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Ichthyophthirius multifiliis is a ciliated protozoan parasite and is known to infect many freshwater teleosts. Characterizing the immune system in epithelial tissues, where the parasites penetrate and settle, is key to understanding host–parasite interactions. This study examined local immune responses in vivo to the infective stage (theront and trophont) of the parasites using intra-fin administration, which has been developed to analyze in vivo immune responses using fish fin. CD8α+ and CD4+ T-cell compositions were increased significantly in the fin cavity injected with theront or trophont antigens. The expression of GATA-3 and T-bet mRNA, which regulate differentiation of helper T-cells, was upregulated significantly in leukocytes from the trophont antigen–injected site. In contrast, the percentages of macrophages and neutrophils, which are innate immunity components, were decreased significantly in the injection sites. These results suggest that I. multifiliis antigens inhibit the migration of macrophages and neutrophils, and T-cells are the first responders to I. multifiliis. Thus, to better understand the interaction of host immunity and I. multifiliis, further studies should focus on exploring the inhibitory factors from I. multifiliis or examining innate functions of teleost T-cells.

Although a previous study using ginbuna crucian carp suggested that cell-mediated immunity can be induced by the oral administration of inactivated viruses, which are exogenous antigens, there is no direct evidence that CD8+ cytotoxic T cells (CTLs) in teleost fish are generated by vaccination with exogenous antigens. In the present study, we investigated whether antigen-specific CD8+ CTLs in ginbuna crucian carp can be elicited by intestinal immunization with an exogenous antigen without any adjuvant. The IFNγ-1 and T-bet mRNA expressions were up-regulated in intestinal leukocytes following the administration of formalin-inactivated crucian hematopoietic necrosis virus (FI-CHNV), whereas the down-regulation of these genes was observed in kidney leukocytes. Furthermore, an increase in the percentage of proliferating CD8+ cells was detected in the posterior portion of the hindgut, suggesting that the virus-specific CTLs are locally generated in this site. In addition, cell-mediated cytotoxicity against CHNV-infected syngeneic cells and the in vivo inhibition of viral replication were induced by immunization with FI-CHNV. Unexpectedly, intraperitoneal immunization with FI-CHNV induced a type I helper T cell (Th1)-response in the intestine, but not in the kidney; however, its effect was slightly lower than that reported after intestinal immunization. These findings suggest that the posterior portion of the intestine is an important site for generating virus-specific CTLs by vaccination with the inactivated vaccine.

Fish mucosal surface is a significant interface for pathogens to infect from an aqueous environment. In addition to mucosal innate and adaptive immune factors, epithelial cells are considered as a significant physical barrier against microbial invasion. Previously, we identified a mammalian CD46-like complement regulatory protein (Tecrem) in teleost and detected its expression on epithelial cells derived from fin, suggesting its physiological role on the fish surface. This study examines the homeostatic roles of Tecrem in maintaining the fish epithelium, by analyzing the expression behavior of Tecrem on the fin-derived epithelial cell lines (KF-1 from the common carp and CFS from ginbuna crucian carp) using monoclonal and polyclonal anti-Tecrem antibodies. Expression of KF-1 protein was associated with the adhesion of KF-1, and the adhesion was enhanced by anti-Tecrem treatments of the cells. Stimulation of the epithelial cells with anti-Tecrem enhanced wound healing, protein expression of tight-junction proteins, and cell density of the KF-1 and CFS monolayer culture. These results suggest that Tecrem on epithelial cells play a homeostatic role in maintaining intactness of the surface epithelial barrier, implying that modification of Tecrem expression may develop a novel tool to improve the first-line defense against pathogens in aquaculture.

Cytotoxic T cells are known to have the ability to kill microbe-infected host cells, which makes them essential in the adaptive immunity processes of various vertebrates. In this study, we demonstrated innate cell-mediated cytotoxicity of CD8+ T cells against protozoan parasites found in the ginbuna crucian carp. When isolated effector cells such as CD8+, CD4+ (CD4-1+), or CD8- CD4- (double-negative, DN), from naive ginbuna crucian carp were co-incubated with target parasites (Ichthyophthirius multifiliis), CD8+ cells from the kidney and gill showed the highest cytotoxic activity. On the other hand, DN cells, which include macrophages and CD4- CD8- lymphocytes, showed the lowest cytotoxic activity against I. multifiliis. Additionally, the cytotoxic activity of CD8+ cells was found to significantly decrease in the presence of a membrane separating the effector cells from I. multifiliis. Furthermore, the serine protease inhibitor 3,4-dichloroisocoumarin and perforin inhibitor concanamycin A significantly inhibited the cytotoxic activity of CD8+ cells. These results demonstrate that CD8+ T cells of ginbuna crucian carp can kill extracellular parasites in a contact-dependent manner via serine proteases and perforin. Therefore, we conclude that CD8+ T cells play an essential role in anti-parasite innate immunity of teleost fish.

