Steve Bird

Evolution of interleukin-1β

Abstract All jawed vertebrates possess a complex immune system, which is capable of anticipatory and innate immune responses. Jawless vertebrates posses an equally complex immune system but with no evidence of an anticipatory immune response. From these findings it has been speculated that the initiation and regulation of the immune system within vertebrates will be equally complex, although very little has been 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 the number of cytokine sequences available from primitive vertebrates growing rapidly. The identification of cytokines, which are mammalian homologues, will give a better insight into where immune system communicators arose and may also reveal molecules, which are unique to certain organisms. Work has focussed on interleukin-1 (IL-1), a major mediator of inflammation which initiates and/or increases a wide variety of non-structural, function associated genes that are characteristically expressed during inflammation. Other than mammalian IL-1β 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-1β gene has changed through vertebrate evolution and whether its role and regulation are conserved within selected non-mammalian vertebrates.

The interleukins of fish

Interleukins are a subgroup of cytokines, molecules involved in the intercellular regulation of the immune system. The term interleukin was first coined in 1979 to refer to molecules that signal between different leucocyte types, although not exclusively restricted to leucocyte communication. Whilst it is now known that interleukins are produced by a wide variety of cell types, nevertheless many are synthesised by CD4(+) T helper cells, macrophages/monocytes and endothelial cells. The nomenclature is relatively straightforward, with interleukin 1 the first discovered and interleukin 2 the second, etc. However, whilst 35 interleukins are currently described in mammals, several are in fact terms referring to subfamilies of more molecules, as with the IL-1 family where 11 members (IL-1F1-IL-1F11) are present, and the IL-17 family where 6 members (IL-17A-IL-17F) are present. So the total is much higher and splice variants and allelic variation increase this diversity further. This review will focus on what is known about interleukins in fish, and will refer to the major subfamilies rather than try to work through 35 descriptions in a row. It is clear that many direct homologues of molecules known in mammals are present in fish, but that not all are present and some novel interleukins exist that may have arisen from fish specific gene duplication events.

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

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

Characterisation and expression analysis of interleukin 2 (IL-2) and IL-21 homologues in the Japanese pufferfish, Fugu rubripes , following their discovery by synteny

This investigation provides the first conclusive evidence for the existence of the interleukin 2 (IL-2) and IL-21 genes in bony fish. The IL-2 and IL-21 sequences have been determined in Fugu rubripes by exploiting the conservation of synteny that is found between regions of the human and Fugu genomes. The predicted 149-amino acid IL-2 homologue contains the IL-2 family signature, has a predicted secondary structure of three α helixes and has the two cysteines important in disulphide-bond formation. It shows low amino acid identities (24–34%) with other known IL-2 sequences. The predicted 155-amino acid IL-21 homologue has a predicted secondary structure of four α helixes and has the four cysteines important in disulphide-bond formation. It shows low amino acid identities (29–31%) with other known IL-21 sequences. The gene organisation of Fugu IL-2 and IL-21 and the level of synteny between the human and Fugu genomes has been well conserved during evolution, with the order and orientation of the genes matching exactly to human Chromosome 4. Phytohaemagglutinin stimulation of Fugu kidney cells resulted in a large increase in the Fugu IL-2 and IL-21 transcripts. In vivo stimulation of Fugu with LPS and poly I:C showed IL-21 expression to be localised within mucosal tissues. The discovery of IL-2 and IL-21 in fish will now allow more detailed investigations into T-helper cell responses.

Identification of a Novel IL-1 Cytokine Family Member in Teleost Fish

A novel IL-1 family member (nIL-1F) has been discovered in fish, adding a further member to this cytokine family. The unique gene organization of nIL-1F, together with its location in the genome and low homology to known family members, suggests that this molecule is not homologous to known IL-1F. Nevertheless, it contains a predicted C-terminal β-trefoil structure, an IL-1F signature region within the final exon, a potential IL-1 converting enzyme cut site, and its expression level is clearly increased following infection, or stimulation of macrophages with LPS or IL-1β. A thrombin cut site is also present and may have functional relevance. The C-terminal recombinant protein antagonized the effects of rainbow trout rIL-1β on inflammatory gene expression in a trout macrophage cell line, suggesting it is an IL-1β antagonist. Modeling studies confirmed that nIL-1F has the potential to bind to the trout IL-1RI receptor protein, and may be a novel IL-1 receptor antagonist.

Cloning and expression analysis of three striped trumpeter (Latris lineata) pro-inflammatory cytokines, TNF-α, IL-1β and IL-8, in response to infection by the ectoparasitic, Chondracanthus goldsmidi.

This study reports the cloning and sequencing of three striped trumpeter (Latris lineata Forster) pro-inflammatory cytokines, TNF-alpha, IL-1beta and IL-8, as well as their differential expression in response to an infection by the ectoparasite Chondracanthus goldsmidi. The striped trumpeter TNF-alpha transcript consisted of 1093 bp, including a 759 bp ORF which translated into a 253 aa transmembrane peptide. The sequence contained a TACE cut site, that would produce a 167 aa soluble peptide containing the TNF ligand family signature. The IL-1beta sequence consisted of 963 bp, including a 774 bp ORF which translated into a 258 aa protein. The protein lacked both a signal peptide and an ICE cleavage site, but did contain the IL-1 family signature. The sequence for the chemokine IL-8 contained 906 bp, with an ORF of 297 bp, which translated into a 99 aa protein. The protein lacked an ELR motif as is common with many teleost IL-8 sequences. The differential expression of the three cytokine genes in parasitized fish was investigated via quantitative real-time PCR. A significant up-regulation of all three pro-inflammatory cytokines was found in the gills, which were the site of parasite attachment. Examination of head kidney cells revealed a significant up-regulation of TNF-alpha, but not IL-1beta or IL-8. Conversely, the spleen cells showed significant up-regulation of both IL-1beta and IL-8, but not TNF-alpha. These findings allow for more detailed investigations of the striped trumpeter immune response.

