immunity is found and is shown to havea more ancient origin. From these studies it can be specu-lated that the initiation and regulation of the immune systemwithin vertebrates will be equally complex.

The evolution of the immune system has awarded cy-tokines a key role as co-ordinators of the immune system.The important role of cytokines in fine-tuning and control-ling immune responses[3,4] raises the question of whetheror not these molecules have been highly conserved throughevolution. Very little has been done to look at the evolution ofcytokine genes within vertebrates, despite well-known bio-logical activities throughout the vertebrates and invertebrates[5]. Which cytokines will be found within primitive verte-brates and how far back in the vertebrate phylogeny thesemolecules go is only beginning to be addressed. It may bethat cytokines important in anticipatory responses will proveto be unique to jawed vertebrates whereas cytokines impor-tant in non-specific defences may be more universal. Re-cently, cytokines which have been well characterised withinmammals[6] have begun to be cloned and sequenced withinnon-mammalian vertebrates. Cytokines have now been se-quenced within amphibians, birds, bony fish, cartilaginousfish and jawless fish (Tables 1 and 2), with the numberof cytokine sequences available from primitive vertebratesgrowing rapidly. Molecules related to vertebrate cytokinereceptors are also being cloned in many vertebrate groups[5,7–9].

2. Interleukin-1 family

Interleukin-1� was initially discovered within mice andhumans and over the last 10 years has been characterisedwithin a wide variety of mammalian species (Table 2). Theinterleukin-1 (IL-1) family of cytokines has four main mem-bers: IL-1�, IL-1�, IL-1 receptor antagonist (IL-1ra) andIL-18, with a number of newly discovered molecules show-ing clear homology to this group[81–87]. IL-1 is a majormediator of inflammation and in general initiates and/or in-creases a wide variety of non-structural, function associatedgenes that are characteristically expressed during inflamma-tion, particularly other cytokines. It is one of the key media-tors of the body’s response to microbial invasion, inflamma-tion, immunological reactions and tissue injury. Both in vivoand in vitro experiments have shown that IL-1� and IL-1�have similar if not identical multiple biological effects whichhave been well catalogued[81,88–91]and both forms affectnearly every cell type and share a common receptor on tar-get cells. Recent work however has shown that the endoge-nous roles of IL-1� and IL-1� are different where IL-1� butnot IL-1� is a potent activator of the humoral immune re-sponse[92]. IL-1� and IL-1� are produced by many differ-ent cell types[88] including neutrophils, natural killer cells,B-lymphocytes, T-lymphocytes and cells of the central ner-vous system, but are made mainly by blood monocytes andtissue macrophages[93–96]. The monocyte/macrophage isan important source because of its strategic locations, itsability to synthesise large amounts of IL-1 and to processthe IL-1 precursor more effectively than other cells.

Members of the IL-1 family belong to what is nowknown as the�-trefoil superfamily due to the presence of12 �-sheets in their mature protein structure which fold

486 S. Bird et al. / Cytokine & Growth Factor Reviews 13 (2002) 483–502

Table 2Table of known IL-1� sequences

Organism Gene GenBank accession number Reference

Homo sapien(Human) IL-1� mRNA M15330 [55]IL-1� gene M15840 [56]

Cercocebus torquatus(Red-Crowned Mangabey) IL-1� mRNA U19837 [57]Macaca fascicularis(Crab-Eating Macaque) IL-1� mRNA D63353 [58]Macaca mulatta(Rhesus Monkey) IL-1� mRNA U19845 [57]Macaca nemestrina(Pig-Tailed Macaque) IL-1� mRNA U19853 [57]

Mus musculus(Mouse) IL-1� mRNA M15131 [59]IL-1� gene X04964 [60]

Rattus norvegicus(Norway Rat) IL-1� mRNA M98820Sigmodon hispidus(Hispid Cotton Rat) IL-1� mRNA AF421387Equus callus(Horse) IL-1� mRNA D42147 [61]Bos taurus(Cow) IL-1� mRNA M35589 [62]Cervus elaphus(Red Deer) IL-1� mRNA U20500Ovis aries(Sheep) IL-1� mRNA X54796 [63]

Sus scrofa(Pig) IL-1� mRNA M86725 [64]IL-1� gene X74568 [65]

Capra hircus(Goat) IL-1� mRNA D63351Felis domesticus(Cat) IL-1� mRNA M92060Oryctolagus cuniculus(Rabbit) IL-1� mRNA D21835 [66]Tursiops truncatus(Bottle-Nosed Dolphin) IL-1� mRNA AB028216 [67]Trichosurus vulpecula(Brush-Tailed Possum) IL-1� mRNA AF071539 [68]Ornithorhynchus anatinus(Duck-Billed Platypus) IL-1� gene AJ239048 [69]Mesocricetus auratus(Golden Hamster) IL-1� mRNA, partial AB028497Canis familiaris(Dog) IL-1� mRNA, partial Z70047

Gallus gallus(Chicken) IL-1� mRNA Y15006 [70]IL-1� gene AJ245728

Xenopus laevis(African Clawed Frog) IL-1� mRNA AJ010497 [71]IL-1� gene AJ314758 [69]

Dicentrarchus labrax(European Seabass) IL-1� mRNA AJ269472 [72]IL-1� gene AJ311925 [73]

Oncorhynchus mykiss(Rainbow Trout) IL-1� 1 mRNA AJ223954 [74]IL-1� 1 gene AJ004821 [75]IL-1� 2 mRNA AJ245925 [76]IL-1� 2 gene AJ245925 [76]