Cytotoxic T cells are known to have the ability to kill microbe-infected host cells, which makes them essential in the adaptive immunity processes of various vertebrates. In this study, we demonstrated innate cell-mediated cytotoxicity of CD8

Kalliklectin is a unique fish-specific lectin, whose sequence is similar to the heavy chain of mammalian plasma kallikrein and coagulation factor XI. In this study, we aimed to evaluate dynamic expression profiles of the lectin gene, during early developmental stages, in fugu, Takifugu rubripes. Reverse transcription-polymerase chain reaction (RT-PCR) showed that the kalliklectin gene was not expressed until 14 h post-fertilization (hpf), while the mRNA was detected after 30 hpf. In real-time quantitative PCR (qPCR), the gene was first expressed at 10.5 hpf; then, the expression level increased with a peak at 30 hpf and then gradually decreased. On the other hand, western blotting with specific antibody detected the lectin protein at all tested stages, including the unfertilized egg, which suggests that the lectin detected in the early stages was a maternal factor. Immunohistochemistry demonstrated that kalliklectin was localized at the basement membranes of the newly hatched larvae, while the lectin was widely detected in epidermal cells in larva at 5 dph. A 40-kDa lectin was partially purified from unfertilized eggs using mannose-affinity chromatography, and the lectin was determined as kalliklectin by liquid chromatography with quadrupole time-of-flight tandem mass spectrometry (LC/Q-TOF-MS) analysis, which indicated that the lectin is functional in the eggs. The egg lectin can bind to Gram-positive bacterial pathogens of fish, such as Lactococcus garvieae and Streptococcus iniae. We conclude that fugu kalliklectin might be an important immunocomponent, transferred from mother to offspring.

Although a previous study using ginbuna crucian carp suggested that cell-mediated immunity can be induced by the oral administration of inactivated viruses, which are exogenous antigens, there is no direct evidence that CD8

Complements have long been considered to be a pivotal component in innate immunity. Recent researches, however, highlight novel roles of complements in T-cell-mediated adaptive immunity. Membrane-bound complement regulatory protein CD46, a costimulatory protein for T cells, is a key molecule for T-cell immunomodulation. Teleost CD46-like molecule, termed Tecrem, has been newly identified in common carp and shown to function as a complement regulator. However, it remains unclear whether Tecrem is involved in T-cell immune response. We investigated Tecrem function related to T-cell responses in ginbuna crucian carp. Ginbuna Tecrem (gTecrem) proteins were detected by immunoprecipitation using anti-common carp Tecrem monoclonal antibody (mAb) and were ubiquitously expressed on blood cells including CD8α(+) and CD4(+) lymphocytes. gTecrem expression on leucocyte surface was enhanced after stimulation with the T-cell mitogen, phytohaemagglutinin (PHA). Coculture with the anti-Tecrem mAb significantly inhibited the proliferative activity of PHA-stimulated peripheral blood lymphocytes, suggesting that cross-linking of Tecrems on T-cells interferes with a signal transduction pathway for T-cell activation. These findings indicate that Tecrem may act as a T-cell moderator and imply that the complement system in teleost, as well as mammals, plays an important role for linking adaptive and innate immunity.

Recent studies on fish immunity highlighted the significance of gills as mucosal immune tissues. To understand potential of gills as vaccination sites for inducing adaptive systemic immunity, we investigated virus-specific cell-mediated and humoral immune responses following a "per-gill infection method", which directly exposes virus only to gills. The viral load in crucian carp hematopoietic necrosis virus (CHNV)-infected gills decreased after peaking at a particular time point. Furthermore, the viral titers in the gills following the secondary infection were lower than that after the primary infection, indicating that local adaptive immunity helped the elimination of virus. Gene expression analysis demonstrated that IFN-γ in gills and perforin in kidney were increased after the gill infection. CD8(+) cells in kidney leukocytes increased after the secondary infection, whereas IgM(+) cells decreased. These results suggest that IFN-γ and CTL contribute in controlling CHNV-replication in gills and kidney. Gill infection could induce specific cell-mediated cytotoxicity of peripheral blood leukocytes (PBL) and secretion of CHNV-specific IgM in serum, indicating that local priming of the gill site can generate adaptive systemic immunity. Thus, the gills could be prospective antigen-sensitization sites for mucosal vaccination.

Ulcer disease, caused by atypical Aeromonas salmonicida, is a serious concern in ornamental koi carp, because it induces skin ulceration, disfiguring ornamental fish and causing economic loses. The present study aimed to establish a novel prophylaxis with chicken egg yolk immunoglobulin, IgY, against ulcer disease and to assess its feasibility in the ornamental fish industry. Addition of egg yolk powder containing anti-A. salmonicida IgY to rearing water provided significant protection against an A. salmonicida bath infection, whereas administration of non-specific IgY did not. Consecutive immersion of fish into rearing water containing specific IgY completely prevented ulcer disease resulting from cohabitation infection, indicating that this prophylaxis could prevent infection from such type of contact. Thus, passive immunization induced by immersing fish into aquarium water containing specific IgY is a prospective prophylaxis against diseases caused by pathogens that invade the skin and gills.

