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¹ Department of Animal Physiology, Institute for Neuroscience, Radboud University, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands
² Department of Cell Biology and Immunology, Wageningen University, Wageningen, The Netherlands

(Requests for offprints should be addressed to M O Huising; Email: m.huising{at}science.ru.nl)

	Abstract

Top Abstract Introduction Class-I helical cytokines share... The modular make-up of... Intracellular signalling... Redundancy and specificity in... Class-I helical cytokines in... 'Short-chain' class-I helical... Vertebrates outdated: insect... Summary and perspectives References

 
The class-I helical cytokines constitute a large group of signalling molecules that play key roles in a plethora of physiological processes including host defence, immune regulation, somatic growth, reproduction, food intake and energy metabolism, regulation of neural growth and many more. Despite little primary amino acid sequence similarity, the view that all contemporary class-I helical cytokines have expanded from a single ancestor is widely accepted, as all class-I helical cytokines share a similar three-dimensional fold, signal via related class-I helical cytokine receptors and activate similar intracellular signalling cascades. Virtually all of our knowledge on class-I helical cytokine signalling derives from research on primate and rodent species. Information on the presence, structure and function of class-I helical cytokines in non-mammalian vertebrates and non-vertebrates is fragmentary. Consequently, our ideas about the evolution of this versatile multigene family are often based on a limited comparison of human and murine orthologs. In the last 5 years, whole genome sequencing projects have yielded draft genomes of the early vertebrates, pufferfish (Takifugu rubripes), spotted green pufferfish (Tetraodon nigroviridis) and zebrafish (Danio rerio). Fuelled by this development, fish orthologs of a number of mammalian class-I helical cytokines have recently been discovered. In this review, we have characterised the mammalian class-I helical cytokine family and compared it with the emerging class-I helical cytokine repertoire of teleost fish. This approach offers important insights into cytokine evolution as it identifies the helical cytokines shared by fish and mammals that, consequently, existed before the divergence of teleosts and tetrapods. A ‘fish–mammalian’ comparison will identify the class-I helical cytokines that still await discovery in fish or, alternatively, may have been evolutionarily recent additions to the mammalian cytokine repertoire.

	Introduction

Top Abstract Introduction Class-I helical cytokines share... The modular make-up of... Intracellular signalling... Redundancy and specificity in... Class-I helical cytokines in... 'Short-chain' class-I helical... Vertebrates outdated: insect... Summary and perspectives References

 
Class-I helical cytokines are considered a monophyletic group, although they share little primary sequence identity (Bazan 1990b). This implies that a single ancestral gene that expanded by successive gene duplication events (largely in the vertebrate lineage) is at the basis of the contemporary multigene class-I helical cytokine family. All class-I helical cytokines fold into a bundle of four -helices and signal via related receptors that share molecular signatures (Bazan 1990a, Thoreau et al. 1991); moreover, following ligand binding, these receptors activate similar intracellular signalling pathways (Taga & Kishimoto 1997, Gadina et al. 2001, Vosshenrich & Di Santo 2002, Heinrich et al. 2003). Our knowledge about class-I helical cytokines is largely based on mammalian studies and therefore offers only a limited view on the evolutionary history of this large and important family of protein signals. Spurred by the recently acquired knowledge about the genome sequences of different bony fish species such as pufferfish (Takifugu rubripes), spotted green pufferfish (Tetraodon nigroviridis) and zebrafish (Danio rerio) (Aparicio et al. 2002, Jaillon et al. 2004), several fish class-I helical cytokines have recently been discovered. This now allows us to compare the class-I helical cytokine repertoires of different vertebrate classes. All fish class-I helical cytokines described to date share limited primary sequence identity with their mammalian orthologs. In contrast, the gene structure of orthologous cytokines is conserved throughout vertebrates, a characteristic that is instrumental in the identification of orthologous relationships within the multigene cytokine family that is characterised by poor primary sequence conservation. The instrumental importance of conserved exon size and intron phase is also true for class-I and -II helical cytokine receptor genes, as was already recognised over a decade ago (Lutfalla et al. 1992, Nakagawa et al. 1994). Intron/exon structures have also been used as a criterion for the identification of fish orthologs to mammalian class-II helical cytokines and their receptors (Lutfalla et al. 2003). Even though the fish helical cytokine repertoire is as yet not completely known, among the fish helical cytokines characterised to date are several examples of conspicuous differences between fish and mammalian orthologs that illustrate the eventful history of vertebrate class-I helical cytokines. Following an introduction on the principles of class-I helical cytokine receptors and their intracellular signalling that is based on mammalian studies, we separately introduce each mammalian class-I helical cytokine and discuss the evidence for their presence in early vertebrates.