Sparus aurata(Gilthead Seabream) IL-1� mRNA AJ277166 [77]IL-1� gene AJ419178 [77]

Carrassius auratus(Goldfish) IL-1� 1 mRNA AJ249136 [69]IL-1� 1 gene AJ419848 [69]IL-1� 2 mRNA AJ249137 [69]IL-1� 2 gene AJ419849 [69]

Cyprinus carpio(Common Carp) IL-1� 1 mRNA AB010701 [78]IL-1� 1 gene AJ245635 [79]IL-1� 2-1 mRNA AJ401030IL-1� 2-2 mRNA AJ401031

Scophthalmus maximus(Turbot) IL-1� mRNA, partial AJ295836Pleuronectes platessa(Plaice) IL-1� mRNA, partial AJ010640 [69]Danio rerio (Zebrafish) IL-1� mRNA, partial AI330756Scyliorhinus canicula(Small Spotted Catshark) IL-1� mRNA AJ251201 [80]

IL-1� gene AJ295947 [80]

Publications cited where available.

S. Bird et al. / Cytokine & Growth Factor Reviews 13 (2002) 483–502 487

to form a trefoil-like structure[97,98]. Other members ofthis superfamily include the fibroblast growth factors[82].IL-1�, IL-1� and IL-1ra contain the IL-1 family signaturepattern or motif, taken from a selected conserved region inthe C-terminal section with the following consensus pattern;[FC]-x-S-[ASLV]-x(2)-P-x(2)-[FYLIV]-[LI]-[SCA]-T-x(7)-[LIVM] as found in the PROSITE database[99]. IL-1�and IL-1� differ substantially in relation to localisation,maturation and secretion. Both forms are translated fromthe messenger RNA (mRNA) into the immature forms(31 kDa), pro-IL-1� and pro-IL-1�, which due to the lackof a hydrophobic leader peptide sequence remain in thecystol and do not accumulate in any organelles[100]. Bothforms can undergo cleavage by enzymes, to produce ma-ture forms, which augments the activity of these peptides.Pro-IL-1� but not pro-IL-1� can be cleaved by calciumdependent membrane proteases called calpains[101,102]to give a 17 kDa mature peptide. IL-1� is rarely found inthe circulation or extracellular biological fluids and only incases of serious illness when it is thought to have origi-nated from lysed cells[103] and cleavage by extracellularproteases[104]. Pro-IL-1� is just as active as the matureform, and appears to remain intracellular and act at thislevel [105,106]. Pro-IL-1� can also be found on the surfaceof several cells, especially monocytes and B-cells, where itremains biologically active.

Pro-IL-1� remains primarily cytosolic and is not fullyactive until it is proteolytically cleaved and transported outof the cell. A cysteine protease, IL-1� converting enzyme(ICE), also known as caspase-1, has been identified as thespecific enzyme responsible for this cleavage of pro-IL-1�[107–109]. Caspases are primarily involved in apopto-sis [110,111], and over expression of ICE itself can leadto increased apoptosis in cells transfected with this gene[112,113]. The ability of both ICE and pro-IL-1� to coexistwithout mature IL-1� being formed suggests ICE activitymust be regulated[114–116]. Also, it has been shown thatICE has five isoforms generated by alternative splicing ofthe mRNA[117], each with varying degrees of activity. Togenerate mature IL-1�, ICE specifically cleaves the aspar-tate (amino acid position 116)–alanine (amino acid position117) bond[118]. It is generally thought that cleavage ofpro-IL-1� is an obligatory step for IL-1� release, sincetreatment with most ICE inhibitors fails to cause detectablelevels of extracellular pro-IL-1� [118]. This is not absoluteand some ICE inhibitors can cause pro-IL-1� to be se-creted instead of the mature form[118,119]. The processesinvolved in secretion of the mature form of IL-1� are illdefined. It has been suggested that pro-ICE present in thecytoplasm is activated, assembled and inserted into the cellmembrane where it would cleave pro-IL-1� and release themature form into the extracellular environment[120]. It isthought that mature IL-1� is released by a novel secretorypathway as it has been shown that IL-1� is contained, in part,within intracellular vesicles that protect it from proteasedigestion and active secretion of IL-1� requires its translo-

cation across the membrane[121]. More recently it hasbeen shown that release of IL-1� is mediated by adenosine5′-triphosphate (ATP) and requires a priming step by LPSwhich is independent of caspase-1 cleavage[122]. In addi-tion, alternative mechanisms are present for the processingof the pro-IL-1� [123]. There are several sites in pro-IL-1�which have been shown to be vulnerable to cleavage byenzymes in the vicinity of alanine 117. Proteolytic enzymesincluding chymase, elastase, cathepsin G, collagenase andmatrix metalloproteinases have all been shown to cleavepro-IL-1� into a form similar in size and specific activityas the ICE processed IL-1� [123,124]. These proteases arereleased at inflammatory and tissue damage sites by a va-riety of immune cells[125,126]. Crystal structure analysishas confirmed site directed mutagenesis studies, showingthat both mature IL-1� and IL-1� bind to the IL-1 receptor(IL-1RI) at two sites, contacting the first two domains ofthe receptor (site A) or the third domain (site B)[127].