In the complement system, the regulators of complement activation (RCA) play crucial roles in controlling excessive complement activation and in protecting host cell from misdirected attack of complement. Several members of RCA family have been cloned from cyclostome and bony fish species and classified into soluble and membrane-bound type as in mammalian RCA factors. Complement-regulatory functions have been described only for soluble RCA of lamprey and barred sand bass; however, little is known on the biological function of the membrane-bound RCA proteins in the lower vertebrates. In this study, a membrane-bound RCA protein, designated teleost complement-regulatory membrane protein (Tecrem), was cloned and characterized for its complement-regulatory roles. Carp Tecrem, an ortholog of a zebrafish type 2 RCA, ZCR1, consists of four short consensus repeat modules, a serine/threonine/proline-rich domain, a transmembrane region, and a cytoplasmic domain, from the N terminus, as does mammalian CD46. Tecrem showed a ubiquitous mRNA expression in carp tissues, agreeing well with the putative regulatory role in complement activation. A recombinant Chinese hamster ovary cell line bearing carp Tecrem showed a significantly higher tolerance against lytic activity of carp complement and less deposition of C3-S, the major C3 isotypes acting on the target cell, than control Chinese hamster ovary (mock transfectant). Anti-Tecrem mAb enhanced the depositions of carp C3 and two C4 isotypes on autologous erythrocytes. Thus, the present findings provide the evidence of complement regulation by a membrane-bound group 2 RCA in bony fish, implying the host–cell protection is an evolutionarily conserved mechanism in regulation of the complement system.

Cytotoxic T-cells (CTLs) play a pivotal role in eliminating viruses in mammalian adaptive immune system. Many recent studies on T-cell immunity of fish have suggested that teleost CTLs are also important for antiviral immunity. Cellular functional studies using clonal ginbuan crucian carp and rainbow trout have provided in vivo and in vitro evidence that in many respects, virus-specific CTLs of fish have functions similar to those of mammalian CTLs. In addition, mRNA expression profiles of CTL-related molecules, such as CD8, TCR and MHC class I, have shown that in a wide range of fish species, CTLs are involved in antiviral adaptive immunity. These findings are a basis to formulate possible vaccination strategies to trigger effective antiviral CTL responses in teleost fish. This review describes recent advances in our understanding of antiviral CTL functions in teleost fish and discusses vaccination strategies for efficiently inducing CTL activities.

Zymosan, a yeast cell wall preparation that binds activated forms of complement C3, is a useful model target to activate the complement system. In our trial to analyze C3 diversity in Nile tilapia at the protein level using zymosan, we found that a novel 240-kDa serum protein (ZBP-240) also bound to zymosan in addition to C3-derived fragments. In the present study, we aimed to characterize tilapia C3 and ZBP-240, focusing on their immune-related functions. Four distinct C3 isoforms were purified from tilapia serum and shown to possess an intrachain thioester bond. ZBP-240 was also isolated from tilapia serum and examined for its binding properties to various microbial targets. As a result, ZBP-240 showed a wide spectrum of binding to Gram-positive and Gram-negative bacteria and yeasts. Amino acid sequence analysis of CNBr fragments of ZBP-240 suggested that this is a novel protein with no homologous sequence in protein databases. It was also suggested that the binding of ZBP-240 to microbes largely depends on hydrophobic interactions in a divalent-cation-independent manner, and that there may be a divalent-cation-dependent factor that enhances the binding of ZBP-240 in tilapia serum. Interestingly, ZBP-240 showed opsonic activity for tilapia kidney phagocytes at a level comparable to that of C3, implying that ZBP-240 is a novel teleost opsonic serum protein.

Regulators of complement activation (RCA) play a role in protecting cells from excessive complement activation in humans. cDNA corresponding to three isoforms of teleost membrane-bound RCA protein (gTecrem) have been identified in the ginbuna crucian carp. gTecrem-1 consists of seven short consensus repeats (SCRs), whereas gTecrem-2 and gTecrem-3 have four SCRs. While gTecrem-1 possesses a tyrosine phosphorylation site in its cytoplasmic region, gTecrem-2 and gTecrem-3 lack the site. Tissue distribution analysis showed that gTecrem-1 and gTecrem-2 mRNAs were expressed in almost all tissues examined, whereas gTecrem-2 expression was not significantly detected in gill, liver, or intestine. Furthermore, analysis showed that gTecrem-1 was expressed in both peripheral blood leukocytes (PBLs) and erythrocytes and was also expressed in T cell subsets such as CD4(+), CD8(+) T cells, and IgM(+) B cells. gTecrem-2 expression was not detected in either PBLs or erythrocytes, whereas gTecrem-3 was expressed only in erythrocytes. These results suggested that gTecrem isoforms may serve different functional roles; gTecrem-1, which is expressed in T cells and possesses a tyrosine phosphorylation site, may act as a complement regulator and a cellular receptor in adaptive immunity.