	Class-I helical cytokines share a common fold

Top Abstract Introduction Class-I helical cytokines share... The modular make-up of... Intracellular signalling... Redundancy and specificity in... Class-I helical cytokines in... 'Short-chain' class-I helical... Vertebrates outdated: insect... Summary and perspectives References

 
The tertiary structure of class-I helical cytokines is characterised by a bundle of four tightly packed -helices, designated helix A–D (Fig. 1); their primary amino acid sequences share little to no sequence similarity. Their common fold separates class-I helical cytokines from other cytokines. For example, class-II helical cytokine molecules (interferons (IFNs), interleukin (IL)-10 and IL-20) contain over four -helices each (Ealick et al. 1991, Zdanov et al. 1995). The IL-1 family, which includes IL-1ß and IL-18, is characterised by a fold rich in ß-strands (Veerapandian et al. 1992). A unique aspect of the class-I helical cytokine fold is that the four -helices are arranged in an ‘up-up-down-down’ fashion as a result of the anti-parallel orientation of two consecutive pairs of helices. In most class-I helical cytokines this four-helix bundle fold is stabilised by up to three disulphide bridges. In IL-11, which lacks conserved cysteine residues, the four-helix bundle is stabilised solely by hydrophobic interactions that result from buried hydrophobic and exposed charged residues (Czupryn et al. 1995).

View larger version (39K): [in this window] [in a new window]   Figure 1 Class-I helical cytokines fold into a bundle of four tightly packed -helices. On the basis of their helix length, class-I helical cytokines are characterised as (A) long chain, such as IL-6, or (B) short chain, such as IL-4. Many long-chain class-I helical cytokines are also recognisable by an additional short -helix in the AB or CD loop, indicated in red in (A). In some short-chain class-I helical cytokines, these loops both contain a stretch of ß-strand that connect to form a ß-sheet (B).

	The modular make-up of helical cytokine receptors

Top Abstract Introduction Class-I helical cytokines share... The modular make-up of... Intracellular signalling... Redundancy and specificity in... Class-I helical cytokines in... 'Short-chain' class-I helical... Vertebrates outdated: insect... Summary and perspectives References

 
Class-I helical cytokines exert their actions via cell surface receptors that share a similar modular make-up. The extracellular domain of all class-I helical cytokine receptors includes at least one cytokine-binding domain of approximately 200 amino acids (made up of a tandem of fibronectin type-III (FnIII) domains) and is often associated with additional FnIII or immunoglobulin domains (Fig. 2). The FnIII domain that makes up the membrane proximal half of the cytokine-binding domain usually contains a characteristic WSXWS signature (Bazan 1990a, Thoreau et al. 1991, Taga & Kishimoto 1997). The WSXWS motif has in some cases been shown to be required for proper receptor folding, but is not directly involved in ligand binding (Yawata et al. 1993, Taga & Kishimoto 1997). Whereas some class-I helical cytokine receptors have long intracellular domains, the cytoplasmatic tails of others are short, and incapable of intracellular signalling. The receptor complexes for all class-I helical cytokines contain at least one receptor chain with a long intracellular domain (Fig. 2). Some class-I helical cytokines share a single signalling receptor chain. The best example is provided by gp130, which is a class-I helical cytokine receptor chain that participates in the receptor complexes of several helical cytokines (Ernst & Jenkins 2004). For that reason, the corresponding class-I helical cytokines are collectively referred to as ‘gp130 cytokines’. This group includes IL-6, IL-11, ciliary neu-rotrophic factor (CNTF), leukaemia inhibitory factor (LIF), oncostatin M (OSM), cardiotrophin-1 (CT-1) and cardiotrophin-like cytokine (CLC) (Fig. 2). The ligand specificity of the receptor is in these instances determined by a second receptor chain that participates in the formation of the receptor complex (Bravo & Heath 2000, Heinrich et al. 2003). The IL-6 receptor complex provides a prototypical example: IL-6 binds with high affinity to its specific receptor chain (IL-6R) and the IL-6/IL-6R complex subsequently recruits gp130 to convey an intracellular signal (Simpson et al. 1997). The stoichiometry of this receptor complex indicates a hexamer consisting of doublets of the IL-6, the IL-6R-chain and gp130 (Boulanger et al. 2003). The IL-11R and CNTF receptor (CNTFR) resemble the IL-6Ra in that they are short and cannot signal intracellularly. A characteristic feature of the CNTFR is the lack of a transmembrane domain and its anchoring via a glycosylphosphatidy-linositol (GPI) anchor. The IL-11 and CNTF receptor complexes are hexamers, similar in conformation to the IL-6 receptor (Hirano et al. 1997, Barton et al. 2000, Heinrich et al. 2003). The CNTF receptor complex contains a single gp130 chain. Instead, the second gp130 chain is replaced by the LIF receptor (LIFR) (Fig. 2), a signalling chain similar to gp130. The other gp130 cytokines, OSM, LIF and CT-1, bind directly and with high affinity to receptor chains that possess long intracellular domains. Their receptor complexes consist of a combination of gp130 and LIFR (for LIF and CT-1), or gp130 and OSM receptor (OSMR) (for OSM) (Fig. 2) (Heinrich et al. 2003).