The IL-1ra is a specific antagonist of IL-1, in that it bindsthe IL-1 receptor without transmitting any signal into thecell [128]. Directed mutagenesis results have shown that,unlike IL-1�/�, IL-1ra has only one binding site for theIL-1 receptor, explaining the absence of signal transmission[129]. The IL-1ra cDNA encodes a 177 amino acid proteinwhich contained a 25 amino acid signal peptide and allowedthe protein to be secreted into the extracellular environment,named secretory IL-1ra (sIL-1ra). Upon stimulation withLPS, human blood monocytes initially express this gene[130], but after 24 h the primary transcript in these cells is anintracellular form of IL-1ra (icIL-1ra) which lacks a leaderpeptide[100]. This form arises due to alternative splicingof the sIL-1ra mRNA which modifies the exon encoding forthe signal sequence[131,132]. A second form of icIL-1rais also generated by alternative splicing of the mRNA andhas been termed icIL-1ra type II[133–135]. It is thoughtthat these intracellular isoforms may represent a reservoir ofIL-1ra, released upon cell death, whose function is to limitthe pro-inflammatory action of cell debris.

3. IL-1 in vertebrates

Due to the importance of IL-1 in the immune systemof mammals and evidence to suggest its biological activ-ity in the immune system of non-mammalian vertebrates,work has focussed on isolating this gene in selected species.Other than mammalian IL-1� sequences there are now fullcDNA sequences available from chicken,Xenopus, trout,carp, goldfish, seabass and catshark (Table 2). Many of thesegenes have been obtained using an homology cloning ap-proach. The genomic organisation for these IL-1� geneshave also been obtained within selected species. In the caseof Ornithorhynchus anatinus(Duck-Billed Platypus), onlythe genomic sequence is available, due to this organism be-ing a protected species, with the predicted platypus IL-1�transcript being obtained with the aid of software, which

488 S. Bird et al. / Cytokine & Growth Factor Reviews 13 (2002) 483–502

searches for intron–exon boundaries[69]. To date, no se-quence data is available for IL-1� or IL-1ra, other than inmammals whilst IL-18 is known in birds as well as mam-mals (see later).

4. Known vertebrate IL-1� sequences

IL-1� has been characterised within a large number ofmammals, including a representative from the marsupials[68] and monotremes (GenBank accession no. AJ239048,[69]), which are known to be the most primitive mammalgroups. More recently IL-1� has been cloned from selectednon-mammalian vertebrates, which include chicken[70],Xenopus[71], trout[74], carp[78], seabass[72] and catshark[80]. When these genes are aligned amino acid homologyis found to be highest in regions containing the secondarystructure of the IL-1� molecule. This suggests the presenceof 12 �-sheets in each of these genes giving the proteinssimilar folding patterns and making each a member of the�-trefoil family [98]. The secondary structure dictates howthe molecule will fold, which gives the molecule its tertiarystructure. The correct folding of the IL-1� molecule is im-portant if it is to interact with the receptor and induce a signalinto the target cell. An early study showed that a histidineresidue, (position 146 in the human precursor protein) wascritical for this molecule binding to its receptor[136]. Thisamino acid residue appears to be conserved in the platypusand chicken molecules. The use of site specific mutagene-sis allowed the identification of another seven amino acids(Arg4, Leu6, Phe46, Ile56, Lys93 Lys103 and Glu105) in hu-man mature IL-1� that are essential for binding to the type IIL-1 receptor[137]. On the three-dimensional structure, theyform a cluster localised in one region of the IL-1� molecule.These amino acids are strongly conserved in other placen-tal mammal IL-1� proteins, partially conserved in platypusand possum IL-1� [68] and not conserved in the knownnon-mammalian sequences. In both the possum and platy-pus IL-1� five of the seven positions contain identical or re-lated amino acids. These amino acid differences may explainwhy placental mammal IL-1� recombinant protein does notcross-react well in possum and vice versa[68,138]. A morerecent comparison has identified two specific receptor bind-ing sites (sites A and B) which have been determined in hu-man IL-1� using the crystal structure of IL-1� complexedwith its receptor[139]. These regions in the non-mammaliansequences show little conservation in the alignment.

The IL-1 family signature, which spans�-sheets 9–11of IL-1�, is reasonably well conserved within each of thenon-mammalian genes (Fig. 1). All mammalian sequences,including the possum and platypus, contain this consensussequence. Both the chicken and theXenopussequences alsohave this signature pattern, but whether this will be true forall bird and amphibian species remains to be seen. Differ-ences begin to be found in the motif within the bony and car-tilaginous fish and slight changes are required to the motif

to allow the inclusion of these sequences to the IL-1 family(Fig. 1). In common, as well, is the presence of numerouscopies of an mRNA instability motif (attta) in the 3′ UTRof each IL-1� gene, which is typical of genes coding forinflammatory mediators[140].

Although the IL-1� sequences share the IL-1 family sig-nature and show the presence of possible�-sheets the over-all nucleotide and amino acid identities of these moleculeswithin vertebrates is quite low.Fig. 2shows that some of thehighest homologies are found between the mammals (hu-man, possum and platypus) whereas the lowest values arefound between the catshark and other vertebrates. This is notunexpected as this sequence represents the earliest knownIL-1� sequence isolated to date. All known IL-1� aminoacid and nucleotide sequences have also been comparedusing different methods of phylogenetic comparison to con-struct trees to look at the relationship of this molecule withinvertebrates. Methods used included neighbour joining[141],maximum parsimony[142] and maximum likelihood[143],although only the tree from neighbour joining is shown as thetrees constructed were fairly congruent (Fig. 3, [69]). In allcases the mammalian and bony fish form two separate cladeswith the relationship between them unresolved. Wheremethods attempted to resolve the relationship between thevertebrates, the data was not supported (for example themaximum likelihood tree obtained from amino acid datahas a bootstrap value of 55%). In each tree, the relation-ships of the bony fish are well supported, with the carp andgoldfish genes forming a separate group to the trout andseabass. The placement of the chicken and theXenopuschanges within the different trees, but in the majority ofcases they branch with the mammals. The bootstrap val-ues in the maximum parsimony tree and neighbour-joiningtrees obtained from amino acid data did not support thisrelationship. Increasing the representatives from each ofthese groups would probably help in resolving the group-ing of these organisms. Including all the IL-1� genesindicated that the platypus and possum appear to be an-cestral to all the mammals. The genetic distance betweenthe bony fish sequences from the catshark was less thanthe mammals, indicating a closer relationship with thissequence.