Previous studies have suggested that anti-viral cytotoxic effector cells induced by infection with a sublethal dose of crucian carp hematopoietic necrosis virus (CHNV) correspond to mammalian cytotoxic T-lymphocytes (CTLs), because the mRNA expression patterns of the effector cells are similar to those of mammalian CTLs. To further characterize the effector population in cell-mediated cytotoxic (CMC) activity, we isolated the effector cells using an anti-CD8α monoclonal antibody, a density gradient and plastic adherence. As expected, the purified CD8α-positive cells killed CHNV-infected cells, indicating that the fish CTLs are one of anti-viral effector cells similar those to in mammals. However, it appeared that cytotoxic cells other than CTLs were the dominant effectors, because CTL-depleted peripheral blood leukocytes (PBL) exhibited significant cytotoxic activity against CHNV-infected cells. In addition, the adoptive transfer of CTL-depleted PBL provided as efficient protection against CHNV-infection as the transfer of PBL containing CTLs. Further analyses showed that sIg/CD8α-negative cells and monocyte-enriched effectors possessed activities that were comparable to or were higher than that of CD8α-positive cells, suggesting that natural killer (NK)-like cells and monocytes are among the dominant effector cells. CMC inhibition assays with concanamycin A suggested that CTLs and CD8α-negative lymphocytes lysed virus-infected cells by a perforin-based cytotoxic pathway. These results indicate that CMC induced by viral-infection is executed by not only CTLs but monocytes and CD8/IgM-negative lymphocytes.

The central component of complement, C3, plays a versatile role in innate immune defense of vertebrates and some invertebrates. A notable molecular characteristic of this component is an intra-chain thioester site that enables C3 to bind covalently to its target. It has been reported that the binding preference of the thioester to hydroxyl or amino groups is primarily defined by presence or absence of the catalytic histidine residue at position 1126 in human C3. In teleosts, a unique C3 (non-His type) has been found, in addition to the common His type C3. These distinct C3 isoforms may provide diversity in the target-binding attributable to the different binding specificities of their thioesters. In the present study, we examine the hypothesized correlation of the catalytic histidine with the binding spectra of two major C3 isotypes of carp towards various model and natural targets. The results reveal that non-His type C3, rather than His type C3, has a wider range of binding spectrum, despite the binding specificity of its thioester being limited to amino groups. It is therefore hypothesized that the binding spectra of C3 isotypes are not defined by the binding specificity of the thioester but is more affected by differences in microbe-associated molecular patterns that activate complement. Overall, the present data imply that non-His type C3 plays a significant role against bacterial infections in the fish defense system.

Clonal ginbuna crucian carp is, a naturally gynogenetic fish, and is a useful model animal for studying T-cell-mediated immunity. To gain molecular information on MHC class I molecules from this species, we have identified four types of MHC class I (caauUA-S3n, caauUF-S3n, caauZE-S3n, and caauZB-S3n) and five beta 2-microglobulin (β(2)m) (caauβ2m-1a, caauβ2m-1b, caauβ2m-2, caauβ2m-3a and caauβ2m-3b) by an expressed sequence tag (EST) analysis and using homology cloning with degenerated primers. Like UA class I genes in other cyprinid fish, the caauUA-S3n shows features of classical MHC class I, such as conservation of all key amino acids interacting with antigenic peptides, and ubiquitous tissue expression. A phylogenetic analysis shows that the β(2)m-1 and β(2)m-2 isoforms are clustered with those of other cyprinid fishes, while β(2)m-3 isoforms make a cluster that is separated from a common ancestor of salmonid and cyprinid fishes. This finding suggests that the β(2)m isoforms of ginbuna cruician carp comprise two lineages and may possess different functions. The MHC class I and β(2)m sequences from one clonal strain will facilitate our understanding of the interaction of MHC class I with β(2)m in teleosts.

The presence of helper and cytotoxic T cells in fish has been suggested, although T cell subsets have yet to be identified at the cellular level. In order to investigate the functions of CD4 and CD8α positive T cells we attempted to produce and characterize monoclonal antibodies (mAbs) against teleost CD4 and CD8α. Here we report the successful production of mAbs against CD4 and CD8α in clonal ginbuna crucian carp Carassius auratus langsdorfii and the function of CD4 positive T cells. In this study we demonstrate the presence of teleost CD4- and CD8α-positive T cell subsets with morphology, tissue distribution and gene expression similar to those of mammalian CD4- and CD8-positive T lymphocytes. Using mAbs we found that CD4/CD8 double positive T cells are only present in the thymus, suggesting that it is the site of T cell development. We further demonstrated in vitro proliferation of CD4 positive T cells by allogeneic combination of mixed leukocyte culture and antigen-specific proliferation of CD4 positive T cells after in vitro sensitization with OVA. In our previous study we showed that CD8α-positive lymphocytes are the primary cell type showing specific cytotoxicity against allogeneic targets. Collectively, these findings suggest that CD4 and CD8α positive T cells in ginbuna are equivalent to helper and cytotoxic T lymphocytes (CTL) in mammals, respectively. This is the first report to show the characteristics and functions of CD4 positive T cells in fish and these findings shed light into the evolutionary origins and primordial functions of helper T cells.

