Graft-versus-host reaction (GVHR) in clonal amago salmon, Oncorhynchus rhodurus.

The graft-versus-host reaction (GVHR) was demonstrated in a salmonid model system of clonal diploid and triploid amago salmon. Triploid operculum grafts on clonal diploid evoked an acute rejection within 12 days. Grafts exchanged among triploid amago salmon exhibited prolonged survival for 18 days. In contrast, diploid grafts on triploid, and allografts among clonal diploid amago salmon were accepted. A typical GVHR was induced in triploid recipients by intraperitonal injection of head kidney cells from sensitised diploid donors. The clinical signs of graft-versus-host disease (GVHD) were observed in the recipients after 1 week of cell injection as a loss of appetite and appearance of solid faeces, followed by haemorrhage, local swelling of ventral skin and an enlarged spleen. Three of six fish died within 1 month. Water temperature and frequency of sensitisation are critical to induce GVHR. Diploid donors had to be sensitised three times at 20 degrees C to induce the typical GVHR. GVHR was most effectively induced by head kidney cells, followed by peripheral blood leucocytes (PBL) and spleen cells. Ploidy analysis by flow cytometry revealed that the donor head kidney cells greatly increased in the recipient liver, head kidney and spleen, and reached the peak after 9 days of donor cell injection. The results in the present study are quite similar to the findings in ginbuna and ginbuna-gold fish hybrid system, suggesting the presence of T cells in salmonid as well as cyprinid fish.

Cytotoxic T cell function in fish.

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.

Differential expression of two tumor necrosis factor genes in rainbow trout, Oncorhynchus mykiss.

A second TNF gene (TNF2) has been cloned and sequenced in rainbow trout. In common with the first TNF gene isolated (TNF1), this gene is more TNF alpha-like than TNF beta-like. The full length cDNA is 1519bp, containing a 765bp open reading frame. The gene has four exons, of 380, 49, 60 and 1030bp, respectively. Analysis of the 5' flanking regions of TNF1 and TNF2 reveals several interesting differences in identified transcriptional regulatory elements, with a CATAAA box present 26bp upstream of the transcription start in both genes. Expression analysis in LPS stimulated macrophages has shown a much stronger expression of TNF2 relative to TNF1, with expression being detected earlier and lasting longer.

Exogenous antigens and the stimulation of MHC class I restricted cell-mediated cytotoxicity: possible strategies for fish vaccines.

An MHC class I restricted cytotoxic T lymphocyte (CTL) activity assay has recently been established for rainbow trout. MHC class I restricted cytotoxicity probably plays a critical role in immunity to most viral diseases in mammals and may play a similar role in fish. Therefore, it is very important to investigate what types of vaccines can stimulate this immune response. Although logical candidates for vaccine components that can stimulate an MHC class I restricted response are live attenuated viruses and DNA vaccines, these materials are generally not allowed in fish for commercial vaccine use due to potential safety issues. In mammals, however, a number of interesting vaccination strategies based on exogenous antigens that stimulate MHC class I restricted cytotoxicity have been described. Several of these strategies are discussed in this review in the context of fish vaccination.

Cloning and characterisation of tandem-repeat type galectin in rainbow trout (Oncorhynchus mykiss).

Fish beta-galactoside binding lectin (galectin) cDNA was cloned from the cDNA library of rainbow trout (Oncorhynchus mykiss) head kidney. The clone contained a single open reading frame encoding 341 amino acids (aa) (38 kDa protein), including the initiator methionine. Significant sequence homology to mammalian galectin-9 (40-55% identity) was observed. Its amino acid sequence showed two distinct N- and C-terminal domains (148 and 130 aa, respectively) connected by a peptide linker (63 aa). The galectin contains two consensus WG-E-R/K motifs thought to play an essential role in sugar-binding, indicating that this lectin is a member of the tandem-repeat type galectins which have not been identified in fish. The 1.6 kDa mRNA of the lectin was found by Northern blot analyses to be widely expressed in the spleen, head kidney, thymus, peritoneal exudate cells, ovary, gills and heart. Southern blot analyses with the probe for C-terminal of the lectin showed the existence of two hybridising genes. These results suggest that rainbow trout has at least one tandem-repeat type galectin as well as proto-type galectin.