Within the trout a second IL-1� gene (IL-1�2) has beenfound [76]. It has a similar intron–exon organisation to thefirst trout IL-1� gene sequenced (IL-1�1) [75], except thatthere are small differences in the sizes of the introns and ex-ons. Multiple copies of IL-1� genes have also been foundin the carp (GenBank accession nos. AJ401030, AJ401031)and in the goldfish[69]. The presence of multiple copiesof IL-1 family genes is not just restricted to fish. Multiplegene have been found within various mammals. In humans,a number of novel homologue members of the IL-1 fam-ily have been found[83,144]. The roles of these at presentare speculative, with three being similar to IL-1ra and onebeing more related to either IL-1� or IL-18. The receptors,signalling pathways, biological activities and tissue expres-

S. Bird et al. / Cytokine & Growth Factor Reviews 13 (2002) 483–502 489

Fig. 1. Alignment of the IL-1 family signatures found to date in mammals, birds, amphibians, bony fish and cartilaginous fish IL-1� sequences. Abovethe alignment is the signature, which is found within all mammalian sequences. Below are the changes (shown in red) required to incorporate all thenon-mammalian sequences.

sion of these homologues have yet to be investigated. It isthought that these homologues may bind to some IL-1R-likereceptors that have yet to be paired with ligands. In pig twogenes for IL-1� have also been identified which share 86%amino acid identity[145]. The findings of two IL-1� genesin trout, carp and goldfish are not totally unexpected as thesefish have a tetraploid ancestry and are quite likely to havemultiples of certain genes. What will be of interest is to de-termine how these genes are regulated and what role, if any,these genes play in the fishes immune system.

What remains to be determined is whether polymorphismexists for IL-1� within non-mammalian vertebrates. In mam-

Fig. 2. Amino acid (yellow) and nucleotide (white) identities of IL-1� sequences from selected mammals and non-mammals.

mals, a certain amount of polymorphism exists for IL-1members since two allelic forms have been described forIL-1� [146], IL-1ra [147] and several for IL-1� [148]. Thesepolymorphisms may be of clinical importance since a spe-cific IL-1� allele is associated with juvenile polyarthritis[149], a rare IL-1� allele is related to the severity of rheuma-toid arthritis erosions[150] or certain types of diabetes[151]and a IL-1ra allele may contribute to susceptibility to sepsis[152]. A recent study has also shown that the IL-1 genotypeis significantly associated with the severity of adult peri-odonitis[153]. These finding could prove particularly usefulfor commercially important species, such as trout or seabass,

490 S. Bird et al. / Cytokine & Growth Factor Reviews 13 (2002) 483–502

Fig. 3. Rooted phylogram showing the phylogenetic relationships between all known IL-1� genes that have been sequenced within the vertebrates.Phylogeny was estimated using amino acid sequences with the neighbour-joining method and the resulting tree was bootstrapped 1000 times. Bootstrapvalues are shown as a percentage. Scale bar represents an estimate of the number of amino acid substitutions per site.

where a certain genotype could prove advantageous againsta particular disease or infection.

5. Processing of IL-1� in vertebrates

All vertebrate IL-1� genes found contain no apparentsignal peptide, as deduced by plotting hydrophobicity ofthe amino acids and using signal P v1.1[154], indicatingthat these molecules are secreted through a non-classicalpathway not involving the golgi/endoplasmic reticulumroute. Differences are found in how the mammalian andnon-mammalian pro-IL-1� molecules are processed intotheir mature form. The ICE cut site is present within allmammalian IL-1� sequences found to date, including theplatypus, although it remains to be tested experimentallythat this is the actual cut site within this primitive mammal.Throughout all other vertebrate groups in which IL-1� has

been cloned and sequenced, no ICE cut site is present.Fig. 4depicts the possible cut site within these peptides based onan alignment of all known non-mammalian vertebrates withselected mammalian sequences. The cut site for the maturepeptide has been predicted within the chicken[70], carp[155], and trout[156,157]and active recombinant peptidesproduced (Fig. 5). The existence in non-mammalian verte-brates of an ICE homologue and whether IL-1� requiresenzymatic cleavage to become biologically is under inves-tigation. ICE enzymes are likely to be present in lower ver-tebrates, since apoptosis is a well characterised event in fish[158] and related genes have been found in invertebrate’s[159]. Recent isolation of a caspase-1 enzyme sequencein chicken[160], numerous caspase homologs in zebrafish[161] and caspase-6 in trout[162] indicate that a homologfor caspase-1 could be present within all vertebrates. Studieshave also shown that the mammalian precursor IL-1� canbe cleaved at different amino acid residues and by different

S. Bird et al. / Cytokine & Growth Factor Reviews 13 (2002) 483–502 491

Fig. 4. Alignment of the cut site region of IL-1� sequences. The ICE cut site (aspartic acid) seen in all the mammalian sequences is in red. The potentialcut site regions in non-mammalian sequences are highlighted white. Amino acids in green relate to�-sheets.