View larger version (55K): [in this window] [in a new window]   Figure 2 Cytokine receptors share a modular make-up. Class-I helical cytokines signal via a multimeric receptor complex that usually consists of several cytokine receptor chains. All receptor chains contain at least one cytokine-binding domain, formed by two fibronectin type-III (FnIII) domains, of which the membrane-proximal one in most cases contains a consensus WSXWS motif. Many cytokine receptor chains contain an immunoglobulin domain as well as several FnIII domains in addition to the pair that forms the cytokine-binding domain. The typical cytokine receptor complex is composed of a ligand-specific receptor chain which in some cases lacks intracellular signalling capacity, and a second receptor chain which is shared by multiple receptor complexes and is responsible for the transduction of an intracellular signal.

The remaining long-chain class-I helical cytokines (granulocyte-colony stimulating factor (G-CSF), leptin, EPO, thrombopoietin (TPO), GH and PRL) all signal via homodimers of receptor chains that combine high affinity ligand binding with intracellular signalling. The receptors for G-CSF and leptin are similar to gp130 in size and extracellular composition. In contrast, the receptors for GH, PRL, EPO and TPO are notably shorter and consist of a single cytokine-binding domain connected to a long cytoplasmatic tail (Fig. 2). Despite these differences, the early studies that addressed the complex of GH and its receptor (GHR) are at the basis of our knowledge of class-I helical cytokine receptor complex formation and stoichiometry (Simpson et al. 1997, Bravo & Heath 2000), as the crystal structure of the GH–GHR complex was the first helical cytokine/receptor complex that was solved (de Vos et al. 1992).

	Intracellular signalling pathways: phosphorylation is the message

Top Abstract Introduction Class-I helical cytokines share... The modular make-up of... Intracellular signalling... Redundancy and specificity in... Class-I helical cytokines in... 'Short-chain' class-I helical... Vertebrates outdated: insect... Summary and perspectives References

 
Intracellularly, the recruitment of the multimeric cytokine receptor complex upon cytokine binding brings together the long intracellular signalling domains of two receptor chains, leading to the activation of several distinct intra-cellular signalling cascades. Each cytoplasmatic domain of gp130 and related signalling chains is constitutively associated with a tyrosine kinase of the JAK family (Janus kinase; after the two-faced, Roman god Janus, referring to JAK’s dual (pseudo)-catalytic domains). Dimerisation of class-I helical cytokine receptor chains with an intracellular signalling domain leads to JAK activation via the transphosphorylation of both JAKs (Chen et al. 2004). Phosphorylated JAKs, in turn, phosphorylate several intracellular membrane-distal tyrosine residues in the cytoplasmatic domain of the receptor chain. The phosphorylated tyrosines serve as a docking site for members of the signal transducer and activator of transcription (STAT) family that bind to phosphorylated tyrosine through their Src homology-2 (SH2) domain (Gadina et al. 2001). The mammalian STAT family consists of seven members (STAT1, 2, 3, 4, 5a, 5b and 6) that are phosphorylated upon docking to a phosphorylated tyrosine. Phosphorylated STATs dissociate from the receptor chain and form STAT homo- or heterodimers that are translocated into the nucleus to initiate transcription (Darnell 1997, Levy & Darnell 2002, O’Shea et al. 2002). Which genes are transcribed depends on the composition of the STAT dimer. As untimely or prolonged JAK/STAT signalling can shift the balance from adequate immune regulation and host defence to a derailed immune response with potentially serious consequences, multiple inhibitory mechanisms control JAK/STAT activity.

One of these inhibitory mechanisms is formed by the SH2 domain containing tyrosine phosphatases (SHP), such as SHP1 and SHP2. These phosphatases dephosphorylate the tyrosines of key signalling components such as JAKs, STATs and helical cytokine receptors, thereby abrogating cytokine signalling (Symes et al. 1997, Heinrich et al. 2003, Shuai & Liu 2003, Chen et al. 2004, Wormald & Hilton 2004).

The protein inhibitors of activated STAT (PIAS) constitute a second class of JAK/STAT signalling inhibitors. Currently, five PIAS members have been identified (PIAS1, PIAS3, PIASx, PIASxß and PIASy). By binding to activated STAT dimers, PIAS1 and PIAS3 directly prevent association with DNA, whereas PIASx or PIASy prevent activated STAT from transcription without directly interfering with DNA binding (Chen et al. 2004).

Among the many target genes that are transcribed in response to JAK/STAT activation is one particular group of genes that encodes members of the suppressor of cytokine signalling (SOCS) family. Currently the SOCS family consists of eight members (SOCS1–7 and cytokine-inducible SH2-containing domain (CIS)); the role of SOCS4–7 in cytokine signalling has received limited attention. The SOCS proteins exert a classical negative feedback. Like STATs, SOCS proteins contain a central SH2 domain that allows them to bind phosphorylated tyrosine residues (Heinrich et al. 2003). SOCS proteins suppress in several ways: SOCS1 directly binds to phosphorylated JAKs and inhibits JAK catalytic activity. CIS, SOCS2 and SOCS3 bind directly to phosphorylated helical cytokine receptor tyrosines, effectively blocking STAT binding (Cooney 2002, Larsen & Ropke 2002, Chen et al. 2004, Wormald & Hilton 2004).

