ISJ 6: S46-S57, 2009 ISSN 1824-307X REVIEW Focusing on Ciona intestinalis (Tunicata) innate immune system. Evolutionary implications N Parrinello Laboratory of Marine Immunobiology, Department of Animal Biology, University of Palermo, Palermo, Italy Accepted March 13, 2009 Abstract Phylogenetic analyses based on molecular data provide compelling evidence that ascidians are of critical importance for studying chordate immune system evolution. The Ciona intestinalis draft genome sequence allows searches for phylogenetic relationships, gene cloning and expression of immunorelevant molecules. Acidians lack of the pivotal components of the vertebrate recombinatory adaptive immunity, i.e., MHC, TCRs and dimeric immunoglobulins. However, bioinformatic sequence analyses recognized genic elements indicating the essential features of the Ig superfamily and ancestor proto-MHC genes, suggesting a primitive pre-duplication and pre-recombination status. C. intestinalis genes for individuality in the absence of MHC could encode diverse molecular markers, including a wide panel of complement factors that could be responsible for self-nonself discrimination. Genome analysis reveals a number of innate immunity vertebrate-like genes which encode Toll-like and virus receptors, complement pathways components and receptors, CD94/NK-receptor-like, lectins, TNF, IL1-R, collagens. However, pure homology seeking for vertebrate-specific immunorelevant molecules is of limited value, and functional screening methods may be a more promising approach for tracing the immune system evolution. C. intestinalis, which displays acute and chronic inflammatory reactions, is a model organism for studying innate immunity genes expression and functions. Key words: immunoevolution; genome; Ciona intestinalis; ascidians; innate immunity; inflammatory response; gene expression Evolutionary relevance of ascidian immunity studies Ascidians (Urochordata), including cosmopolitan compound and non-colonial species, occupy a key position in the phylogenetic line leading to the vertebrates (Swalla et al., 2000; Zeng and Swalla, 2005; Delsuc et al., 2006), therefore they have attained importance for immunity evolution studies recently promoted by available genome sequences (Dehal et al., 2002b; Satou et al. 2002; Satoh, 2003; Yokobori et al., 2003; Kasahara et al., 2004; Litman and Cooper, 2007; Ben-Shlomo, 2008). Bioinformatic approach and extensive in silico search of immunorelevant genes have been in part validated by expression patterns and biological properties of their products (Davidson and Swalla, 2002; Nonaka and Miyazawa, 2002; Fujita, 2002; Azumi et al., 2003; ___________________________________________________________________________ Corresponding author: Nicolò Parrinello Laboratory of Marine Immunobiology Department of Animal Biology University of Palermo via Archirafi 18, 90123 Palermo, Italy E-mail: nicpar@unipa.it Shida et al., 2003; Terajima et al., 2003; Fujita et al., 2004; Du Pasquier, 2004; Kasahara et al., 2004; Litman and Cooper, 2007). Botryllids provided of allorecognition reaction (De Tomaso and Weissman, 2004; De Tomaso et al. 2005; Ballarin , 2008; Gasparini et al., 2008) and Ciona intestinalis which displays acute inflammatory responses (Parrinello, 1981; Parrinello and Patricolo, 1984; Parrinello et al., 1984) are model organisms for studying chordate evolution. The whole genome of C. intestinalis has been sequenced and analyzed, genes have been annotated and some expression studies carried out, whereas adequate botryllid genome sequencing is lacking. Adaptive immunity pivotal genes In vertebrates, the emergence of the adaptive immune system is linked to the acquisition of the enzyme machinery encoded by the recombination activating genes (RAG) that provide to the rearrangement of immunoglobulin (Ig) and T cell receptor (TCR) genes. Both B and T cells carry S46 mailto:nicpar@unipa.it receptor molecules to recognize and respond to the antigens. B cell receptor is a prototype of the antibody, and TCR on the surface of T lymphocytes is responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. These receptors are composed of two polypeptides of the Ig superfamily (Ig heavy H and light L chains; TCR α and β or γ and δ chains) with recognizable constant (C) and variable (V) domains. The C domain of C1-set characterizes only Ig, TCR, and MHC class II, class I and class I-related molecules, whereas the C2 set domain occurs in both vertebrates and invertebrates (see Kasahara et al., 2004). The domain provided with variability is generated by somatic recombination of multiple elements scattered in the genic locus. In lymphocytes, RAGs cut DNA at the recombinant signal sequence (RSS) for mediating recombination of V(D)J regions through the joining gene segments. Somatic hypermutation, RAGs and activation-induced cytidine deaminase (AID), regulators of secondary Ig diversification, are crucial components of vertebrate adaptive immunity (Manis et al., 2002; Bransteitter et al., 2003) producing an unlimited repertoire of diversity that enables the cells to recognize and respond to unpredictable pathogens. Analysis of C. intestinalis genome sequences did not reveal the pivotal genes and molecules for adaptive immunity, such as MHC genes, TCRs, or dimeric Igs (Dehal et al. 2002b; Azumi et al., 2003; Shida et al., 2003). Nevertheless, bioinformatic sequence analyses have recognized two Ig domain- containing regions, key V regions, the essential feature of an Ig superfamily VC1-like core trait, presumptive proto-MHC regions scattered throughout the genome, and three types of genes with receptor-like V-C architecture (Du Pasquier, 2004). In addition, there are indicators of V domains that could