cleavage enzymes to obtain a biologically active molecule.Granzyme A but not granzyme B, which are released fromcytotoxic T-lymphocytes, cleaves at Arg120 [163]. Elastase,trypsin and chymotrypsin, with chymotrypsin producing themost biologically active IL-1�, can cleave precursor IL-1�[164]. A mast cell chymase[126] and various extracellu-lar matrix metalloproteinases (MMP)[124] have also beenshown to produce biologically active IL-1�. These proteasesare found in inflammatory fluids, at sites of neutrophil,lymphocyte, or macrophage infiltration, and can therefore

Fig. 5. Northern blot showing the effect of LPS and rainbow trout recombinant IL-1� on IL-1� gene expression in rainbow trout macrophages.

participate in the generation of active IL-1�. As pro-IL-1�can be released from live cells or passively from deadcells [165], it is possible that extracellular processing ofpro-IL-1� by inflammatory proteases takes place. Evidencehas also been found in human plantar stratum and psoriaticscales where biologically active IL-1� has been activatedin a process not involving ICE[166]. Thus non-mammalianvertebrate IL-1� may be activated by cleavage by an enzymedistinct from an ICE homologue. The exact cut site, how thepro-peptides are processed and what protease is involved in

492 S. Bird et al. / Cytokine & Growth Factor Reviews 13 (2002) 483–502

non-mammalian vertebrates remains to be determined. Thequestion also remains as to the origin of the ICE cut site inIL-1� and due to the unavailability of reptilian sequencesfor IL-1� it is unknown if the ICE cut site is present withinthis group of vertebrates. If not then the use of ICE forthis purpose would appear to be an exclusively mammalianevent.

6. Vertebrate IL-1� gene organisation

The intron and exon arrangement of IL-1� genes havebeen determined within selected vertebrate species andcompared (Figs. 6 and 7). The precursor sequence of hu-man, mouse and pig IL-1� were the first to be sequencedand are found to span seven exons separated by six introns[56,60,65]. The first non-mammalian vertebrates to havetheir IL-1� genomic sequence discovered were trout, carpand chicken ([75,79]; GenBank accession no. AJ245728),followed by seabass[73] and Xenopus(GenBank acces-sion no. AJ314758,[69]). A monotreme mammal, theDuck-Billed Platypus (GenBank accession no. AJ239048,[69]), is also known.

Similar to the known mammalian IL-1� genes, carp con-sists of six introns and seven exons, with exon sizes similarto those seen in the human IL-1� gene. The chicken, troutand catshark IL-1� genes possess a different organisationcontaining only five introns, although trout appears to havelost a coding exon where chicken and catshark have lost a5′ UTR exon. The introns for the carp, trout and chickengenes are considerably smaller than the mammalian genes.In contrast, the catshark introns are much larger. The platy-pus andXenopussequence has the same intron/exon organi-sation to mammals, although the sizes of the introns do vary.The recently sequenced seabass gene organisation has a dif-ferent organisation again, containing four introns and fiveexons, and like trout appears to be missing an exon at the 5′end. Exons 4–7 appear to be relatively conserved betweenspecies whereas exons 1–3 appear in some organisations tobe spliced together or completely missing. On discovery ofthe trout IL-1� gene it was hypothesised tentatively that themammalian IL-1� had evolved by the introduction of extrasequence at the N-terminus[75]. With the introduction ofother non-mammalian sequences this still may hold true, al-though in view of the larger size of the coding region of thecatshark gene, the ancestral IL-1� gene may have lost cod-ing sequence as it evolved within some vertebrate groups.

There seems to be a lot of variation in the number ofintrons and where they are placed in the genomic organi-sation of IL-1�, which is especially seen in the bony fishwhere each species studied to date shows a different or-ganisation. However, this is not an unusual event as intronshave been shown to frequently occur in different positionsand/or different numbers in homologous genes of differ-ent species[167]. This is thought to be due to either gains(“introns-late”) or losses (“introns-early”) of introns, or per-

haps both. Evidence exists to support both the introns-earlyand introns-late hypotheses[168–170]. Furthermore, it hasbeen proposed that introns may speed up the evolution ofgenes by increasing recombination events between exons[168]. The sequence and size of introns may be evolutionaryimportant in directing the molecular organisation of genes.