	Redundancy and specificity in helical cytokine signalling

Top Abstract Introduction Class-I helical cytokines share... The modular make-up of... Intracellular signalling... Redundancy and specificity in... Class-I helical cytokines in... 'Short-chain' class-I helical... Vertebrates outdated: insect... Summary and perspectives References

 
Many class-I helical cytokines, such as IL-6 and IL-11, and LIF and OSM, display considerable overlap in their spectrum of biological actions. This redundancy is explained by the shared use of the gp130 signalling chain that funnels the intracellular response that follows receptor activation along the same signalling cascade. The cell’s response is eventually determined by integration of a plethora of biological signals that may differ among cell types. The redundancy that stems from the shared use of gp130 is illustrated by the embryonically lethal phenotype of gp130 knockout mice, which is considered to result from the cumulative effects of the loss of signalling capacity for a large group of helical cytokines (Yoshida et al. 1996).

Besides the overlap in cytokine receptor conformation, redundancy in helical cytokine signalling is achieved by the promiscuity of components of the JAK/STAT signalling pathway. Four JAKs (JAK1–3 and tyrosine kinase 2 (Tyk2)) and seven STATs have been identified in mammals. Together they constitute the intracellular mechanism that is responsible for signalling in response to at least 30 different class-I helical cytokines. This can only be achieved when different cytokines share an intracellular signalling cascade. The involvement of the various JAKs and STATs in signalling in response to specific (groups of) class-I helical cytokines has been defined by assessing the phenotypes of mice lacking specific components of JAK/STAT signalling. The absence of JAK1 leads to a complex perinatally lethal phenotype that is characterised by neurological as well as lymphoid defects (Chen et al. 2004). JAK1^–/– mice fail to respond to IFNs and lack signalling capacity via the c-receptor chain, which is the receptor chain responsible for intracellular signal transduction in the receptor complex of many short-chain class-I helical cytokines (Gadina et al. 2001). The phenotype of JAK3 knockout mice in part resembles that of the JAK1 knockout, but is less severe. JAK3^–/– mice are viable, although they suffer from severe combined immunodeficiency (Shuai & Liu 2003, Chen et al. 2004). This immunological defect is caused by abrogated c-receptor signalling and, since the c-receptor^–/– and JAK3^–/– phenotypes are identical, it follows that JAK3 is only employed by the short-chain class-I helical cytokines that signal via this common receptor chain (Suzuki et al. 2000). JAK2 is employed by all class-I helical cytokines that signal via a homodimeric receptor. This group includes leptin, PRL, GH, EPO, TPO and G-CSF (Gadina et al. 2001, Vosshenrich & Di Santo 2002). Phenotypically, JAK2^–/– mice are characterised by defective erythropoiesis as a consequence of silenced EPO signalling (Shuai & Liu 2003, Chen et al. 2004). This renders JAK2 knockouts embryonically lethal, before a phenotype of defective signalling by any of the other helical cytokines that depend on JAK2 becomes manifest. The Tyk2 knockout is viable; this is a mild phenotype compared with the phenotypes described earlier for JAK1–3. Tyk2 can be activated in response to IFN-/ß, IL-10 and various gp-130 cytokines, but appears only to be critically required for IL-12 signalling (Gadina et al. 2001, Vosshenrich & Di Santo 2002, Shuai & Liu 2003). Due to this defective IL-12 signalling and possibly diminished IFN signalling capacity, Tyk2^–/– mice are susceptible to (viral) pathogen infection, but appear to be otherwise normal (Karaghiosoff et al. 2000, Shimoda et al. 2000).

STAT3 is the principle signal transducer that is activated in response to gp130 cytokines as well as leptin and G-CSF. The class-I helical cytokines that bind receptors with short extracellular domains (GH, PRL, EPO and TPO) all engage STAT5 (Gadina et al. 2001, Vosshenrich & Di Santo 2002). Duplicated and highly similar STAT5 genes, designated STAT5a and STAT5b, are encoded in both the human (locus 17q11.2) and the mouse (locus 11D) genome (Ambrosio et al. 2002). Although both are activated by the helical cytokines that depend on STAT5 in vitro, STAT5a^–/– and STAT5b^–/– mice each have a specific phenotype. Female STAT5a knockouts have impaired mammary gland development and fail to lactate as a consequence of abrogated PRL signalling (Liu et al. 1997). In contrast, STAT5b^–/– mice display growth defects due to defective GH signalling (Udy et al. 1997).