be targets for RAG-mediated transposition. These genes belong to two families with recognizable homologs in vertebrates: the cortical thymocyte marker of Xenopus/Junctional adhesion molecules family (Ci-CTX/JAM) and the nectin Ci-Nec2 family. Signal of ancestor proto-MHC genes can be drawn from the human MHC genome paralogy (Kasahara, 2000). More than 100 genes are located in the human leukocyte complex (HLA) and about 40 of them have paralogous copies on 1, 9 and 19 chromosomes. C. intestinalis genome contains a single copy gene with features of a precursor of multiple human MHC paralogous suggesting the existence of a pre-duplication region (Bodmer, 1972; Kasahara et al., 2004). Chromosome duplications could be took place before the emergence of a common ancestor of vertebrates as indicated by MHC paralogous emergence. MHC- based allorecognition seems to be a unique feature of jawed vertebrates, and finding of variable lymphocyte receptors in agnates (Pancer et al., 2004) indicate that different groups of molecules could be responsible for similar functions. Accordingly, allorecognition machinery in urochordates has nothing in common with the MHC- based histocompatibility reaction of vertebrates (De Tamaso et al., 2005; Klein, 2006), and observations indicate that different ascidian species could have evolved their own independent allorecognition strategies. In example, the hystocompatibility Fu/Hc receptor of B. schlosseri has no direct homologs neither in vertebrates nor in C. intestinalis, indicating that self-nonself discrimination systems have branched off into a variety of unique and specialized systems during evolution. In the absence of vertebrate-like adaptive immunity, Ciona‘s genes reflect a primitive pre- duplication and/or pre-recombination status leading to the genesis of MHC, TCR and Ig (Yu et al., 2005). Moreover, it is generally accepted that C. intestinalis activating and inhibitory receptors of immunocytes have MHC-independent functions. Presumptive molecular codes for sef-nonself discrimination in the absence of MHC Although most animals are able to distinguish self from non-self (Buss, 1987; Chadwick-Furman and Rinkevich, 1994; Cadavid et al., 2004), allorecognition molecular mechanisms do not seem of monophyletic origin and remain to establish if they evolved independently. Different groups of invertebrates could have developed their own histocompatibility system and could express taxon- specific self-nonself determinants. Ciona is a valuable model for deepening the question. It is hermaphrodite producing eggs and sperm simultaneously, but self-fertilization normally does not occur because the follicle cells accept only allogeneic sperm (Rosati and De Santis, 1978; Pinto et al., 1995; Murabe and Hoshi, 2002). It can be assumed that a receptor, potentially involved in self- incompatibility, should have sequence and expression variable in oocytes from different individuals. Based on this assumption, novel models have been proposed upon a C. intestinalis molecular code for individuality in the absence of MHC. To search for molecular markers of individuality, a suppression subtractive hybridisation approach has been used for comparing the somatic transcriptomes of two Ciona individuals and for identifying individually variable cDNAs (Khalturin et al., 2005). The results show that two genes, CiMETA2 and CiS7, encode two classes of soluble proteins which exhibit high degree of inter- and intra-individual variability and contain multiple domains suitable for protein-protein interaction. Both classes of individually variable proteins are coded in the genome by several gene copies organized in clusters. One class consists of secreted protein thrombospondin type 1-like domains (thrombospondin type 1 repeat, TSR superfamily), the second one consists of secreted proteins with multiple epidermal growth factor (EGF)-like domains. Although the functional significance is unknown, the authors suggest that these gene loci may participate in controlling non-self recognition. CiS7 and CiMETA2 isoforms with a high degree of aminoacid sequence variations are expressed in hemocytes and gametes which are mediators of recognition events. Individuals have several genes and transcribe several mRNAs coding for similar but S47 not identical proteins of each gene family. Apparently each individual carries an unique repertoire (haplotype) in each locus, probably established by crossing over events during gamete maturation and/or fertilization. Individuality may be encoded by the haplotype and the individual-specific combination of genes in the genome locus. This phenomenon resembles the variability of vertebrate killer cell Ig-like receptors (KIR), and fit with the predictions of self-nonself recognition molecules provided with individual variability, allorecognition and block of self-fertilization. To shed light on C. intestinalis specific receptors for allorecognition and self fertilization avoidance, Kurn and colleagues (2007) subctracted gonadal cDNA from three genetically unrelated C. intestinalis individuals by suppression subtractive hybridisation. Individual- specific genes encoding variable transmembrane complement receptor-like protein (vCRL) have been identified. Interestingly, they contain several complement controlling protein domains (SCR/CCP). One of these genes reveals a high degree of inter-individual vCRL amino acid variation, and it is expressed in follicle cells and hemocytes. Diverse vCRL1s, highly variable between individuals, show sequence similarity varying from 70 % to 93 %. Each animal has its own version of vCRL1, therefore cells in different individuals are marked by non-overlapping receptors and corresponding ligands. Intra- individual