7. Vertebrate IL-1� expression and regulation

The involvement of IL-1� in the non-mammalian verte-brate immune response is indicated by expression studies.In mammals, IL-1� is produced in response to many stimuliwhich include bacterial LPS, numerous microbial products,cytokines (TNF, IFN-�, GM-CSF and IL-2), T-cell/antigenpresenting cell interactions and immune complexes[171].Similar patterns of expression have also been seen in tissuesfound to contain lymphomyeloid tissue in chicken,Xeno-pus, bony fish and cartilaginous fish. In chicken, IL-1� isquickly induced in blood monocyte derived macrophagesreaching optimal levels within 1 h after LPS treatment[70].The XenopusIL-1� transcript was inducible in vivo fol-lowing injection with LPS[71]. Expression was clearlydetectable by RT-PCR in the kidney, liver and spleen, withthe brain showing the lowest expression levels. Northernblot analysis confirmed that the transcript was not constitu-tively expressed in the spleen and that there was a three-foldincrease post injection. Stimulation of splenocytes in vitrowith LPS also produced a significant increase in transcriptexpression. In carp, administration of PMA and LPS tophagocytes isolated from the head kidney induced expres-sion of IL-1� in these cells after 2 h stimulation[79]. Also,fish injected with immunopotentiators, sodium alginate andscleroglucan 48 h earlier showed increased IL-1� mRNAlevels in peritoneal and head kidney cells when comparedto saline injected fish[78]. In trout, head kidney leucocytesand isolated macrophages express IL-1� after stimulationwith LPS for 4 h ([74,172], Fig. 5). A total of 5�g/ml LPSis shown to produce maximal expression and was first de-tectable 1–2 h post stimulation with maximal induction at4 h. Culture temperature has been shown to have a markedeffect on trout IL-1� expression[172]. Using LPS a cleardose dependent induction of the trout IL-1�2 is shown incultured trout leucocytes[76]. Also, a trout mononuclearphagocyte cell line, RTS-11 cells express IL-1�. Stimu-lating these cells with LPS led to increased expression ofIL-1� [173]. More recently, LPS stimulation of seabassgill and head kidney cells in vitro for 4 h has also re-sulted in an increase in IL-1� expression, as seen usingRT-PCR and Northern blotting[72]. Expression studies inS. caniculusindicate that the IL-1� molecule is biologi-cally relevant to cartilaginous fish immune responses toGram-negative bacteria[80]. IL-1� was shown to be in-ducible in vivo following injection with LPS, 24 h earlier.Expression was clearly detectable by RT-PCR in the spleenand testes, tissues known to contain a variety of leucocytes

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Fig. 6. Genomic organisation of vertebrate IL-1� genes. Intron and exon numbering relates to mammalian genes. Non-coding regions of the exons arehighlighted in white (? denotes exact size unknown).

Fig. 7. Comparison of the gene organisation and intron/exon sizes between known vertebrate IL-1� genes.

494 S. Bird et al. / Cytokine & Growth Factor Reviews 13 (2002) 483–502

Fig. 8. Comparison of mammalian and non-mammalian vertebrate IL-1� promoters (? denotes exact size unknown).

[174]. Stimulation of splenocytes in vitro with LPS from0 to 8 h also resulted in a significant increase in transcriptexpression with maximal expression seen after 5 h. North-ern blot analysis confirmed that there was a seven-foldincrease in the transcript after 5 h incubation with LPS[69,80].

Within the seabass[72], trout [172] and carp[79] the ex-pression of incompletely spliced variants of the IL-1� genehas been shown. They appear to be expressed at lower levelsin these fish relative to the mature transcript and are not uni-versally present in all tissues that express IL-1�. It is thoughtthat the incompletely spliced forms of these cytokines maybe involved in the regulation of the mature transcript, as hasbeen seen in mammals in certain situations[175].

The regulation of the non-mammalian IL-1� genes hasalso been investigated with the sequencing of promotersfrom selected species, which include trout[176] and cat-shark (GenBank accession no. AJ295947). The IL-1� pro-moter has been examined extensively in humans and mice[177–185]and homologous positive and negative regulatoryelements for transcription factors have been found in thenon-mammalian IL-1� promoters. A TATA box is present24–27 bp upstream of the transcription start site in all speciesexamined. An NF-�B element, which is known to be cru-cial for activity within the immediate upstream promoter ofmammalian IL-1� [186] and the more upstream enhancer[184], has also been identified (Fig. 8). In trout an NF-�Bsite is located 19–21 bp (trout IL-1�1 and IL-1�2) upstreamof the TATA box whereas in the catshark it is 91 bp up-stream. It is known that IL-1� is a strong inducer of NF-�Band is thought to positively autoregulate its own synthesis,which appears to hold true in fish as the addition of troutrecombinant IL-1� to trout macrophages induces IL-1� ex-pression (Fig. 5). Preliminary studies using the trout IL-1�1promoter confirm that the NF-�B transcription factor site

is required for expression of the trout IL-1�1 gene[176].Other potential elements for transcription activators in thetrout and catshark promoters include sites for AP1, SP1 andCEBP.

8. Vertebrate IL-1 receptors

IL-1 receptors have been isolated within a few selectnon-mammalian species. Within trout a homologue of themammalian IL-1 type II receptor has been isolated[187],which is known as a ‘decoy’ receptor and thought to rep-resent a way of negatively regulating IL-1 activity. This re-ceptor gene is induced in trout head kidney leukocytes afterstimulation by LPS and TNF-�, consists of a signal peptide,transmembrane region, extracellular region with three Ig-likedomains and a short cytoplasmic tail which is characteris-tic of the mammalian IL-1 type II receptor. Homologues ofthe mammalian IL-1 type I receptor gene have been clonedin chicken[188] and salmon[189]. In the chicken the im-portance of this receptor has been shown as IL-1 activityis blocked when this receptor is produced in soluble form[190]. This receptor has also been shown to transduce a sig-nal into target cells as IL-1 activity is decreased when thisreceptor is blocked using an antibody.

9. Interleukin-18

IL-18 or IFN-� inducing factor (IGIF) was cloned in micefrom partial amino acid sequences deduced from the purifiedproteins[191] and later in humans[192]. Analysis of the pri-mary amino acid sequence of both human and mouse IL-18indicated the presence of an IL-1 signature-like sequence andit was proposed that IL-18 contained 12 strands of�-sheets

S. Bird et al. / Cytokine & Growth Factor Reviews 13 (2002) 483–502 495

Fig. 9. Amino acid alignment of the IL-18 cut site region from mammalian and chicken sequences with the aspartic acid coloured red. Identical (∗) andsimilar (: or .) residues are indicated.

forming the�-trefoil fold, typical of the IL-1 family[193].IL-18 exists as an inactive pro-IL-18 molecule until it iscleaved by ICE to generate the mature active form of IL-18[194,195]. It appears that the correct folding of IL-18 oc-curs prior to processing with ICE, at the level of pro-IL-18[196]. The structure and processing of this molecule suggestthat IL-18 and IL-1 originate from a common ancestor, al-though the target specificity’s and the receptor systems arecompletely different. It has also been reported that the IL-1receptor related protein is the functional component of theIL-18 receptor[197].