Despite the general redundancy of intracellular signalling pathways, the use of some STATs is highly restricted to a single helical cytokine or a small group of related class-I helical cytokines. These restrictions are so specific that activation of these STATs has become synonymous for the activation of distinct immunological pathways. This is pre-eminently illustrated by the specific role of STATs in the determination of T helper 1 (Th1) and Th2 immune responses. The Th1/Th2 dogma states that the modality of an immune response depends on and is tailored for the effective eradication of the eliciting pathogen. Th1 and Th2 refer to the extremes of a continuous spectrum of immune responses and are, as a rule of thumb, associated with intracellular and extracellular pathogens respectively. Both Th1 and Th2 responses are associated with a panel of ‘signature’ cytokines (Mosmann et al. 1986, Mosmann & Coffman 1989). IL-12 is a key cytokine in the determination of Th1 responses that, when released from activated macrophages, induces IFN production in T cells (Agnello et al. 2003, Watford et al. 2003). A Th2 response, in contrast, is associated with the release of IL-4, IL-5 and IL-13, a group of related short-chain class-I helical cytokines that utilises similar receptors (Jarnicki & Fallon 2003, O’Byrne et al. 2004). Th1 cytokines inhibit the expression of Th2 cytokines and vice versa, thus the balance between Th1 and Th2 cytokines determines the outcome of the overall immune response. And whereas most STATs are promiscuous to some degree, Th1 and Th2 signalling each involve a unique STAT: IL-4 and IL-13 activate STAT6 whereas IL-12 relies solely on STAT4 (Gadina et al. 2001, O’Shea et al. 2002, Vosshenrich & Di Santo 2002). Consequently, the activation of STAT4 or STAT6 is a tell-tale for the type of immune response unfolding (Agnello et al. 2003).

	Class-I helical cytokines in teleosts

Top Abstract Introduction Class-I helical cytokines share... The modular make-up of... Intracellular signalling... Redundancy and specificity in... Class-I helical cytokines in... 'Short-chain' class-I helical... Vertebrates outdated: insect... Summary and perspectives References

 
In the following paragraphs we separately introduce the long-chain class-I helical cytokines and discuss evidence for orthologous non-mammalian cytokines. Helical cytokines that are found on the same locus in mammals (e.g. LIF and OSM) are discussed together, as are helical cytokines that share distinct structural characteristics and/or functional properties such as the heterodimeric cytokines. Also the order in which the various (pairs of) helical cytokines are covered is loosely determined by shared features such as gene structure and shared cysteine residues.

IL-6

The pleiotropic nature of IL-6 is aptly illustrated by the history of its discovery. The cloning and characterisation of IL-6 by four separate groups (Haegeman et al. 1986, Hirano et al. 1986, May et al. 1986, Zilberstein et al. 1986) revealed that a variety of factors known as B-cell stimulatory factor 2, B-cell differentiation factor, hybridoma-plasmacytoma growth factor, IFN-ß2, hepatocyte-stimulating factor, T cell replacing factor-like factor and monocyte-granulocyte inducer type 2 were in fact one and the same. IL-6 is an important inducer of antibody production, although the finding that IL-6-deficient mice display reduced IgG, but normal early IgM responses to Vaccinia virus infection indicates that IL-6 is not equally important for the production of all Ig isotypes (Kopf et al. 1994). IL-6 also has the capacity to stimulate T cells, and participates in haematopoiesis by inducing the proliferation of pluripotent progenitor cells. In addition, IL-6 stimulates the differentiation of myeloid progenitors into granulocytes and macrophages and promotes mega-karyocyte maturation (Taga & Kishimoto 1997). In the liver, IL-6 is one of the major inducers of the acute phase reaction, in concert with IL-1ß and tumour necrosis factor- (TNF) (Suffredini et al. 1999). An interesting property of IL-6 is that it is one of several class-I cytokines that can modulate the hypothalamic–pituitary–adrenal axis, thus directly influencing the stress response (Chesnokova & Melmed 2002). The IL-6 gene in humans and mice consists of five exons (Tanabe et al. 1988) and the four-helix bundle topology of IL-6 is stabilised by two internal disulphide bridges. The elucidation of the pufferfish genome has revealed the existence of IL-6 in fish (Fig. 3). The pufferfish IL-6 gene is similar in organisation to that of mammalian IL-6 and G-CSF genes. Pufferfish IL-6 shares two conserved cysteine residues with mammalian IL-6 and G-CSF sequences but lacks their N-terminal cysteine pair (Fig. 4). In vitro stimulation of pufferfish head kidney cells with the T cell activator phytohaemagglutinin rapidly upregulates IL-6 expression, demonstrating its expression and upregulation within the leukocyte compartment (Bird et al. 2005b).

