Invertebrate and fish cytokines

ARTICLE

CYTOKINE-LIKE ACTIVITIES IN INVERTEBRATES

The pivotal role played by cytokines in innate and acquired defence system, and the similarity of their properties among different animal groups, indicates that these proteins may be factors conserved through evolution. Recognition between distant cells is a phenomenon almost as old as eucaryotic organisms themselves, dating back one billion years. A clear example of cell-to-cell recognition can be found in protozoa, during sexual reproduction when recognition and signalling occurs between cells having a cell surface-associated set of "permissive" molecules, allowing conjugation and exchange of genetic material between individual cells. In a species of marine protozoa, it has been elegantly shown that secreted soluble mediators inducing sexual conjugation display striking structural similarities with mammalian IL-2, suggesting a conservation of this molecular structure in cell signalling during evolution [1].

In metazoan invertebrates, some data have been recently reported indicating the presence of genes or biological activities resembling those of cytokines and cytokine-associated molecules. For example, in nematodes a gene coding for a TNF receptor-associated factor (TRAF) has been recently identified [2], a finding which suggests the existence of a TNF receptor and of its ligand.

In protostomes (e.g., annelids, molluscs and insects), which display cell-mediated and humoral defence reactions [3], cells responsible for the defence mechanisms against non-self are a complex population present in body fluids, collectively called hemocytes [4]. In snails, exposure of hemocytes to mammalian IL-1alpha, IL-1beta, TNF-alpha or TNF-beta resulted in profound alteration of their ability to release biogenic amines [5]. However, no further data are provided in this work on the mechanisms by which these cytokines exerted their biological activity. In other molluscs (e.g., mussels), immunocytochemical analysis of the nervous system showed that glial cells were positive to staining with polyclonal antisera raised against mammalian IL-1alpha and IL-6 [6, 7]. In mammals, genes coding for inflammatory cytokines such as IL-1, IL-6 and IL-8 can be controlled by the activation of NFkappaB. Molecular events triggering NFkappaB activation are in turn controlled by the immune function-related protein Toll [8, 9]. The Toll gene has been shown to be present in insects and to be involved in the embryogenesis and defence functions of Drosophila [9-11]. Despite the difference in their ligand-binding ectodomains, the cytoplasmic domains of Toll and of receptors for IL-1 and IL-18 show striking similarities in their amino acid sequence [12, 13]. Indeed, molecules involved in the signal transduction pathway initiated by IL-1R and IL-18R (MyD88, IRAK, NFkappaB/IkappaB) all have homologous counterparts in the Toll signaling pathway (Tube, Pelle, Dorsal/Cactus) [9, 14-16]. These similarities raise the question of whether co-evolution of intracellular signalling pathways might be driven by conservation of the ligand molecule binding to the extracellular domain of the receptor. In earthworms, a defence molecule activates prophenoloxidase activity in response to microbial invasion [17]. This molecule displays activities similar to mammalian TNF-alpha, although no genetic relationship can be found between the two mediators, suggesting that they emerged independently during evolution. Very recently, evidence has accumulated indicating the evolution of analogous mechanisms of defence against pathogens in invertebrates and vertebrates. In mosquitos, bacterial infection can induce the expression of a proteinase (Easter) whose substrate is the molecule Spätzle (the ligand for Toll), which in turn is activated and induces anti-microbial peptide synthesis [18]. Similarily, in Drosophila antifungal defence reactions are controlled by the Spätzle/Toll/Cactus gene group [10, 19, 20]. These studies show that mutations within genes of this group can cause the constitutive expression of serine proteinase(s), which in turn induce the continuous production of antimicrobial peptides. Moreover, the first structurally characterised invertebrate protein belonging to the cystine knot superfamily was discovered in horseshoe crabs [21], this protein being a coagulogen greatly similar to the insect morphogen Spätzle [22]. From these reports it is tempting to hypothesize a common origin for some processing proteinase cascades in arthropods. The similarity between these proteinases and the vertebrate IL-1 processing enzyme ICE (see below) and caspases exemplifies the divergent evolution of proteinase cascades with different functions during evolution.

In protochordates (ascidians), characterisation of IL-1-like activity was reported in blood cells of a tunicate [23]. This IL-1-like activity induced increased vascular permeability in rabbit skin and was neutralised by a polyclonal anti-human IL-1 antiserum. In another protochordate, mRNA coding for IL-1beta was found by in situ hybridisation in the nervous system [24].