variants most likely are due to the two alleles of a single locus, and several alternative intracellular vCRL1 domains may be produced by alternative splicing. The cells within one individual are appropriately marked and will be referred to as “self” whereas any cell of genetically different individual will be distinguished as “non self”. In mammals, the complement components are not variable between individuals. In Ciona, the number of genes encoding complement system components is greatly expanded compared to mammals and, together the vCRL high variability, allow to suppose that the Ciona allorecognition reactions involve complement- related receptors in a “missing self” mechanism. Intriguingly, complement receptors are also expressed in human gametes and are involved in sperm/oocyte interaction (Seya et al., 1999). The CiFLRT transmembrane receptor gene expressed in hemocytes (Kurn et al., 2007) was also analyzed. The corresponding protein consists of a signal peptide, nine leucine-rich repeats (LRRs, protein interaction modules), fibronectin type III domain (mammalian FNIII repeats on cell surface and extracellular) and a transmembrane domain (Khalturin et al. 2005). The CiFLRTs from two individuals were amplified, sequenced and compared to sequences reported by Khalturin and colleagues (2005). The distance between the CiFLRT proteins of the four individuals is much smaller than that between vCRL1. The high degree of cCRL1 variation clearly exceeds the naturally occurring variations normally found in Ciona genes. Toll-like receptors The Toll-like receptor (TLR) multigene family encodes recognition receptors of innate immune system conserved in invertebrates and vertebrates (Kaisho and Akira, 2000; Medzhitov and Janeway, 2000; Imler and Hoffmann, 2001; Vasselson and Detmers, 2002). TLRs recognize a variety of endogeneous and exogeneous ligands, they are pattern recognition receptors that bind molecules broadly shared by pathogens collectively referred to as PAMPs, many of which are conserved molecules essential for pathogen survival (Zhong and Kyriakis, 2007). C. intestinalis genome sequence analysis disclosed three distinct TLRs expressed genes and the corresponding signal transduction cascades (Azumi et al., 2003; Terajima et al., 2003; Roach et al., 2005; Shida et al., 2005). In invertebrates, Toll- like receptors mediated antibacterial, antifungal and antiviral systems. Recently, C. intestinalis inflammatory responses challenged by LPS have been reported. The triggering mechanism presumably includes Toll-like receptors (TLRs) which bind LPS (a component of the PAMPs) initiating the inflammatory reactions. Responses stimulated by LPS including complement activation and products, galectin- cytokine-like and CD94-NK-C-type lectin-like receptor genes expression, and collagen synthesis have been reported (see below). Genes for intracellular immunoreceptors, tyrosine-based inhibition motifs (CiITIMs) and tyrosine-based activation motifs (CiITAMs) may be responsible of the cell signaling pathways (Azumi et al. 2003; Shida et al., 2003). Vertebrate ITIMs and ITAMs are short conserved sequences in the cytoplasmic tails of many immune cells inhibitory and activating receptors (including TCRs), respectively. Virus receptors In C. intestinalis genome, homologs to vertebrate adhesion molecules members of membrane Ig superfamily show ancestral features of antigen receptors (Du Pasquier, 2004). They include the junction adhesion molecule (reovirus receptor) and the cortical thymocyte marker of Xenopus (adenovirus receptor) which are members of CiCTX/JAM family, and the poliovirus receptor (PVR) members of the CiNec family. PVR genes contain one distal V domain followed by two C domains, transmembrane and cytoplasmic segments. Vertebrate PVRs are known to allow endocytosis of different viruses through the interaction with V domain. Since in humans 4 paralogous groups exist, the Ciona set of genes could correspond to a preduplication status. It has been suggested that the virus binding property of the members of this family were recruited in the vertebrate immune system following the introduction of the somatic rearrangement machinery. Complement system The innate complement system evolved long S48 before the origin of vertebrate somatically re- arranging antibodies. In vertebrates, there are about 30 complement protein components and three distinct pathways by which the complement system can be activated (Nonaka and Yashizaki, 2004a, b): 1. classical, antibody-mediated pathway; 2. lectin mediated pathway; 3. alternative pathway, triggered the pathogen surface. All the activation mechanisms converge to the C3 component proteolysis, and generate a same set of activation products. C3 is the central component, equipped with an unique intramolecular thioester bond which, upon activation, is exposed to the molecular surface and forms a covalent bond with invading microorganisms (Lambris, 1990). C3 proteolysis enhances the phagocytosis (opsonin) of pathogens through some C3b fragment and the chemotaxis by the C3a-fragment which binds to macrophage and leucocyte receptors, furthermore C3b contribute in forming C5 convertase that activates C5-C9 late complement components leading to the formation of a cytolytic complex (membrane attack complex or MAC). A large number of complement components are conserved between higher vertebrates and urochordates. In C. intestinalis, a wide and detailed architecture of the primitive complement system has been described (Nonaka and Yoshizaki, 2004a, b; Fujita et al., 2004), and complement-like genes, most of which are transcriptionally active, indicate potential activity of lectin and alternative pathways. The Ciona lectin mediated complement