View larger version (99K): [in this window] [in a new window]   Figure 3 Phylogeny of the vertebrate class-I helical cytokines. For most mammalian class-I helical cytokines, non-mammalian orthologs have been identified to date. The amino acid sequence alignment obtained by T-Coffee (Notredame et al. 2000) was refined by hand to correct for overt alignment mismatches. Phylogenetic analysis of such a large number of amino acid sequences (>250 sequences), which are only weakly similar both within a single species (paralogs) as well as between distantly related species (orthologs), is sensitive to artifactual topologies. To prevent this from occurring, constraints (indicated by dashed lines) were introduced for the deep topology of the tree based on the information regarding gene organisation, chromosomal location and conserved cysteine pairs, which is summarised in Fig. 4. The phylogeny was reconstructed on the basis of an amino acid differences (p-distance) in MEGA3 (Kumar et al. 2004) using the neighbour-joining algorithm. The tree obtained via the minimum evolution algorithm had an essentially identical topology (not shown). Branch lengths reflect the extent of the genetic distance between sequences. The confidence level of 1000 bootstrap replications is indicated by the size of the dots at the branch nodes. Clusters of mammalian, avian, reptilian, amphibian, and teleostean cytokines are indicated in red, yellow, blue, green and lilac respectively. Note that some clusters are notably more compact than others, reflecting a higher degree of primary sequence conservation. Accession numbers are as follows: human IL-6, P05231 [GenBank] ; rhesus macaque IL-6, P51494 [GenBank] ; crab-eating macaque IL-6, P79341 [GenBank] ; sooty mangabey IL-6, P46650 [GenBank] ; Ma’s night monkey IL-6, AF014510 [GenBank] ; squirrel monkey IL-6, AF294757 [GenBank] ; lemurine night monkey IL-6, AF097323 [GenBank] ; black-headed night monkey IL-6, AF097322 [GenBank] ; noisy night monkey IL-6, AF014505 [GenBank] ; mouse IL-6, P08505 [GenBank] ; rat IL-6, P20607 [GenBank] ; rabbit IL-6, AAF86660 [GenBank] ; hamster IL-6, BAA78766 [GenBank] ; cotton rat IL-6, AF421389 [GenBank] ; woodchuck IL-6, O35736 [GenBank] ; cow IL-6, P26892 [GenBank] ; pig IL-6, P26893 [GenBank] ; sheep IL-6, P29455 [GenBank] ; horse IL-6, Q95181 [GenBank] ; camel IL-6, AB107656 [GenBank] ; llama IL-6, AB107647 [GenBank] ; goat IL-6, Q28319 [GenBank] ; cat IL-6, P41683 [GenBank] ; dog IL-6, P41323 [GenBank] ; killer whale IL-6, Q28747 [GenBank] ; beluga whale IL-6, AAD42929 [GenBank] ; harbour seal IL-6, Q28819 [GenBank] ; sea otter IL-6, AAB01428 [GenBank] ; chicken IL-6, CAC40812 [GenBank] ; Xenopus IL-6, AW637075 [GenBank] ; pufferfish IL-6, Q6 L6X6; mouse G-CSF, P09920 [GenBank] ; rat G-CSF U37101 [GenBank] ; human G-CSF, P09919 [GenBank] ; cat G-CSF, NM_001009227; cow G-CSF, AF092533 [GenBank] ; pig G-CSF, U68481 [GenBank] ; chicken myelomonocytic growth factor, NM_205279 [GenBank] ; human IL-11, P20809 [GenBank] ; crab-eating macaque IL-11, P20808 [GenBank] ; mouse IL-11, P47873 [GenBank] ; rat IL-11, AAK29623 [GenBank] ; carp IL-11, AJ632159 [GenBank] ; halibut IL-11, AU090873 [GenBank] ; pufferfish IL-11a, BN000713 [GenBank] ; pufferfish IL-11b, BN000714 [GenBank] ; green puffer IL-11a, BN000715 [GenBank] ; green puffer IL-11b, AY374508 [GenBank] ; zebrafish IL-11a, BN000717 [GenBank] ; zebrafish IL-11b, BN000718 [GenBank] ; trout IL-11, AJ535687 [GenBank] ; horse EPO, AB100030 [GenBank] ; cow EPO, P48617 [GenBank] ; dog EPO, P33707 [GenBank] ; cat EPO, P33708 [GenBank] ; human EPO, P01588 [GenBank] ; crab-eating macaque EPO, P07865 [GenBank] ; rhesus macaque EPO, Q28513 [GenBank] ; mouse EPO, P07321 [GenBank] ; rat EPO, P29676 [GenBank] ; sheep EPO, P33709 [GenBank] ; blind mole rat EPO, AJ715792 [GenBank] ; pig EPO, NM_214134 [GenBank] ; pufferfish EPO, AY303753 [GenBank] ; zebrafish EPO, DQ278896 [GenBank] ; carp EPO, AJ831393 [GenBank] ; green puffer EPO, AY374507 [GenBank] ; grouper EPO, AY735012 [GenBank] ; human IL-12p35, P29459 [GenBank] ; rhesus macaque IL-12p35, P48091 [GenBank] ; mouse IL-12p35, P43431 [GenBank] ; rat IL-12p35, AAD51364 [GenBank] ; hamster IL-12p35, AB085791 [GenBank] ; woodchuck IL-12p35, X97018 [GenBank] ; guinea pig IL-12p35, AB025723 [GenBank] ; cotton rat IL-12p35, AF421396 [GenBank] ; cow IL-12p35, P54349 [GenBank] ; water buffalo IL-12p35, AY232819 [GenBank] ; deer IL-12p35, U57751 [GenBank] ; pig IL-12p35, Q29053 [GenBank] ; horse IL-12p35, Q9 XSQ6; sheep IL-12p35, Q9TU27; goat IL-12p35, O02814 [GenBank] ; dog IL-12p35, Q28267 [GenBank] ; cat IL-12p35, O02743 [GenBank] ; carp IL-12p35, AJ580354 [GenBank] ; zebrafish IL-12p35, AB183001 [GenBank] ; chicken IL-12p35, AY262751 [GenBank] ; pufferfish IL-12p35, AB096265 [GenBank] ; green puffer IL-12p35a, AY374509 [GenBank] ; green puffer IL-12p35b, AY374510 [GenBank] ; human IL-23p19, AF301620 [GenBank] ; chimpanzee IL-23p19, AY412450 [GenBank] ; mouse IL-23p19, AF301619 [GenBank] ; rat IL-23p19, NM_130410 [GenBank] ; deer mouse IL-23p19, AY259629 [GenBank] ; guinea pig IL-23p19, AB058509 [GenBank] ; horse IL-23p19, AY704416 [GenBank] ; mouse IL-27p28, AY099297 [GenBank] ; human IL-27p28, AY099296 [GenBank] ; pig