Interleukin-8 (IL-8) is a CXC-type chemokine with a chemotactic activity mainly on neutrophils and plays a key role in promoting inflammation. In teleosts, several CXC-chemokines have been cloned and characterized as being IL-8-like. Phylogenetic data however indicate that the reported teleost IL-8-like chemokines are substantially remote from mammalian IL-8, forming a fish-specific clade of IL-8-like chemokines distinct from that of tetrapod IL-8. In the present study, a novel IL-8-like chemokine, designated CaIL-8, has been found in the expressed sequence tags of carp gills and identified as an orthologue of mammalian IL-8. The CaIL-8 transcript encodes 99 amino acids containing a typical CXC motif but lacks an ELR motif, as in most teleost IL-8-like chemokines. Phylogenetic tree constructed by the maximum likelihood method suggests a closer relationship of CaIL-8 with mammalian IL-8 than with other teleost CXC-chemokines reported to be IL-8-like. In a normal unstimulated carp, CaIL-8 mRNA was detected by RT-PCR only in gills, kidney, spleen, heart and peripheral blood leukocytes, in contrast to a previously reported carp IL-8-like chemokine CXCa, which shows ubiquitous basal expression. The results, taken together, are strongly indicative of the presence of two major IL-8-like lineages of CXC-chemokines in teleost.

The clonal triploid ginbuna crucian carp Carassius auratus langsdorfii, a naturally occurring gynogenetic fish, is a useful model for studying T-cell-mediated immunity. CD4, a T-cell receptor (TCR) coreceptor, is a membrane-bound glycoprotein found on helper T-cells, and assists in the binding of major histocompatibility complexes. In the present study, full-length cDNAs encoding the CD4 molecule from the S3n strain of ginbuna crucian carp were cloned and characterized. 5'-rapid amplification of cDNA ends (RACE) and 3'-RACE yielded two distinct cDNA clones of CD4 homolog from the ginbuna, and these sequences share 95% identity at the amino acid level. These ginbuna CD4 molecules consisted of a signal peptide, immunoglobulin superfamily (IgSf) like domains, a transmembrane domain, and a cytoplasmic domain similar to other known CD4. A tyrosine protein kinase p56(lck) binding motif is conserved in the cytoplasmic tail of ginbuna CD4. Phylogenetic tree analysis indicated that ginbuna CD4 sequences are closely related to CD4L-1 from other fish species. Expression of ginbuna CD4 mRNA was detected in the gill, thymus, head kidney, trunk kidney and peripheral blood leukocytes, indicating that its expression pattern is similar to that of ginbuna TCR beta mRNA. The results suggest that ginbuna CD4 sequences are useful as molecular probes for helper T-cells.

An evolutionarily ancestral complement system is considered to be an opsonic system that seems to be a combination of the lectin and alternative pathways known in mammalian complement. The ancestral system is equipped with lectin-like pattern-recognition molecules linked with specific proteases homologous to the mannose-binding lectin-associated serine protease (MASP) and factor B, which cleave C3 into physiologically active forms with inflammatory and opsonic functions, as seen in primitive invertebrate species. In vertebrates, two rounds of whole-genome duplication allowed the occurrence of two additional reaction cascades, the classical and lytic pathways, providing jawed vertebrates with much more specific and efficient ability of pathogen elimination. A trace of molecular and functional evidence that represents a transient status from invertebrate prototypic complement to a full equipped system can be found in extant jawless species such as lamprey. A striking diversity of complement component isoforms unique to teleost fish is also discussed in evolutionary and functional points of view.

We have cloned cDNAs encoding the alpha and beta chains of CD8 from the tiger pufferfish (fugu), Takifugu rubripes. The cDNA sequences encode a putative leader peptide, extracellular immunoglobulin variable region-like domain, stalk region, transmembrane region, and cytoplasmic tail. A protein tyrosine kinase p56lck binding motif was not found in the putative fugu CD8alpha cytoplasmic tail. O-linked glycosylation sites were found in the stalk of both CD8 chains, suggesting possible stalk formation. Phylogenetic analysis showed that fugu CD8alpha and CD8beta chains cluster with other vertebrate CD8alpha and CD8beta chains, respectively. The fugu CD8 genes comprise six exons separated by five introns. The genes are tandemly aligned 3.6 kb apart and are in the same transcription orientation. Quantitative RT-PCR analysis demonstrated that fugu CD8 is expressed predominantly in lymphoid tissues. In situ hybridization showed that fugu CD8 genes are expressed in thymocytes and lymphocytes within lymphoid organs. Molecular characterization of CD8 in fish provides the basis for development of specific antibodies to identify T-cell subsets, as well as potentially important insights into the evolution of CD8 and the adaptive immunity.