pathway has been revealed by genome sequence analysis, EST, cloning and expression studies that identified nine mannose-binding lectins (CiMBL), nine ficolins and four CiMBL-associated serine proteases (CiMASPs C1r/C1s-like). The carbohydrate-binding collectin pathway (CiMBLs/ficolins as pattern recognition proteins), initiated by recognition of PAMPs and activated by CiMASPs, leads to CiC4, CiC2 and CiC3 cleavage. Several soluble components and receptors (four CiMASPs, CiC3 and corresponding CiCR3/CR4 receptors) have been recognized in the genome, and ESTs disclosed transcripts in hemocytes (Fujita, 2002; Azumi et al., 2003; Fujia et al., 2004; Wakoh et al., 2004). CiMASPs could exert trypsin- like activity, and the identified CiMBL collectin genes encode proteins composed of the collagen and lectin-like domains. Recently a CiMBL has been cloned and sequenced, and its expression was promptly enhanced by LPS (Bonura et al., submitted). The deduced amino acid sequence (221 aa) showed a N-cysteine-rich terminal domain, a type 2 collagen domain, and a C-type mannose/glucose-specific CRD. Comparative analysis reveals 56.5 % similarity and 37.8 % identity with human MBL. Marino and colleagues (2002) cloned two CiC3-like genes (CiC3-1 and CiC3-2 cDNA) from Ciona hemocyte mRNA. The deduced aminoacid sequences of both CiC3 proteins show an overall similarity to the C3 molecules from vertebrate species, and exhibit a canonical processing site for α- and β-chains, including a typical thioester site with the His residue required for nucleophilic activation. LPS activates the complement, presumably via CiMBL and CiC3-1 proteolysis leading to the CiC3a fragment which is a pro-inflammatory peptide akin to the vertebrate anaphylatoxin (Pinto et al., 2003). The inflammatory challenge upregulates the CiC3-1 expression in hemocytes and CiC3-1a production. The recombinant CiC3-1a exerts in vitro chemotactic effect on hemocytes interacting with a receptor molecule CiC3aR coupled with Gi protein and homologous to the mammalian receptor (Melillo et al., 2006). An ancestor of the two CiC3 seems to have diverged from a common ancestor of vertebrate C3/C4/C5 and has duplicated in two genes. Accordingly, sequence phylogenetic analysis indicates that CiMASPs have diverged from the common ancestor of vertebrate MASP/C1r/C1s. In addition, the genome presents two α2Macroglobulin-like (Ciα2M) genic elements (Azumi et al., 2003). Mammalian α2M is able to inactivate an enormous variety of proteinases, inhibits complement activation by MASP and it is conceivable that it plays a regulatory role in MBP- derived complement activation (Terai et al., 1995). C3/C4/C5 and α2M form a molecular family different from other complement components and they do not show clear domain structure. In mammals, the factor B (Bf) is the central serine esterase of the alternative pathway of complement activation, and in the active form (Bb) is component of C3 convertase. In Ciona three Bf- like genes, supported by cDNA evidence, have also been identified and they, presumably responsible for the alternative pathway, encode predicted proteins with domain structures similar to the vertebrate Bf/C2 gene family basic domain structure (Fujita, 2002; Azumi et al. 2003; Fujita et al. 2004). Bf and C2 are catalytic subunits of the C3 convertases of the alternative and vertebrate classic pathways respectively. The CiBf genes are longer than those of jawed vertebrates, the deduced amino-acid sequences of CiBf1-3 contain the usual domains of Bf and, in addition, three extra domains at the N-terminus. Similarly to vertebrate MASP-2, MASP-3 and C1r/C1s, CiBf presents one additional short consensus repeat domain, and two low-density lipoprotein receptor (LDLR) domains. Overall deduced amino-acid identity between CiBf-1 and CiBf -2 was 88 %, whereas CiBf-3 showed 49 % identity to both CiBf-1 and CiBf-2. The CiBf serine protease domain shared characteristic features with the complement components C1r/C1s, MASP-2 and MASP-3, and the active site appears to be of the AGy codon type for the catalytic serine residue, and lacks of a histidine loop disulfide bridge (Fumiko et al., 2005; Yoshizaki et al., 2005). Phylogenetic analysis indicates that CiBf genes could be the result of duplication and gene conversion occurred after the divergence of the urochordate lineage from the vertebrate subphylum as also shown by genomic organization and intron/exon composition. Presumably they have diverged from the common ancestor of vertebrate Bf S49 and C2 before the divergence of Bf and C2. These results indicate that complement genes have evolved through extensive exon shuffling events in the early stage of chordate evolution. Exons 3 and 5 of the three CiBf genes show an extremely high degree of nucleotide identity, indicating duplication and gene conversion, since its divergence from the vertebrate Bf/C2 gene. The Ciona genome analysis also allowed the identification of eleven presumptive genes with MAC/perforin domain, nine of them exhibit domain structures similar to those of late complement components. Although, activation mechanism and a functional linkage between CiC3 and the possible lytic components have not been demonstrated, the lytic function is strongly suggested by the presence of the MAC/perforin-like domain. Combination of several domains assign the lytic components to the C6-C9 family. However, CiC6 and CiC7 lack the several C-terminal domains of the corresponding human components, and their functional link to CiC3 remain unclear. Finally, a group of 132 presumptive genes with complement control module (SRC domain) have been identified (Azumi et al., 2003). In mammals, regulators for inhibiting undesirable complement activation