IL-27p28, AY788913 [GenBank] ; human CT-1, Q16619 [GenBank] ; chimpanzee CT-1, XM_523348 [GenBank] ; mouse CT-1, Q60753 [GenBank] ; rat CT-1, Q63086 [GenBank] ; dog CT-1, XM_843979 [GenBank] ; chimpanzee neuropoitin (NP), Q6R2R2; mouse NP, P83714 [GenBank] ; rat NP, AY518205 [GenBank] ; cow NP, XM_609151 [GenBank] ; red-backed salamander plethodontid receptivity factor (PRF), AY926884 [GenBank] ; Appalachian salamander PRF, AY926937 [GenBank] ; human leptin, P41159 [GenBank] ; chimpanzee leptin, O02750 [GenBank] ; gorilla leptin, Q95189 [GenBank] ; orang utan leptin, Q95234 [GenBank] ; rhesus macaque leptin, Q28504 [GenBank] ; mouse leptin, P41160 [GenBank] ; rat leptin, P50596 [GenBank] ; cow leptin, P50595 [GenBank] ; sheep leptin, Q28603 [GenBank] ; pig leptin, Q29406 [GenBank] ; dog leptin, O02720 [GenBank] ; cat leptin, AB041360 [GenBank] ; dunnart leptin, AF159713 [GenBank] ; Xenopus leptin, AY884210 [GenBank] ; salamander leptin, CN054256 [GenBank] ; medaka leptin, AB193548 [GenBank] ; zebrafish leptin, BN000830 [GenBank] /GENSCAN00000007598; pufferfish leptin, AB193547 [GenBank] ; green puffer leptin, AB193549 [GenBank] ; carp leptin-I, AJ830745 [GenBank] ; carp leptin-II, AJ830745 [GenBank] ; human CLC, AF176912 [GenBank] ; mouse CLC, Q9QZM3; rat CLC, NM_207615 [GenBank] ; chicken CLC, XM427323; Xenopus CLC, CR762259 [GenBank] ; human CNTF, P26441 [GenBank] ; rat CNTF, P20294 [GenBank] ; mouse CNTF, P51642 [GenBank] ; pig CNTF, O02732 [GenBank] ; cow CNTF, XM_607445 [GenBank] ; rabbit CNTF, P14188 [GenBank] ; chicken CNTF, Q02011 [GenBank] , carp M17, AY102632 [GenBank] ; green puffer M17, CAF99247 [GenBank] ; zebrafish M17, NW_634687; pufferfish M17, GENSCANSLICE00000010963/CAAB01000021.1; cow LIF, Q27956 [GenBank] ; human LIF, P15018 [GenBank] ; mouse LIF, P09056 [GenBank] ; rat LIF, P17777 [GenBank] ; mink LIF, O62728 [GenBank] ; pig LIF, Q9 GKZ8; hamster LIF, AY171245 [GenBank] ; possum LIF, AF303448 [GenBank] ; cow OSM, P53346 [GenBank] ; human OSM, P13725 [GenBank] ; mouse OSM, P53347 [GenBank] ; human GH1, P01241 [GenBank] ; human GH2, P01242 [GenBank] ; mouse GH, P06880 [GenBank] ; rat GH, P01244 [GenBank] ; rhesus macaque GH1, P33093 [GenBank] ; rhesus macaque GH2, Q07370 [GenBank] ; squirrel monkey GH, AF339060 [GenBank] ; cow GH, V00111 [GenBank] ; dog GH, Z23067 [GenBank] ; cat GH, U13390 [GenBank] ; water buffalo GH, X72947 [GenBank] ; mink GH, X56120 [GenBank] ; giant panda GH, AF540936 [GenBank] ; guinea pig GH, AF233853 [GenBank] ; hamster GH, S66299 [GenBank] ; possum GH, AF052192 [GenBank] ; opossum GH, AF312023 [GenBank] ; pig GH, AY536527 [GenBank] ; goat GH, Y00767 [GenBank] ; chicken GH, P08998 [GenBank] ; turkey GH, M33697 [GenBank] ; duck GH, X07079 [GenBank] ; goose GH, AY149895 [GenBank] ; Xenopus GH1, P12855 [GenBank] ; Xenopus GH2, P12856 [GenBank] ; bullfrog GH, AY251538 [GenBank] ; giant toad GH, AF062746 [GenBank] ; carp GH, P10298 [GenBank] ; pufferfish GH, O12980 [GenBank] ; goldfish GH1, O93359 [GenBank] ; goldfish GH2, O93360 [GenBank] ; trout GH1, P09538 [GenBank] ; trout GH2, P20332 [GenBank] ; sockeye salmon GH1, Q91222 [GenBank] ; sockeye salmon GH2, Q91221 [GenBank] ; chum salmon GH, P07064 [GenBank] ; Russian sturgeon GH, AY941176 [GenBank] ; sturgeon GH1, P26773 [GenBank] ; sturgeon GH2, P26774 [GenBank] ; African lungfish GH, AF062745 [GenBank] ; human PRL, P01236 [GenBank] ; rhesus macaque PRL, U09018 [GenBank] ; mouse PRL, P06879 [GenBank] ; rat PRL, P01237 [GenBank] ; hamster PRL, AAB20367 [GenBank] ; vole PRL, AF178933 [GenBank] ; cat PRL, U25974 [GenBank] ; rabbit PRL, Q28632 [GenBank] ; deer PRL, AY373035 [GenBank] ; sheep PRL, X13483 [GenBank] ; goat PRL, X76049 [GenBank] ; cow PRL, V00112 [GenBank] ; horse PRL, AY373339 [GenBank] ; dog PRL, AY741405 [GenBank] ; mink PRL, P29234 [GenBank] ; giant panda PRL, AY161285 [GenBank] ; pig PRL, NM_213926 [GenBank] ; opossum PRL, AF067726 [GenBank] ; possum PRL, AF054634 [GenBank] ; chicken PRL, P14676 [GenBank] ; quail PRL, AB162003 [GenBank] , duck PRL, AB158610 [GenBank] ; turkey PRL, U05952 [GenBank] ; goose PRL, AY993962; leopard gecko PRL, AB182277 [GenBank] ; bullfrog PRL, X16063 [GenBank] ; salamander PRL, AY332494 [GenBank] ; Xenopus PRL, BC075216 [GenBank] ; pufferfish PRL, newsinfrut00000132442/CAAB01004482.1; carp PRL, X12543 [GenBank] ; Nile tilapia PRL1, A07820 [GenBank] ; Nile tilapia PRL2, A07824 [GenBank] ; noble carp PRL, X61049 [GenBank] ; zebrafish somatolactin (SL), AJ867249 [GenBank] /ENSDARP00000023510; goldfish SL, P79697 [GenBank] ; green puffer SL, AY374504 [GenBank] ; pufferfish SL, GENSCANSLICE00000000291/CAAB01000976.1; red drum SL, AF062520 [GenBank] ; rabbitfish SL, AB026186 [GenBank] ; black seabream SL, AY714370 [GenBank] ; gilthead seabream SL, L49205 [GenBank] ; sole SL, U06753 [GenBank] ; medaka SL, AY530202 [GenBank] ; Atlantic cod SL, D10639 [GenBank] ; European eel SL, U63884 [GenBank] ; channel catfish SL, AF267991 [GenBank] ; lumpfish SL, L02118 [GenBank] ; grouper SL, AY129310 [GenBank] ; perch SL, AY332490 [GenBank] ; chum salmon SL, D10640 [GenBank] ; Atlantic halibut SL, L02117 [GenBank] ; bastard halibut SL, M33696 [GenBank] ; African lungfish SL, O73847 [GenBank] ; white sturgeon SL, AB017200 [GenBank] .