In contrast to what is observed with IL-1�, constitutivegene expression of IL-18 has been seen within various celltypes including unstimulated, freshly isolated human PBMCand murine splenocytes[198], and haemopoietic cell lines ofmyelomonocytic origin[199]. It has been shown that the pro-moter for IL-18 does not contain a TATA box and that pro-moter activity upstream of exon 2 acts constitutively[200].It is also found that the 3′ UTR of human IL-18 lacks theAUUUA destabilisation sequence, allowing for more sus-tained levels of the polyadenylated species and translationinto protein. The presence of preformed pro-IL-18 in thecell would allow for a very rapid production of the mature,active molecule once the cell is activated.

Recently, IL-18 was cloned within chicken[11], the onlynon-mammalian vertebrate to date known to have this cy-tokine. Sequence comparison reveals a putative ICE cleav-age site at aspartic acid 29 (Fig. 9). Synthesis of IFN-� incultured primary chicken spleen cells was detected when in-cubated with the chicken mature recombinant IL-18 usingthe predicted cut site. Thus the presence of ICE in chickenswould appear crucial for IL-18 processing in this species.

10. Cytokine and IL-1 evolution

The evolution of the genes that encode the current spec-trum of known mammalian cytokines and receptor com-plexes involved multiple duplications from a smaller set ofgenes, followed by the divergence of sequence and product

function [201]. To date, very little in fact is known aboutthe evolution of cytokine genes and much is inferred frommammalian sequences due to the lack of data from othervertebrate groups. A number of studies have attempted topredict the possible evolutionary history of several cytokinesusing mammalian nucleotide data.

In general cytokines have been found to be some of themost rapidly evolving genes known. A survey of 1196 or-thologous human and mouse genes found that 7 out of the 25most divergent genes were cytokines and cytokine receptors(Fas, IFN-�, IFN-�R, IL-4, IL-4R, IL-6, IL-6R), sharing aslittle as 51% nucleotide sequence identity and 41% aminoacid identity[202]. Using knockout mice a number of geneshave been termed essential and non-essential genes depend-ing on the survivability of these mice. It was found that alarge proportion of the non-essential genes are immune sys-tem genes, including cytokines, which appear to be evolvingfaster than the essential ones[203]. This rapid evolution ofcytokine genes is reflected in the finding that cross-reactivityof IL-1� between marsupial and placental mammals is low.IL-1 in marsupials was first demonstrated when a moleculewith molecular weight of 15–17 kDa was obtained fromopposum LPS stimulated macrophages and keratinocytes[138]. This IL-1 seemed to be non-stimulatory to mouse thy-mocytes and mouse and human IL-1 did not stimulate oppo-sum thymocytes. Recent work has isolated the IL-1� genefrom possum LPS stimulated lung alveolar macrophages[68]. The deduced amino acid sequence has 43–46% iden-tity to placental mammals and contained an ICE cut site. Thepossum recombinant IL-1� when given intravenously hada profound effect on the haematology of possums. A largetransient decrease in circulating leucocytes and primary neu-trophils followed by a large increase in numbers of both neu-trophils and lymphocytes was found. Also when used as anadjuvant with KLH, serum antibody levels were increasedsix-fold at 6 weeks after immunisation relative to unadju-vanted, immunised animals[204]. In agreement with earlierwork, there was little cross-reactivity between possum IL-1�and placental mammal leucocytes. These cytokine genesmay be under directional selection because of host-parasite

496 S. Bird et al. / Cytokine & Growth Factor Reviews 13 (2002) 483–502

co-evolution. One reason put forward for this high rate ofevolution for antimicrobial host defence proteins, such ascytokines is said to be due to pathogenic microorganisms us-ing molecular mimicry[205–208]. For example, numerousherpes viruses and pox viruses encode chemokine mimicsable to block chemokine action. Mutations in these immuneregulatory genes become favoured because they result in atemporary advantage until it is counteracted by mutations inthe pathogen genome.

Knowledge on the structure and function of cytokinesfrom different species is beginning to accumulate withthe use of such recombinant cytokines. Preliminary resultsfrom cross-reacting cytokines from different mammalianspecies has shown that there is an apparent threshold ofaround 60% amino acid identity[209]. Below this iden-tity, it is unlikely that cross-reactivity between cytokinesfrom different species will be found. Cross-reactivity ofdifferent cytokines can also be correlated to the foldingfamilies that the cytokines belong to. Cytokines belongingto the short 4-�-helical bundle cytokine family are the leastcross-reactive whereas cytokines belonging to the�-sheetbased folds are the most cross-reactive and also the mostconserved amongst the species studied. This may show thatcertain families are able to accumulate more differences intheir amino acid composition and still produce a proteinable to fold in the desired way.