against self cells are composed of repeat of short consensus repeats SRC domain. In brief, three functional units may be distinguished in activating CiC3 and active factors production: 1. collectins, composed of collagen and lectin-like domains which recognize miscroorganisms, associated with serine proteases that presumably activate CiC3 leading to a chemotactic product; 2. CiBf and CiC3, components of the vertebrate alternative pathway, and the activation product may have an opsonic activity; 3. presumptive CiC6-C9 molecules forming the MAC/Perforin complex with cytolytic activity. Cytokine-like molecules and receptors Pleiotropic and multifunctional proinflammatory cytokines (tumour necrosis factor TNF, interleukins IL1 and IL6) play a pivotal role in innate immune responses, in cell proliferation, differentiation, apoptosis, and stimulation of the collagen synthesis in wound healing and tissue repair. In invertebrates, cytophilic humoral molecules with functional similarities to vertebrate cytokines have been reported (Beck, 1998; Beshin et al., 2001; Ottaviani et al., 2008). In ascidians, cytokine-like molecules active in stimulating cell proliferation, phagocytosis and opsonisation, have been revealed by immunological and biochemical methods (see Beshin et al., 2004). Genome sequencing, cDNA/EST derived from C. intestinalis hemocytes, and identification of the corresponding genes in genome sequences (Shida et al., 2003; Terajima et al., 2003) revealed the existence of CiIL1 receptor, CiIL17 receptor genes and an ectodysplasin/TNF-like multigene family. The presumptive IL6 gene was also identified. Three interleukine-1-recepor-like (CiIL-1R) genes present an extracellular Ig and an intracellular TIR domain which is the conserved Toll/IL-1 receptor (TIR) domain of the two families of receptors (Tong, 2005). This domain was first characterized due to the homology between the intracellular region of the mammalian IL-1Rs and the Drosophila TLRs. Like TLRs, IL-1R signaling pathways are key mediators of the innate immune response to bacteria (LPS), fungi, cytokines and growth factors. In mammals, the signalling pathways mediated by TLR, IL-1R and TNFR share common components. The possibility exists that these signalling cascades are indeed functioning in C. intestinalis hemocytes. Hemocyte CiTNFα gene expression is challenged by LPS Vertebrate TNFα is a component of a wide TNF family, it is a type II transmembrane protein with an extracellular homotrimeric C-terminal domain. A membrane-bound form may be cleaved, and the mature cytokine may be released as soluble form by a variety of cell types including macrophages, monocytes, granulocytes, NK-cells. TNFs are promptly expressed, and regulate inflammatory reactions by interacting with other pro-inflammatory cytokines recruiting and activating inflammatory cells (Arika et al., 1990). In the Ciona genome, one CiTNF-like and three CiTNF-receptors–like genes have been identified (Terajima et al., 2003). The C. intestinalis TNFα-like cDNA (CiTNFα) has been cloned from the pharynx excised at 4 h after LPS inoculation (Parrinello et al., 2008). Comparative analysis of the deduced aminoacid sequence discloses that the cloned CiTNFα-like clusters at a phylogenetic position close to vertebrate TNFα, whereas a considerable distance separates CiTNFα from Drosophila melanogaster TNF-related Eiger isoforms and earthworm CCF. Like the vertebrate TNFα, CiTNFα is constitutively expressed in the hemocytes and it is promptly (4 h) increased by LPS both in the pharynx after in vivo inoculation and in hemocytes challenged in vitro. Western blot analysis with monoclonal antibodies specific for human recombinant TNFα, showed a cell bound form (43 kDa) in hemocytes and a 15 kDa soluble form in the serum suggesting the role of this cytokine in both local and systemic responses to inflammation. Densitometry analysis of these bands confirms the gene upregulation. In particular the cell bound form is enhanced at 2 h post LPS injection, whereas later (4 h pi.) the soluble form appears to be enhanced in the serum in accordance with the gene expression disclosed by real-time PCR analysis of the pharynx. The anticipated expression of the cell- bound form in hemocytes challenged in vitro appears to be congruent with the presumed maturation process of the soluble one. Similarly to vertebrates, different cell types can secrete the same cytokine. At 4 h after LPS inoculation, amebocytes with large granules, contained in the pharynx vessels and in the connective tissue lining the tunic, as well as circulating hyaline amebocytes and granulocytes express the CiTNFα mRNA as revealed by in situ S50 hybridization and immunohystochemistry with anti-human rTNFα monoclonal antibody. Hemolymph galectins with IL1α epitopes are modulated by LPS The direct homologue of mammalian IL-1 has not been found in the Ciona genome. In mammals, IL-1α and -β interleukines as well as galectins are pro-inflammatory molecules, furthermore many cytokines are bifunctional molecules containing a receptor-binding domain and an evolutionary conserved carbohydrate recognition domain (CRD) that is typical of lectins. The carbohydrate binding is requested for the cytokine biological activity (Beschin et al., 2004). In this regard, IL1α and β can be considered as lectins (Cebo et al., 2001, 2002) directly interacting and contributing to pathogen elimination via opsonization and/or leukocyte activation. Recently, inducible galectin-like molecules with human recombinant IL-1 epitopes and opsonic properties have been found. Parrinello and colleagues (2007) have shown that Ca2+- independent Cigalectin-like molecules, specific for D-galactose and D-galactosides, present human rIL1α