For a comprehensive view, Table 1 summarizes the main reports and studies of invertebrate soluble mediators, including invertebrate molecules detected by cross-reactivity with mammalian cytokine probes.

CYTOKINES AND FISH

Comparative immunology is a field in rapid growth, and knowledge of fish immunology is increasing continuously. However, knowledge on the evolution of vertebrate cytokine genes is comparatively meager, at least with respect to other well known genes such as those coding for immunoglobulins, T cell receptor and MHC. Considerable progress has been made with chicken cytokine genes, whereas in reptiles and amphibians evidence is mostly based on immunocytochemistry with antisera to mammalian cytokines or on bioactivity [25-27].

The most ancient group of living, jawed vertebrates are cartilaginous fish (sharks and rays). It has been supposed that the mechanisms of vertebrate immunity and adaptive responses had their origin in these organisms [28]. Convincing evidence supporting this hypothesis were the discovery of a humoral response in sharks [29], and later, the identification of T cell receptor (TCR) [30] and MHC genes [31]. However, virtually nothing is known about cytokines in cartilaginous fish. On the other hand, in agnathans (jawless vertebrates), represented today by lampreys and hagfish, a homologue of mammalian IL-8, called LFCA-1, has been cloned [32]. This molecule has great similarity to chicken EMF-1 (40%) and to mammalian IL-8 (32-33%), but lacks the ELR motif essential for the neutrophil chemoattractant function of mammalian IL-8-related chemokines.

In fish, most work has been done in teleosts, which are the largest group of vertebrates (about 20,000 species). Teleosts arose around 300 million years ago and display features of the immune system present in modern animals [33]. These features include anatomical organisation [34], presence of functional lymphocytes [35-37], MHC [38], TCR [39] and presence of cytokines [40, 41]. Experimental evidence has accumulated recently showing that gut-associated lymphoid tissue (GALT) of teleosts contains an elevated number of T cells [42] displaying an alphabeta⁺ phenotype [43]. Their presence in fish may represent the first step in the evolution of adaptive mucosal immunity [44].

Teleosts are also important experimental models for the application of immunological studies to biotechnology. Aquaculture is a field in rapid growth, with many freshwater and marine species having been introduced into fish farms. Several diseases can affect fish at all stages of their life cycle, and knowledge of the immune system is of major importance, as this may allow the introduction of treatments (e.g., vaccines and immunostimulants) as alternatives to the use of chemicals and antibiotics which pose a number of environmental concerns.

Early reports of the fish immune system date back to the forties [45]. Later, the antibody response of a fish to a viral antigen was studied [46]. Shortly afterwards, the discovery of T cell functions was reported [47], and later, the identification of T cells [48]. After these pioneering studies, amplifying/regulatory leucocyte products were identified in teleost fish. In fact, supernatants of PHA-activated pronephric leucocytes from carp (the pronephros, or head kidney, is a site of active lymphopoiesis and is functionally similar to the bone marrow in other vertebrates) contain a lymphocyte growth factor which induces proliferation of purified lymphoblasts [49]. The presence in fish of typical inflammatory activities, with exudates containing neutrophils and macrophages, raised the question of the possible presence of inflammatory cytokines [50]. In trout, studies on the elevation of macrophage respiratory burst activity with macrophage-derived supernatants suggested the production of TNF-alpha and transforming growth factor-beta (TGF-beta) factors [51]. TGF-beta belongs to a pleiotropic cytokine family involved in tissue remodelling, wound repair, development and haematopoiesis [52]. Three isoforms of TGF-beta recently successfully identified and cloned in bony fish; TGF-beta1 and TGF-beta2 in teleosts [53-55], and TGF-beta3 in teleosts and chondrosteans [56]. It is interesting to note that whilst these three isoforms are present in fish, birds and mammals, TGF-beta3 is absent from amphibia, suggesting a whole gene deletion event could have occurred in this group of vertebrates. The genomic organisation of the TGF-beta genes is fully known in humans, chickens and amphibians, and shows that each gene contains six introns. The trout TGF-beta gene lacks intron 2 present in other vertebrates, whereas an additional intron is present at the 3'-end, splitting exon 7 in two parts [57]. Despite this, there is a close similarity in the TGF-beta exon sequences among all vertebrates investigated. On the other hand, the introns show lower homology and are smaller in trout compared with birds, the only other animal group where introns are fully sequenced [57].