Tumor necrosis factor alpha (TNFalpha)-like factor produced by macrophages in rainbow trout, Oncorhynchus mykiss.

The presence of a Tumor Necrosis Factor alpha (TNFalpha)-like molecule has been suggested in fish by biological assays and biological and antigenic cross-reactivities with human TNFalpha. In the present study, whether rainbow trout macrophages produce TNFalpha was examined. Murine recombinant TNFalpha (m-rTNFalpha) was used as the standard mammalian TNFalpha. The supernatants were harvested from trout macrophage culture stimulated with lipopolysaccharide (LPS) and then passed through a Polymyxin B column to remove LPS. Results show that trout macrophage culture supernatants exhibit TNF-like activities. The supernatants significantly enhanced neutrophil migration and macrophage respiratory burst activity as assessed by NBT reduction test. The supernatants were also highly cytotoxic to murine L929 cells, which are known to be sensitive to mammalian TNFalpha. The biological activities of TNF in the trout macrophage culture supernatant was determined as 2.6 U ml(-1) in the presence of actinomycin D. This indicates biological cross-reactivity of trout TNFalpha-like factor on mammalian cells. Moreover, these activities were inhibited by a rabbit anti-mTNFalpha antibody. These results suggest that rainbow trout macrophages produce a TNFalpha-like factor that is similar to the mammalian TNFalpha in functions.

Luciferase expression 2 years after DNA injection in glass catfish (Kryptopterus bicirrhus).

Glass catfish (Kryptopterus bicirrhus) have transparent muscles and skin. Intramuscular injection of DNA encoding luciferase into these fish induced luciferase expression that was measurable in vivo with a low light video image analyser. Expression could be detected up to at least 2 years after DNA injection. Although luciferase is not representative of all types of antigen, this study stresses the need for future studies directed to limit the period of antigen expression after DNA vaccination.

Specific cell-mediated immunity in fish.

This review describes the fish immune system, focusing on specific cell-mediated immunity. Specific in vivo cell-mediated immune responses have been shown by allograft rejection, graft-versus-host reaction (GVHR) and delayed hypersensitivity reaction (DTH). Recent in vitro studies also showed specific cell-mediated cytotoxicity against allogeneic target cells. These in vivo and in vitro experiments strongly suggest the presence of cytotoxic T cells in fishes. Also described are current studies on shark and trout MHC class I polymorphism and function that demonstrate strong similarities between fish and mammals.

Conservation and diversification of MHC class I and its related molecules in vertebrates.

The elucidation of the complete peptide-binding domains of the highly polymorphic shark MHC class I genes offered us an opportunity to examine the characteristics of their predicted protein products in the light of the latest advance in the structural studies of the MHC class I molecules. The results suggest that the fundamental characteristics in the T-cell recognition of the MHC class I molecule/peptide complex are expected to have been established at the early stage of the vertebrate evolution. The elucidation of the typical classical class I molecules from fishes and also of some MHC class I-related molecules may help us-to explore the common denominator of the ancient class I molecules.

The graft-versus-host reaction (GVHR) in the ginbuna crucian carp, Carassius auratus langsdorfii.

A model system of clonal triploid ginbuna and tetraploid ginbuna-goldfish hybrids was employed to demonstrate the presence of graft-versus-host reaction (GVHR) in a teleost fish. Tetraploid scale grafts on triploid clone members evoked an acute rejection, whereas the reverse transplants were accepted. When sensitized triploid cells were injected into tetraploid recipients, a typical GVHR was induced, leading to death of the recipients within one month. The onset of illness appeared about one week after cell injection as a loss of appetite and constipation, followed by a scale protrusion, severe haemorrhage, local destruction of the ventral skin and prominent splenomegaly. GVHR was most effectively induced by head-kidney cells and peripheral blood leukocytes (PBL), followed by spleen and thymus cells. Donors had to be sensitized at least twice by scale grafting to induce the reaction. A considerable number of recipients injected with cells from donors which had been sensitized by allogenetically different tetraploids died, suggesting a limited polymorphism or heavy cross-reactions between the alleles of the histocompatibility antigens. Ploidy analyses revealed that donor cells greatly increased in the host liver and spleen, constituting approximately 30% of total cells after 2 3 weeks. Most of these features of acute GVHR observed in this fish system are quite similar to those found in mammals and birds. thereby suggesting the presence of allo-reactive cytotoxic T lymphocytes in teleosts.