View larger version (51K): [in this window] [in a new window]   Figure 4 The gene structure of orthologous cytokines is conserved throughout vertebrates, a feature that is instrumental in the identification of orthologous relationships within the multigene cytokine family, which is characterised by limited primary sequence conservation. Boxes represent exons and are drawn to scale; numbers indicate their size in nucleotides. The underlined numbers underneath each intron indicate intron phase. Note that all introns are phase 0, except when the coding part of the first exon is small (<43 nucleotides), then the first intron is invariably phase 1. Shaded lines indicate conserved cysteine residues, which are generally conserved in their presence and spacing between orthologous cytokines. The italic lettering beneath the human and mouse cytokine names reflect their chromosomal locations. The carp and pufferfish icons reflect the gene structure of cyprinid (Cyprinidae) and puffer (Tetraodontidae) species respectively. Note that the human NP gene is a pseudogene due to an eight-nucleotide deletion in its third exon, indicated by the gap in the third exon.

Circulating neutrophilic granulocytes constitute an essential component of innate immunity and respond rapidly to local pathogenic insults by local margination. The circulating neutrophil population is short lived and is continuously replenished from the bone marrow haematopoietic precursor population. G-CSF is one of the principle regulators of neutrophil differentiation and activation. In the bone marrow, stromal cell-derived G-CSF st