Interferon production in fish has been long established, as exemplified by the ability of leucocytes from the anterior kidney of trout to produce potent antiviral activity when stimulated with poly I:C or cell membrane-associated infectious hematopoietic necrosis virus (IHN) [58]. Whether fish leucocytes can secrete type II or g interferon (IFN-g) activity has also been addressed. Macrophage activating factor (MAF)-containing supernatants, generated by mitogen stimulation of rainbow trout leucocytes, were found to confer antiviral resistance on a rainbow trout epithelial cell line challenged with infectious pancreatic necrosis virus, both biological activities being typically heat-sensitive [59]. Leucocytes from vaccinated Atlantic salmon have also been found to produce MAF activity following stimulation with outer membrane protein antigens of the Gram-negative microorganism Aeromonas salmonicida [60]. These findings were extended to other fish species, since MAF activities were induced in catfish in response to another Aeromonas species [61], and in gilthead sea bream in response to mitogens [62].

Suppression subtractive hybridization is a powerful molecular technique, which has permitted the molecular cloning of several fish homologues of mammalian cytokines, as for instance in carp, where pre-B cell enhancing factor (PBEF), a CC chemokine, CXC chemokine receptors, allograft inflammatory factor-1, natural killer cell enhancing factor and IL-1beta were cloned and sequenced [63-65]. PBEF has been cloned in humans and fish only, the two genes showing a remarkable 86% homology between the deduced peptide sequences, and both lacking a signal sequence. Chemokines are small, inducible proteins which direct the migration of leucocytes, which can be grouped into two major families. CXC chemokines (or chemokines alpha; prototype being IL-8) have a characteristic group of two cysteines near the N-terminal separated by one amino acid residue. On the other hand, in CC chemokines (or chemokines beta) the two cysteines are adjacent. In trout, a putative chemokine (CK-1) belonging to the CC chemokine family has been cloned, whose deduced peptide sequence displays striking similarities to mammalian chemokines beta, in particular with the C6 subfamily [66].

Interleukin-1 (IL-1) is the common name for two distinct proteins, IL-1alpha and IL-1beta, members of a growing family of regulatory and inflammatory cytokines. Biological activities of IL-1 have been extensively reviewed [67]. Along with IL-1 receptor antagonist (IL-1ra) and IL-18, IL-1alpha and IL-1beta play pivotal roles in the regulation of acute inflammation. Both IL-1 molecules are produced as a 31 kDa precursor, share about 23% homology in their peptide sequence and, in common also with IL-1ra, IL-18 and FGF, have a beta-trefoil structure composed of twelve beta-sheets [67]. The inactive IL-1beta precursor must by cleaved intracellularly by the IL-1beta converting enzyme (ICE) to release the biologically active form [68]. IL-1alpha and IL-1beta bind differentially to the two types of IL-1 receptor (IL-1R) [69], by interacting with different domains of the receptor molecules [70]. It has been known for many years that channel catfish macrophages and carp epithelial and macrophage cell lines can produce an IL-1-like bioactivity [71, 72]. More recently, this observation was extended to carp macrophages and neutrophils [73, 74]. Thymocytes of channel catfish were shown to be responsive, in terms of proliferation, to mitogenic stimulation only in the presence of accessory cells (peripheral blood monocytes) or monocyte-derived supernatants, presumably containing IL-1 [75]. In these studies, antisera to human IL-1 could inhibit the biological activity of fish IL-1, suggesting a similarity in structure between fish and human IL-1, if not sequence homology.

Using degenerate primers designed to amplify evolutionarily-conserved regions in the IL-1 molecule, cDNA from cells likely to secrete IL-1 has been used in PCR in the search for fish IL-1. This approach has been successful for the cloning of IL-1beta from rainbow trout [76], the first non-mammalian sequence obtained, and for carp [63]. The molecular mass of biologically active IL-1 in fish has been determined to be in the range of 15 to 70 kDa in catfish [77] and 15-22 kDa in carp [73]. From cloned genes, the full-length IL-1beta precursor in trout [78] and carp [63] is predicted to be 28 kDa. The IL-1beta gene contains seven exons in mammals. Studying this gene in the two fish species (trout and carp), where complete data are available has revealed interesting features. In trout, a smaller gene is present consisting of six exons (exon 1 or 2 is missing) [79], whereas carp possess a more "mammalian-like" gene with seven exons [80]. Thus, despite salmonids being ancient teleosts that preceded the cyprinids (carp) by millions of years in evolution, they appear to have a divergent IL-1beta gene organisation. Furthermore, fish IL-1 activity can elicit IL-2 secretion by mammalian T cell lines.