Thrombocytes are nucleated blood cells in non-mammalian vertebrates, which were recently focused on not only as hemostatic cells but also as immune cells with potent phagocytic activities. We have analyzed the phagocytic activation mechanisms in common carp (Cyprinus carpio) thrombocytes. MACS-sorted mAb(+) thrombocytes showed no phagocytic activity even in the presence of several stimulants. However, remixing these thrombocytes with other anti-thrombocyte mAb(-) leukocyte populations restored their phagocytic activities, indicating that carp thrombocyte phagocytosis requires an appropriate exogenous stimulation. Culture supernatant from anti-thrombocyte mAb(-) leukocytes harvested after PMA or LPS stimulation, but not culture supernatant from unstimulated leukocytes, could activate thrombocyte phagocytosis. This proposed mechanism of thrombocyte phagocytosis activation involving soluble factors produced by activated leukocytes suggests that thrombocyte activation is restricted to areas proximal to injured tissues, ensuring suppression of excessive thrombocyte activation and a balance between inflammation and tissue repair.

The lectin pathway of complement is considered to be the most ancient complement pathway as inferred from identification of ancient homologs of mannose-binding lectin (MBL) and MBL-associated serine proteases (MASPs) in some invertebrates. MBL homologs with galactose selectivity and an MASP3-like sequence also occur in bony fish, linking the evolution of the lectin complement pathway from invertebrates to higher vertebrates. However, these cannot be considered authentic complement components until confirmatory functional evidence is obtained. Here, we report the isolation and characterization of two MBL homologs from a cyprinid teleost, the common carp, Cyprinus carpio. One, designated GalBL, corresponds to the MBL-like molecule with the galactose specificity. The other is an authentic MBL with mannose specificity. Both were found to associate with a serine protease that cleaves native human C4 into C4b but not C4i with a hydrolyzed thioester. Molecular cloning and phylogenetic analysis revealed this C4-activating protease to be carp MASP2, indicating that MASP2 arose before the emergence of bony fish. Database mining of MBL-like genes reveals that MBL and GalBL genes are arranged in tandem in the zebrafish genome and that both lectins are conserved in the distantly related puffer fish. These results imply that bony fish have developed a diverged set of MBL homologs that function in the lectin complement pathway.

Our previous studies have demonstrated that virus-specific cell-mediated cytotoxicity of sensitized leukocytes can be induced using clonal ginbuna crucian carp and their syngeneic cell lines. In the present study, we attempt to determine if virus-specific cytotoxic cell populations of fish express CD8alpha and TCRbeta genes. Leukocytes from ginbuna crucian carp were separated into four fractions by immunomagnetic separation and density gradient centrifugation: Fraction A, leukocytes with a density of 1.08 g/ml (primarily lymphocytes); Fraction B, sIg-negative leukocytes with density of 1.08 g/ml; Fraction C, sIg-positive cells (primarily B-lymphocytes); Fraction D, leukocytes with a density of 1.08-1.09 g/ml (primarily neutrophils). Leukocytes in all fractions from uninfected fish do not exhibit cytotoxic activity against virus-infected syngeneic cells and weakly express CD8alpha and TCRbeta mRNAs. In contrast, leukocytes in fractions A and B from virus-infected fish exhibit a high level of cytotoxic activity and strongly express CD8alpha and TCRbeta mRNAs. In addition, mRNA expressions of CD8alpha and TCRbeta in effector cells are upregulated by cocultivation with virus-infected target cells but not uninfected ones. The present study suggests that fish possess virus-specific cytotoxic cells with phenotype and gene expression pattern similar to those of CTLs in mammals.

Like mammalian leucocytes, white blood cells of fish are able to kill altered (e.g. virus-infected) and foreign (allogeneic or xenogeneic) cells. The existence of natural killer (NK)-like and specific cytotoxic cells in fish was first shown using allogeneic and xenogeneic effector/target cell systems. In addition to in vivo and ex vivo studies, very important contributions were made by in vitro analysis using a number of different long-term cytotoxic cell lines established from channel catfish. In mammals, specific cell-mediated cytotoxicity (CMC) as part of the adaptive immune response requires a number of key molecules expressed on effector leucocytes and target cells. CD8+ T lymphocytes kill infected cells only, if their antigen receptor (TCR) matches the MHC class I with bound peptide of the target cell. Expression patterns of the fish gene homologues for TCR, CD8 and MHC class I, as well as related genes, are in agreement with similar function. Convenient systems for the analysis of specific CMC have only recently become available for fish with the combination of clonal fish with syngeneic or allogeneic but MHC class I matching cell lines. It was demonstrated that both, NK- and cytotoxic T (Tc) cells are involved in the killing of virus infected MHC class I matching and mismatching target cells. Analysis of these lymphocyte subsets is only starting for fish. There is also evidence that the different viral proteins trigger different subsets of killer cells. This review further discusses findings on fish CMC with regard to temperature/seasons and ontogeny.