epitopes. The LPS inoculation challenges a promptly (4 h) enhanced serum concentration of this lectin that has been related to the augmented serum opsonizing and hemagglutinating activities assayed with yeast and rabbit erythrocytes respectively. Furthermore, human IL-1 epitopes are involved in the opsonizing and hemagglutinating processes which are blocked by anti-human recombinant ILα monoclonal antibodies. The western blot pattern showed that, within the initial phase of the inflammatory response (4 h), several serum proteins (59, 37, 30, 23, 15 kDa) cross-reacted with the antibody suggesting an oligomerization process of the opsonin/lectin. CiIL-17 receptor Hemocytes express an interleukine CiIL-17R gene that encode a predicted polypeptide of 769 aminoacid residues (Dehal et al., 2002b; Shida et al., 2003). The C-terminal half of this protein shows homology to the cytoplasmic region of mammalian IL-17R (27 % identity/40 % similarity). The central portion is rich in hydrophobic aminoacid residues presumably correspondent to a trans-membranous region, and 22 residues at the extreme N-terminal portion could be a signal peptide sequence. The N- terminal portion, probably an extracellular region, shows no homology to the corresponding region of mammalian IL-17R. IL-17 is produced by mammalian T-lymphocytes whereas its receptor is expressed in a variety of cell types such as fibroblasts and stromal cells disclosing a wide spectrum of activity. A possible ligand of CiIL17R has not been predicted. Emergence of NK cells receptors Natural killer (NK) cells are critical in the evolution of the innate immune system being active in discriminating and killing “normal” and virus- infected, tumor or allogeneic cells. In mammals, NK cells monitor MHC class I expression on target cells by means of inhibitory NK cell receptors (NKRs) (Lanier, 2000; Vivier et al., 2002). The NKRs transmit an inhibitory signal that cancels a program for cytotoxic action previously triggered by the target cell contact. NKRs belong to two distinct groups of molecules: Ig-like receptors or C-type lectin receptors including human CD94 (Boyington, 1999). C-type lectin receptors are type II transmembrane glycoproteins with a C-type lectin domain (CTLD) in the extracellular region that bind proteins in a Ca2+- independent manner rather than sugars. They are known to be a hallmark of surface markers for NK cells (Biassoni et al., 2007). C-type lectin superfamily includes carbohydrate-binding proteins (lectins) involved in pathogen recognition and neutralization, leukocyte trafficking, phagocytosis, antigen uptake and processing, and apoptosis. The Ca2+-dependent binding of their carbohydrate recognition domain (CRD) characterized the CTDL. However, many C-type lectins included in the superfamily lack critical amino acid residues required for CRD to bind carbohydrates (Rogers and Wong, 2003). In this respect it has been hypothesized that divergent evolution, acting on the CTLD fold, has generated the lectin-like natural killer (NK) receptors that bind proteins in a Ca2+- independent manner, rather than sugars. Hemocyte CiCD94 gene expression modulated by LPS is involved in phagocytosis C. intestinalis CD94 (CiCD94-1) protein containing CiCTLD is a homolog of the Botryllus schlosseri (50/66 % identity/similarity) BsCD94/NKR-P-1 molecule (Khalturin et al., 2003), and human (30/46 % I/S) CD94. CiCD94-1 has been cloned and sequenced, and hemocytes have been stimulated in vitro with LPS (Zucchetti et al., 2008). Even though sequence homology situates the CiCD94-1 molecule close to CD94, several features do not suggest that they are complete orthologs. CiCD94-1 could be considered a C-type lectin which lacks Ca2+-binding property and its carbohydrate- recognition (mannose/galactose and related sugars) capacity, and it could be located along the evolutionary line leading to the NK receptors functionally related to the human CD94 which recognizes peptides in the groove of MHC class I molecules. On the other hand, the lack of MHC genes in C. intestinalis genome should indicate that CiCD94 is functionally similar to the mice NK cells MHC-independent CD94 (Iizuka et al., 2003; McNerney et al., 2005). However the involvement of CiCD94 in cytotoxic mechanism has not been shown, whereas it appears to be involved in phagocytosis of polystyrene latex beads by granular amebocytes inhibited in the presence of anti- CiCD94-1 specific antibodies (Zucchetti et al., 2008). No assays have been reported to disclose a CiCD94-1 dependent cytotoxic activity by unilocular refractile granulocytes which are known to be cytotoxic (Parrinello, 1996; Parrinello et al., 1996). The CiCD94-1 as receptor on phagocytes can be up-regulated by LPS, presumably part of a S51 mechanism of self-nonself recognition (Zucchetti et al., 2008). Interestingly, granular amebocytes, together with compartment cells, are engaged in the production of CiC3-1 and express CiC3a-R following inoculation of LPS. The presence in the genome of a CD94 homolog and molecules with inhibition (CiITIM) and activation (CiITAM) motifs reasonably sustain the activity of precursors of NK cells in Ciona. The CiCD94-1 protein has been found in about 20 % of the granular amebocytes of naïve ascidians suggesting the existence of a cell population that constitutively express CiCD94-1 presumably acting as a self-nonself sentinel. Altogether these results indicate that hemocytes are provided with a complex array of surface receptors and effector molecules, enabling them to be active in several immune responses. Alternatively, distinct hemocyte populations originated from a same hemocyte type (lymphocyte- like cells) could express distinct receptors and exert different activities