Fish IL-1beta lacks the sequence coding for the interleukin-1 converting enzyme (ICE) cleavage site [76], which can however be found in other non-mammalian vertebrates (e.g., chicken, Xenopus). Nevertheless, by alignment with known sequences, the putative initiation site of the mature peptide can be predicted. In trout IL-1beta, the site between Arg⁹⁴ and Ala⁹⁵ appears the most likely cleavage site. Indeed, recombinant proteins beginning with Ala⁹⁵ are biologically active [81]. Very recently, a second IL-1beta gene was cloned and shown to be expressed in rainbow trout [82]. The IL-1beta2 gene displays 82% amino acid sequence similarity to the previously cloned IL-1beta and is similarily composed of six exons and five introns. The biological activity of this second form remains to be determined.

Apart from classical stimuli used to induce expression of IL-1beta genes in trout cells, studies on environmental or biological factors influencing expression of the IL-1beta genes are currently in progress [83]. These studies showed that IL-1beta expression by leucocytes can be upregulated in vitro by LPS and inhibited by low temperature and cortisol.

RT-PCR experiments were run in LPS-stimulated head kidney cells of sea bass with the same set of degenerated primers employed to detect trout IL-1beta. In this way, it has been possible to amplify a DNA having the expected size of IL-1beta DNA. Primer sequences were 5'-GGGAAAGAATCTRTACCTGTCYTG-3' (forward), and 5'-TGAGAGGTGCTGATGAACCAGT-3' (reverse). Controls for the presence of genomic DNA contamination were negative. DNA amplified by RT-PCR was fractionated by agarose electrophoresis and inserted into pCR2.1-topo 3.9 kb vector (Invitrogen Europe, De Schelp, NL). Sequence analysis was performed using the Sequence Analysis Software Package version 10 (1999) at the Wisconsin Genetic Computer Group and the PCGene software from Intelligenetics, Inc (Oxford, UK). Homologies to known genes were determined with the Expasy database using the FASTA [84] and CLUSTAL [85] programs. After sequencing and comparison with databases, the sea bass DNA results were similar to other known IL-1beta sequences, the best scores being with trout IL-1beta (59% amino acid similarity; Figure 1). RT-PCR experiments using the same primers were also performed on RNA extracted from stimulated head kidney and gill leucocytes and from purified T cells from gut-associated lymphoid tissue of sea bass (Figure 2). Expression could only be detected in stimulated kidney cells. These results strongly suggest that we have cloned part of a functional sea bass IL-1beta gene. Experiments are in progress to obtain a full length cDNA suitable for expression studies.

Another member of the beta-trefoil cytokine family has been cloned in fish, FGF-3. In zebrafish, its presence, secretion pathways and biological (mitogenic) activity have been studied and have revealed intermediate properties relative to mouse and Xenopus homologues [86]. By comparing database sequences, a trefoil factor family (TFF)-domain has been found in many non-cytokine proteins, strongly suggesting that this domain arose early in evolution [87].

For a comprehensive summary of fish soluble mediators and cytokines see Table 2.

CYTOKINE RECEPTORS

Even less is known about cytokine receptors in invertebrates and fish than is known about cytokines and cytokine-like activities. Chemokine receptors belonging to families of CXC receptors (CXCR4) and CC receptors (CCR7) have been recently cloned and sequenced in rainbow trout and carp [88, 65]. The sequencing of CCR7 in trout raised the question of whether this receptor may be involved in mechanisms of selection of memory T lymphocytes, as occurs in mammals [91]. As for mammalian chemokine receptors, these putative receptors are G protein-linked molecules of the rhodopsin receptor superfamily. However, no information is available on the binding properties of these molecules for chemotactic ligands. In the puffer fish, both the platelet-derived growth factor receptor beta (PDGFRbeta) and macrophage colony-stimulating factor-1 receptor (CSFIR) have been sequenced from genomic DNA [90]. The two TNF receptors have also been cloned recently in expressed sequence tag (EST) studies in the Japanese flounder [89]. In trout, the common cytokine receptor g chain (CRgC), an essential component of IL-2, IL-4, IL-7, IL-9 and IL-15 receptors, has been cloned [92]. The CRgC receptor displays motifs (e.g., WSXWS) in its gene sequence similar to those of mammalian CRgC and other haematopoietic cytokine receptors. Its importance is illustrated by the impaired T cell and B cell development seen in CRgC-deficient mice [93]. Whilst little is known of cytokine receptor signalling in fish, for the first time a direct involvement of second messengers and protein kinase C in cytokine-induced signal transduction has been observed [94].

CONCLUSION

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