In the present study, we confirmed that cellular immune responses, especially specific cell-mediated cytotoxicity, could be induced in systemic carp leucocytes, following anal administration of antigens. Effector cells isolated from systemic lymphoid tissues (head kidney, spleen and peripheral blood) of carp that were immunised anally with allogeneic cells (EPC or KG cell line) efficiently lysed immunogenic target cells. The lytic activity was increased as a result of secondary sensitisation and peaked around 7 days after the final immunisation. In some aspects, the alloantigen-specific cell-mediated cytotoxicity induced by anal sensitisation was different from that induced by intraperitoneal (i.p.) injection. First, the activity induced by anal immunisation was higher than that resulting from i.p. immunisation when fish were immunised twice with a 7-day interval, whereas similar kinetics of the cytotoxicity were observed after the final immunisation. Second, repeated anal administrations tended to decrease the cytotoxic activity, although repeated i.p. injections increased the activity. These findings indicate that the anal administration of antigens in fish can elicit and modulate cellular immune responses.

The genes and corresponding cDNAs of both alpha and beta chains of the Atlantic salmon (Salmo salar) CD8 molecule have been sequenced and characterized. In addition, the cDNAs for alpha and beta chains of brown trout (Salmo trutta) and for the beta chain in rainbow trout (Oncorhynchus mykiss) have been sequenced. The cDNAs code for signal sequences which are preceded by short 5' UTRs. These are followed by typical immunoglobulin superfamily variable sequences all of which contain two conserved cysteines for the intra-chain disulphide bond. The hinge regions display conserved cysteines for dimerisation and several O-glycosylation motifs for each predicted protein. The domain sharing the highest sequence identity with mammals is the single pass transmembrane domain for all sequences. In salmon, each domain is predominantly coded for by a single exon except the cytoplasmic/3' UTR domains, which are coded for by 3 and 2 exons for the alpha and beta genes, respectively. In the alpha gene, the second cytoplasmic exon may be spliced out to form an alternative shorter transcript which if expressed would exhibit a truncated cytoplasmic tail. A splice variant found for the salmon beta gene introduces a stop codon after only 40 amino acids. Overall amino acid identities between salmonid sequences were higher than 90%, whereas they shared only 15-20% identity with species such as, chicken and human. Analysis of the expression patterns of the two salmon genes using quantitative RT-PCR shows a very high expression in the thymus. This is mirrored by the expression of the TCRalpha gene, which is known to be co-expressed with CD8 on mammalian T cells. This is the first report of a sequence for CD8beta in a teleost and together with the CD8alpha sequence, it encodes the ortholog of the CD8 co-receptor molecule on mammalian T cells.

We cloned and sequenced full-length cDNAs for two types of CD8alpha from the S3n strain of ginbuna crucian carp (Carassius auratus langsdorfii) and quantified the expression of CD8alpha genes after sensitization by scale grafting, employing a model system of clonal triploid ginbuna and tetraploid ginbuna-goldfish hybrids. RT-PCR yielded four different fragments of CD8alpha homologue from the S3n strain of ginbuna and these sequences were classified into two groups. The two types of ginbuna CD8alpha (gbCD8alpha) were also found in other strains of triploid ginbuna and goldfish, which are a subspecies of ginbuna. The gbCD8alpha chains consisted of a signal peptide, Ig superfamily (IgSf) V-like domain, hinge, transmembrane domain, and cytoplasmic domain similar to other known CD8alpha. Phylogenetic analysis indicated that both types of gbCD8alpha are closely related to CD8alpha from other vertebrates. Expression of both types of gbCD8alpha mRNA was detected in the gill, thymus, head kidney, posterior kidney, spleen, intestine and peripheral blood leucocytes. In addition, quantitative real-time PCR analysis demonstrated that copy numbers of both gbCD8alpha gene products in kidney cells increased significantly following grafting with allogeneic but not isogeneic scales, and that regulation of expression correlated with that of TCRbeta. Expression of both gbCD8alpha genes after second scale allografting was elevated compared to that after the first set of grafting. These results suggest that expression analysis of these two gbCD8alpha sequences provides a useful tool to address the involvement of cytotoxic T-lymphocytes during the cell-meditated immune response in fish.

This study was designed to examine the effects of dietary vitamin E (VE) on modulation of immune responses when supplied with two levels of n-3 highly unsaturated fatty acids (n-3 HUFA) in rainbow trout, Oncorhynchus mykiss. Six semipurified diets were prepared containing three levels of dietary VE (0, 100 or 1000 mg alpha-tocopheryl acetate kg(-1) diet) and n-3 HUFA either at 20 or 48% of dietary lipid provided from fish oil or docosahexaenoic acid (DHA) concentrated fish oil respectively. The diets were fed to rainbow trout (100 g initial mean weight) for 15 weeks. The VE, vitamin C (VC) content in plasma and tissues and the nonspecific immune responses, both humoral (alternative complement activity, total immunoglobulin) and cellular (phagocytosis, nonspecific cytotoxicity) were examined. VE contents in the kidney reflected the dietary input but were lower in fish fed 48% n-3 HUFA diets, and could have impaired some of immune responses compared to fish fed 20% n-3 HUFA. VC contents in kidney followed the same pattern as VE. Both humoral and cellular immune functions deteriorated in fish fed VE deficient diets whereas improvement in most of the parameters corresponded to its supplementation. However, the higher dose of dietary VE did not substantially enhance the responses assayed compared to the 100 mg dose. Besides clearly indicating the role of VE in maintaining the immune functions in fish in relation to dietary n-3 HUFA, this study has revealed that optimum health benefits could be achieved when VE is maintained slightly above the levels generally recommended for normal growth.