A number of ideas have been formulated on the evolutionof IL-1 with the accumulation of sequence data and theircomparisons with closely related genes which share the same�-sheet structure. The IL-1 family belongs to the�-trefoilfamily, which also includes molecules, such as IL-18 and theFGF family of proteins. Although the FGF and IL-1 familieswere considered homologous, there was limited evidencebeyond similarity in three-dimensional structures to suggesta common ancestor for any pair of�-trefoil families [210].Each family possesses distinct functions, occur in extra- andintracellular contexts and lack similarities in either the typeof molecules with which they interact or in the location oftheir binding sites on the tertiary structure[211]. It was un-known if the fold arose independently several times withindifferent molecules during evolution, meaning that�-trefoilproteins could be examples of analogous or homologousgenes[212]. More recent work has analysed the significanceof sequence identities observed after structure alignment,which has provided evidence that�-trefoil proteins are ho-mologous, having arisen from a common ancestor[210]. Itis now thought that the�-trefoil fold arose from the dupli-cation of an ancestral gene encoding a homotrimeric singlerepeat protein.

Until recently, only mammalian sequences were availableto infer the possible evolution of the interleukin-1 familymembers. It was initially found that genes for human, mouseand rat IL-1ra, IL-1� and IL-1� have similar intron–exon or-ganisation, indicating that gene duplication events may havebeen important in the creation of this gene family[60,213]. Ithas been suggested that the IL-1ra duplicated from an ances-

tral IL-1 gene first, corresponding to the time of the appear-ance of amphibians (320–400 million years ago). IL-1� andIL-1� separation is thought to have occurred more recently(270–300 million years ago). An alternative hypothesis isthat IL-1� duplicated first from the ancestral IL-1, favouredby the fact that IL-1� has diverged more from IL-1ra andIL-1� than these two proteins have from each other. Whichever duplication took place first, the dates where duplicationsare thought to have taken place may suggest that bony andcartilaginous fish contain only one IL-1 gene. It has been fur-ther proposed that the common ancestor of IL-1� and IL-1rawas an alternatively spliced gene[214]. One transcript couldhave encoded the ancestral IL-1� while the other transcriptencoded the IL-1ra. After gene duplication, one copy of thegene would be freed to encode only the receptor antagonist.With the discovery of IL-1� sequences within vertebrates asancient as cartilaginous fish it is certainly clear that IL-1�had an early origin. However, it remains to be discoveredat what stage other IL-1 family members evolved.

11. Conclusion

The discovery of IL-1� genes in mammals, amphibians,bony fish and cartilaginous fish has major implications forinvestigation into the immune systems of vertebrates. Theidentification of the coding sequences for IL-1� within thesevertebrate groups is only the beginning and studies into thefunctional characteristics of the proteins encoded by thesegenes must now be investigated. Ultimately the productionof recombinant peptides for all non-mammalian genes willallow the bioactivity to be determined and compared be-tween vertebrate groups. The effect of molecules, such asIL-1 on the immune system is in its early stages and requiresintense investigation. Once a better idea of IL-1 mediatedimmune cell activity is established, the use of IL-1 or IL-1inhibitors may be realistically considered as a way to modu-late the immune response possibly at the time of vaccinationin commercially important vertebrate species. Whatever theoutcome, it is likely that IL-1 will be a major regulator ofthe immune responses in these other vertebrates as it is inmammals. In addition to analysing IL-1 ligands and theiractivities in vertebrates it will also be of interest to inves-tigate the signalling mechanisms in some species. Furtherwork needs to be done to detect or isolate IL-1 receptorgenes from tissues within non-mammalian vertebrates anddetermine expression patterns/regulations for each of thesereceptors, in addition to assessing ligand-binding kinetics.

Homology cloning has proved to be a useful tool withininvestigations for isolating and cloning cytokine sequencesfrom a wide variety of vertebrates[43,45,71,74,80,204]. Thecontinuing discovery of more cytokines has been improvedmore recently, using methods such as subtractive hybridi-sation [48,78]. Techniques, like this, are especially usefulwhen looking for molecules that may have diverged dur-ing evolution, which is not uncommon for cytokines. Also,

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with major advances within technology, the sequencing ofcomplete genomes is now becoming a possibility within arelatively short time[215,216]. The identification of allgenes within selected vertebrates other than mammals willimpact research into the evolution of cytokines dramati-cally. It will allow the identification of all cytokines, whichare mammalian homologues, may reveal molecules, whichare unique to certain organisms and will also give a betterinsight into where immune system communicators arose.

With the discovery of IL-1� within cartilaginous fish andthe presence in these vertebrates of components required fora functional innate and adaptive immune system, it givesgood reason to suspect that other cytokines are present at thislevel in vertebrate phylogeny. What is unknown is at whatlevel within evolution did these immune regulators arise,since it is now generally considered unlikely that cytokinesfrom vertebrates will have a homologue within invertebrates[5,217]. Whether IL-1 will be found within an organismmore primitive than a cartilaginous fish is under study withinjawless vertebrates. To date, little success has been had inidentifying cytokines within hagfish and lamprey, althougha homologue of IL-8 has been sequenced within the lam-prey,Lampetra fluviatilis[54]. It may be that cytokines suchas IL-1 arose within the jawed vertebrate lineage. It also re-mains to be determined where IL-1 family members such asIL-1� and IL-1ra arose during vertebrate evolution. It hasbeen speculated that these family members arose during theemergence of the amphibians[213]. There is still much thatremains to be discovered about the evolution of cytokineswithin vertebrates and it will be the focus of many studiesfor years to come.

Acknowledgements

Most of the non-mammalian sequences and the Duck-Billed Platypus sequence presented in this review wereobtained during a BBSRC programme (1/S09641) and aBBSRC studentship to S.B. In the case of the platypus se-quence, many thanks go to Niall Stewart in the collectingand curating of the monotreme tissues. The platypus mate-rial was collected under a permit (to B.M.) provided by theTasmanian Parks and Wildlife Service.

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