In vitro cell-mediated cytotoxicity against allogeneic erythrocytes in ginbuna crucian carp and goldfish using a non-radioactive assay.

Cell-mediated cytotoxicity of clonal ginbuna crucian carp leukocytes against allogeneic erythrocytes is described using a sensitive non-radioactive in vitro assay. Hemoglobin released from target erythrocytes after cell-mediated erythrolysis was detected by tetramethylbenzidine (TMB). TMB assay showed clear correlation with a 51Cr-release assay and even exhibited higher cytotoxicity. The use of erythrocytes as target cells has several advantages over a conventional 51Cr-release assay. Erythrocytes do not have cytotoxic activity, are relatively homogeneous, are available in large numbers and erythrocyte donors need not be killed. Leukocytes from fish sensitized by erythrocyte injection or scale grafting efficiently lysed allogeneic erythrocytes, but did not kill isogeneic or autologous erythrocytes. In contrast, leukocytes from unsensitized fish did not lyse allogeneic erythrocytes and repeated sensitizations by allogeneic grafts were necessary to induce cytotoxic cells. Effector cells isolated from peripheral blood showed a higher cytotoxic effect toward allogeneic target cells than effector cells isolated from kidney. These studies support the hypothesis that fish are capable of a genetically restricted specific cell-mediated cytotoxicity.

Antigen uptake and immune responses after immersion vaccination.

Immersion vaccination is an effective and practical method for mass vaccination of fish and most commercial bacterins are currently administered by this method, even though the exact mechanisms of antigen uptake and protection still remain unknown. Immersion vaccination includes several delivery techniques including spray, direct immersion, hyperosmotic dip and flush exposure. Various factors have been shown to influence the uptake of antigen from a vaccine bath, such as the concentration of vaccine, the length of immersion time, size of the fish, stress, pH and salt concentration of the vaccine solution, the water temperature, anaesthetics, the use of adjuvants, and the physical state (particulate or soluble) of the antigen. Among these, the antigen concentration is the most important factor for antigen uptake and protection. Recently we found that the amount of antigen taken up is correlated with the length of immersion time in dilute vaccine solutions. Most authors have suggested the gills as the main site of antigen entry, but skin, lateral line and the gut have also been suggested. Our quantitative study has shown that the skin is the main site of antigen uptake and that there are no differences in rate of uptake between the lateral line and the remaining skin of the body surface. Not only phagocytes but also several types of epithelial cells are involved in antigen uptake. Cells involved in antigen uptake can be different depending on the physical state of the antigen and the site of antigen entry. In most trials with immersion vaccination, antibodies against pathogens are not detectable in the serum by micro-titration and, even when antibodies are found, the titre does not always correlate with protection. However, some authors have reported elevated specific antibody levels in the serum of fish vaccinated by immersion, and even that protection can be successfully conferred by transferring immune plasma. Thus, the role of humoral immunity on protection mechanisms after immersion vaccination has been controversial and potentially important roles for cell-mediated immunity or local immunity have been implicated.

The most primitive vertebrates with jaws possess highly polymorphic MHC class I genes comparable to those of humans.

We report the isolation and extensive analysis of highly polymorphic MHC class I genes from sharks (Triakis scyllia), which belong to the most primitive vertebrate group with jaws, the cartilaginous fish. Predicted complete peptide-binding domains showed retention of the critical amino acid residues that would interact with antigenic peptide termini and revealed extensive allelic polymorphisms comparable to those of classic human MHC class I molecules. Mosaic structures were apparent in these domains, suggesting recombinational mechanisms to create allelic diversity. The present study demonstrates the establishment of the basic strategy for antigen-presentation employed by MHC class I molecules and documents complete divergence of two polymorphic MHC classes at a phylogenetically primitive stage of vertebrate evolution.