Fish possess immunoglobulins, major histocompatibility complex (MHC), T-cell receptors, and lymphocyte populations analogous to B and T cells and can evoke specific immune responses against a variety of antigens. However, T-cell subsets have yet to be demonstrated and the information on cell-mediated immunity is limited. Here we briefly review our recent studies on specific cell-mediated immunity, particularly on cytotoxic T-cell function employing isogeneic fish and cell lines. Analyses of the graft-versus host reaction (GVHR) and cell-mediated cytotoxicity (CMC) against allogeneic erythrocytes or cell lines show alloantigen-specific cytotoxicity in clonal ginbuna crucian carp. We also describe specific cytotoxicity against virus-infected cells using clonal ginbuna and a syngeneic cell line. Lastly, we report MHC-restriction in CMC against virus-infected cells using homozygous clonal rainbow trout and trout cell line which share the same MHC class I allele. These studies on CMC strongly suggest the presence of antigen specific cytotoxic T cells in teleosts and functional similarities between the immune systems of fish and higher vertebrates. Experimental model systems established in these studies can be applied to the investigation of protective antigens to induce cell-mediated immunity for the development of fish vaccines.

rag1−/− zebrafish have been employed in immunological research as a useful immunodeficient vertebrate model, but with only fragmentary evidence for the lack of functional adaptive immunity. rag1-null zebrafish exhibit differences from their human and murine counterparts in that they can be maintained without any specific pathogen-free conditions. To define the immunodeficient status of rag1−/− zebrafish, we obtained further functional evidence on T- and B-cell deficiency in the fish at the protein, cellular, and organism levels. Our developed microscale assays provided evidence that rag1−/− fish do not possess serum IgM protein, that they do not achieve specific protection even after vaccination, and that they cannot induce antigen-specific CTL activity. The mortality rate in non-vaccinated fish suggests that rag1−/− fish possess innate protection equivalent to that of rag1+/− fish. Furthermore, poly(I:C)-induced immune responses revealed that the organ that controls anti-viral immunity is shifted from the spleen to the hepatopancreas due to the absence of T- and B-cell function, implying that immune homeostasis may change to an underside mode in rag-null fish. These findings suggest that the teleost relies heavily on innate immunity. Thus, this model could better highlight innate immunity in animals that lack adaptive immunity than mouse models.

Thrombocytes, nucleated hemostatic blood cells of non-mammalian vertebrates, are regarded as the functional equivalent of anucleated mammalian platelets. Additional immune functions, including phagocytosis, have also been suggested for thrombocytes, but no conclusive molecular or cellular experimental evidence for their potential ingestion and clearance of infiltrating microbes has been provided till date. In the present study, we demonstrate the active phagocytic ability of thrombocytes in lower vertebrates using teleost fishes and amphibian models. Ex vivo, common carp thrombocytes were able to ingest live bacteria as well as latex beads (0.5–3 μm in diameter) and kill the bacteria. In vivo, we found that thrombocytes represented nearly half of the phagocyte population in the common carp total peripheral blood leukocyte pool. Phagocytosis efficiency was further enhanced by serum opsonization. Particle internalization led to phagolysosome fusion and killing of internalized bacteria, pointing to a robust ability for microbe elimination. We find that this potent phagocytic activity is shared across teleost (Paralichthys olivaceus) and amphibian (Xenopus laevis) models examined, implying its conservation throughout the lower vertebrate lineage. Our results provide novel insights into the dual nature of thrombocytes in the immune and homeostatic response and further provide a deeper understanding of the potential immune function of mammalian platelets based on the conserved and vestigial functions.

Although many recent studies have suggested that CD4 + helper T cell (Th-cell) functions are well conserved among teleost fishes and mammals, there is little evidence that CD4 + Th-cells in fish are actually involved in both humoral and cell-mediated immunity during a secondary immune response. In the present study, adoptive transfer using clonal ginbuna crucian carp and crucian carp hematopoietic necrosis virus (CHNV) was used to investigate the functions of CD4 + cells during humoral and cell-mediated immunity. With regard to humoral immunity, transplanting CHNV-sensitized donor cells, containing CD4 + cells, into naive fish induced more rapid and stronger antibody production than by transplanting non-sensitized donor cells or sensitized donor cells lacking CD4 + cells. During cell-mediated immunity, no significant differences were found in recipients that received sensitized cells regardless of whether the donor cells contained CD4 + cells, although recipients that received both sensitized donor cells (with and without CD4 + cells) exhibited more efficient cell-mediated cytotoxicity than those that received non-sensitized donor cells. These findings suggest that inducing a secondary antibody response requires CD4 + cell help, and secondary cell-mediated immunity can be induced both by CD4 + cells and leukocytes other than CD4 + cells.