following an inflammatory challenge. Ca2+-dependent hemocyte cytotoxicity seems to be CiCD94-independent Apparently, a CiCD94-independent a Ca2+- dependent cytotoxic activity of C. intestinalis hemocytes has been shown. The hemocyte type named unilocular refractile granulocytes (URG, the unique large granule occupies the cytoplasm), constitutively display cytotoxic activity and lyse rabbit erythrocytes and K562 tumour cell line. Zucchetti and colleagues (2008) did not report any URG that express CiCD94-1, furthermore immunocytochemical staining of Percoll gradient separated hemocytes shows that, after a short incubation with LPS, about 80 % granular amebocytes express the CiCD94-1 protein. No signs of the receptor in hyaline amebocytes, that have been reported to be phagocytes (Rowely, 1981), have been found. The receptor involved in hemocyte cytotoxic activity remain unknown. A plaque forming cell assay with rabbit erythrocytes showed that the cell- killing mechanism requires effector-target cells contacts for challenging the release in vitro of soluble cytolysins. The cytolysin is Ca2+-dependent and is inhibited by sphingomyelin and carbohydrates (unpublished). The unique granule displays phenoloxidase activity, and URGs are components of the inflammatory reaction and densely populate the tunic matrix after LPS inoculation (Parrinello, 1996; Parrinello et al., 1996; Cammarata et al., 2008). Although, any evidence exists on the involvement of CiCD94-1-NKR in URG mediated cytotoxicity, in line with the observation that some C-type lectins bind carbohydrates in a Ca2+- independent manner (Brown and Gordon, 2001), carbohydrates could be still be potential ligands for CiCD94-1, and the possibility exists that a coreceptor may be involved. Since lectins have also been claimed as recognition molecules (Quesenberry et al., 2003), presumably different self-nonself recognition molecules characterize ascidian hemocytes and tissues. CiFACIT-collagen expression as a component of the inflammatory reaction Inflammation plays an important role in many processes, protecting organisms against pathogens, and also potentially promoting damage progression (Henson, 2005). Collagens are major structural components of extracellular matrix in tissues of vertebrates and invertebrates, involved in defence and reparative processes (Singer and Clark, 1999). In the mammalian acute inflammatory reaction, collagen fibres bundles are organized for tissue repair during the remodelling phase (Nwomeh et al., 1998), moreover, the total collagens present in normal tissue, is increased from 2- to 9-fold in the chronically inflamed tissue (Narayanan et al., 1983). In this respect, activation of the innate immune response leads to the production of proinflammatory cytokines that can promote collagenolysis. Collagen degradation, and collagen fragments modulate inflammation either augmenting or suppressing interleukine production from peripheral-blood cells (Thomas et al., 2007). A family of non-fibrillar collagens, including type IX (FACIT) collagen, contain short triple helical domains, composed of three genetically distinct polypeptide-chains, interrupted by short non-helical domains (Ricard-Blum et al., 2005). This collagen type does not form fibrils, and interacts with fibrillar collagen (fibril-associated collagen of cartilage extracellular matrix) and with other extracellular matrix partners (Eyre and Wu, 2005). Since type-IX collagen interacts with the cellular receptor integrins it may have an important function as mediator of cell adhesion to collagen fibrils (Käpylä et al., 2004). A C. intestinalis type IX-like collagen cDNA (Ci- typeIX-Col 1α chain), with features of fibril associated collagens formed of interrupted triple helices (FACIT) has been cloned and sequenced (Vizzini et al., 2002). The involvement of this collagen in the inflammatory response has been shown by real-time PCR analysis, ISH assay and immunohistochemical methods. In addition flow cytometry with anti-Ci-typeIX-Col 1α chain specific antibodies, showed a prompt (1-4h) and enhanced collagen expression in the circulating hemocytes treated in vitro with LPS, and in epidermis cells after in vivo LPS inoculation (Vizzini et al., 2008). Morula cells with large granules (morular feature) express this collagen revealing a fibroblast-like role. Conclusions The barrel-shaped sea squirt C. intestinalis (non-colonial ascidian, Tunicata) has its life cycle in shallow sea and ocean waters around the world. One day after an egg is fertilized, it develops into a swimming small tadpole that settles down and metamorphoses into an immobile adult. The settled adult feeds by siphoning seawater using a basket- like filter to capture particulate food and oxygen. Despite the adult humble appearance, the tadpole larva, comprised of about 2,500 cells, is provided of notochord and dorsal neural tube revealing kinship S52 to vertebrates. The importance of tunicates as models for the vertebrate ancestor was recognized by Kowalevsky, who first identified them as chordates, and they have played an important role in various evolution scenarios (see Gee, 1996). Due to the very simple ascidian body plan and an apparently increased body complexity of cephalochordates, ascidians were previously thought to be living form of the earliest chordate lineage. In the December 13, 2002, an issue of the journal Science, an international consortium of researchers reported on the draft sequencing, assembly, and analysis of the C. intestinalis genome (Dehal et al., 2002a). From then on, sequence comparison, cDNAs, EST and gene modulation and function studies provided new insights about the evolution of key vertebrate systems including immune system and