Identification and characterization of the transcription factors involved in T-cell development, t-bet, stat6 and foxp3, within the zebrafish, Danio rerio.

The discovery of cytokines expressed by T‐helper 1 (Th1), Th2, Th17 and T‐regulatory (Treg) cells has prompted speculation that these types of responses may exist in fish, arising early in vertebrate evolution. In this investigation, we cloned three zebrafish transcription factors, T‐box expressed in T cells (t‐bet), signal transducer and activator of transcription 6 (stat6) and fork‐head box p3 (foxp3), in which two transcripts are present, that are important in the development of a number of these cell types. They were found within the zebrafish genome, using a synteny approach in the case of t‐bet and foxp3. Multiple alignments of zebrafish t‐bet, stat6 and foxp3 amino acids with known vertebrate homologues revealed regions of high conservation, subsequently identified to be protein domains important in the functioning of these transcription factors. The gene organizations of zebrafish t‐bet and foxp3 were identical to those of the human genes, with the second foxp3 transcript lacking exons 5, 6, 7 and 8. Zebrafish stat6 (21 exons and 20 introns) was slightly different from the human gene, which contained 22 exons and 21 introns. Immunostimulation of zebrafish head kidney and spleen cells with phytohaemagglutinin, lipopolysaccharide or Poly I:C, showed a correlation between the expression of t‐bet, stat6 and foxp3 with other genes involved in Th and Treg responses using quantitative PCR. These transcription factors, together with many of the cytokines that are expressed by different T‐cell subtypes, will aid future investigations into the Th and Treg cell types that exist in teleosts.

Rainbow trout interleukin-2: cloning, expression and bioactivity analysis.

In this study the rainbow trout (Oncorhynchus mykiss) interleukin-2 (IL-2) cDNA has been cloned, and its expression and bioactivity analysed in head kidney leucocytes. The IL-2 precursor encoded an open reading frame of 429 bp, that translates into a predicted protein of 142 aa, with a 20 aa signal peptide. The trout IL-2 had moderate protein homology (30.9% identity/48.3% similarity) with Fugu IL-2, the only IL-2 homologue identified in fish to date, with lower homology to avian (17.8% identity/23.2% similarity) and mammalian (34.2 identity/46.5% similarity) IL-2s. IL-2 expression was induced by the T cell mitogen PHA and by the mixed leucocyte reaction, where leucocytes from pairs of fish were cultured together for four days. Expression was also induced in vivo during bacterial (Yersinia ruckeri) infection. The Escherichia coli produced recombinant IL-2 was shown to increase the expression of two transcription factors, STAT5 and Blimp-1, known to be involved in IL-2 signalling in mammals, as well as IFN-gamma, gIP and IL-2 itself. The potential signalling pathways involved and possible use as an adjuvant for fish vaccines are discussed.

Molecular cloning of the gene for interleukin-1β from Xenopus laevis and analysis of expression in vivo and in vitro

Abstract The Xenopus cDNA for interleukin-1β (IL-1B) was cloned and sequenced. The gene contains 1462 nucleotides that translate in a single reading frame to give a predicted 283-amino acid IL-1β molecule. The translated molecule contains a single potential glycosylation site, a readily identifiable IL-1 family signature, and has highest homology to chicken IL-1β by phylogenetic tree analysis and sequence similarity. It lacks a signal peptide in common with other known IL-1B genes, and lacks a clear ICE (caspase) cut site in common with other nonmammalian IL-1B genes sequenced to date. RT-PCR was used to study sites of IL-1B transcript expression, 24 h following injection of lipopolysaccharide (LPS). Expression was detected in the brain, liver, kidney, and spleen, with expression weakest in the brain and strongest in the spleen. No transcript expression was detectable following injection of saline. Northern blot analysis was used to quantify the induction of IL-1B expression in splenocytes following in vivo or in vitro stimulation with LPS. The results are discussed in relation to the potential role of IL-1β in amphibian immune responses.

Phylogenetic analysis of vertebrate CXC chemokines reveals novel lineage specific groups in teleost fish

In this study, we have identified 421 molecules across the vertebrate spectrum and propose a unified nomenclature for CXC chemokines in fish, amphibians and reptiles based on phylogenetic analysis. Expanding on earlier studies in teleost fish, lineage specific CXC chemokines that have no apparent homologues in mammals were confirmed. Furthermore, in addition to the two subgroups of the CXCL8 homologues known in teleost fish, a third group was identified (termed CXCL8_L3), as was a further subgroup of the fish CXC genes related to CXCL11. Expression of the CXC chemokines found in rainbow trout, Oncorhynchus mykiss, was studied in response to stimulation with inflammatory and antiviral cytokines, and bacterial. Tissue distribution analysis revealed distinct expression profiles for these trout CXC chemokines. Lastly three of the trout chemokines, including two novel fish specific CXC chemokines containing three pairs of cysteines, were produced as recombinant proteins and their effect on trout leucocyte migration studied. These molecules increased the relative expression of CD4 and MCSFR in migrated cells in an in vitro chemotaxis assay.