development. Recently, taking advantage of the genomes sequencing, Delsuc and colleagues (2006) proposed that tunicates and not cephalochordates are the closest living relatives of vertebrates stimulating further research on Chordates evolution. Simplified form of vertebrate gene families were typically found in Ciona, whereas the lancelet lineage diverged before the tunicates and vertebrates. Genome sequences show that these relationships, reflected at the molecular level, indicates how similar systems and gene sets evolved in different ways from a common ancestor. The close relationship to vertebrates along with its compact genome (about 160 million base pairs, 1/20 the size of the human), makes this sea squirt an ideal model organism for studying chordate evolution. Sequence analysis revealed that C. intestinalis genome contains about 16,000 genes, about 80 % of which are also present in humans and other vertebrates. However, the total number of Ciona genes is only about half the number in vertebrates, presumably due to the fact that it has single copies of a large number of genes whereas they are present in multiple copies in vertebrates (Francino, 2005). Anyway, it can be retained that comparative analysis of genes and knowledge of the immunity genes evolution models are consistent with Darwin’s 1871 suggestion that ascidians and vertebrates diverged from a common ancestor. Increase in the extent of genome resources as well as understanding of transcription at both transcriptosome and spliceosome levels may unveil conserved features on the coding and non coding DNA that sustain genetic stability or promote changes (Litman and Cooper, 2007) either they mutate away and disappear, or they evolve to perform other functions and advance in complexity. Altogether adaptive and innate immune system, form a tremendous complex system to recognize non-self and provide protection from a wide variety of pathogens. The innate immune system is the most ancient of the two systems, and the adaptive immune system appeared more recently developing a high degree of complexity and interconnectivity. Many components of the innate immune system in vertebrates can be reliably traced to urochordates. For example, genome analysis reveals a number of innate immunity vertebrate-like genes, including Toll-like and virus receptor genes, complement pathways components and receptors, CD94/NK- receptor-like, lectins, TNF, IL1-R, collagens. However, pure homology seeking for vertebrate- specific immunorelevant molecules in invertebrates is of limited value, and functional screening methods may be a more promising approach. Accordingly, the expression analysis of humoral and receptorial molecules in Ciona’s tissues and hemocytes following a challenge indicate their involvement in Ciona’s inflammatory response. There is no evidence of MHC orthologs, TCR, Igs in urochordates and agnathans, and no evidence have been reported on the hundreds of key genes involved in vertebrate adaptive immunity. There is a lack of evidence for a gradual transition from the invertebrate innate immune system to the recombinatorial immune system of higher vertebrates (Khalturin et al., 2004) and how the adaptive immune system emerged is still obscure. In the genome of C. intestinalis, genes that encode molecules with membrane receptor features have been found among many members of the Ig superfamily. They contain the V, and C1-like domains typical of vertebrate antigen receptors and MHC class I and II. The human homologs of these genes segregate in a single unit of four paralogous segments on chromosomes 1q, 3q, 11p, and 21q. In these regions there are several genes involved in the adaptive immune system, and MHC paralogs with some related members. Presumably, an ancestral receptor emerged before the RAG- mediated rearrangement originated the ancestor of Ig and TCR provided with a V domain, in which V and J regions were encoded by a single exon. It has been hypothesized that the simplest ancestral receptor could be a single chain made up of V and C domain, every one linked to a transmembrane segment and a short cytoplasmic tail for signaling (Azumi et al., 2003; Du Pasquier, 2004; Kasahara et al., 2004). The status of the urochordate genes reflects perhaps a primitive pre-duplication/pre- recombination-activating gene (RAG) stage that foreshadow the pathway leading to the genesis of the T-cell receptor (TCR) and antibodies (Du Pasquier et al., 2004). The absence of antigen-presenting molecules of the MHC-linked class I and II types, raise questions on the ancestral chordates self-nonself self and allorecognition constituents. Recently novel perspectives have been proposed upon a C. intestinalis molecular code for individuality in the absence of MHC (Khalturin et al., 2005) as well as on the involvement of complement components. Kurn and colleagues (2007) proposed that early during chordate phylogenesis the components of the complement system in addition to their role in pathogen elimination may be involved in allorecognition. So far the phylogenetic analysis of complement components indicates that gene expansion was generated by duplication events that occurred independently in the ascidian and vertebrate lineages. Furthermore, since diverse lectin-CRD repertoires in tunicates mediate broad recognition (Quesenberry et al., 2003), molecular diversity in S53 non self recognition could be due to the glycome code. In addition, mounting evidence indicates that invertebrate immune-type receptors may undergo somatic diversification through elaborate RNA processing mechanism (Zhang et al., 2004; Kalturin et al., 2005; Watson et al., 2005; Sadd and Schimid- Hemplel, 2006). 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