Jason W. Holland

Factors influencing the expression of interleukin-1β in cultured rainbow trout (Oncorhynchus mykiss) leucocytes.

The dose dependency and kinetics of lipopolysaccharide (LPS) induced interleukin-1beta (IL-1beta) mRNA expression in rainbow trout leucocytes has been studied. Northern blot analysis revealed weak hybridisation of a trout IL-1beta probe to RNA from head kidney leucocytes stimulated with 0.1 microg LPS/ml, with a large increase in IL-1beta transcript level seen between 0.1 and 1.5 microg LPS/ml. Using 5 microg LPS/ml expression was first detectable 1-2 h post-stimulation. By 4 h post-stimulation maximal induction was seen but by 24-48 h the level had fallen and by 72 h no transcript was detectable. Culture temperature had a marked effect on IL-1beta expression, with low temperatures inhibiting transcription. Indeed, an eight-fold increase in IL-1beta transcript level was seen between cells cultured at 4 degrees C and 22 degrees C. Preincubation with cortisol was also shown to inhibit LPS-induced IL-1beta expression.

Cloning and expression of the first nonmammalian interleukin-11 gene in rainbow trout Oncorhynchus mykiss

Interleukin (IL)‐11 is a multifunctional cytokine that stimulates hematopoietic progenitor cells and exerts a series of important immunomodulatory effects. It was believed to be restricted to mammals, but here we report the first nonmammalian IL‐11gene, in rainbow trout (Oncorhynchus mykiss). A trout IL‐11 cDNA clone was isolated that contains a 5′‐untranslated region (UTR) of 400 bp, an open reading frame of 612 bp and a large 3′‐UTR of 1924 bp. Analysis of a genomic DNA clone from a trout lambda library revealed that the trout IL‐11 gene has the same five exon/four intron gene organization, as well as the same intron phase, as mammalian IL‐11 genes. The 204 amino acid trout IL‐11 translation has a predicted signal peptide of 26 amino acids and mature peptide of 178 amino acids, with a calculated molecular mass of 20.5 kDa and a theoretical pI of 9.83. The mature peptide contains a cysteine residue and a potential N‐linked glycosylation site that are not present in mammals. Phylogenetic analysis clearly grouped trout IL‐11 with IL‐11 molecules from other species and separated from other members of the IL‐6 family. The IL‐11 gene is highly expressed in intestine and gills in healthy fish and its expression can also be detected in spleen, head kidney, brain, skin and muscle. Bacterial infection of rainbow trout markedly up‐regulates IL‐11 expression in liver, head kidney and spleen. IL‐11 expression is also up‐regulated in RTS‐11 cells (a trout macrophage cell line), which constitutively expressed the lowest level of IL‐11 of the four trout cell lines examined, after stimulation with bacteria, lipopolysaccharide, poly(I:C) and recombinant trout IL‐1β. Only a single transcript of 3.2 kb could be detected in lipopolysaccharide or recombinant IL‐1β‐stimulated RNA samples by northern blotting. The expression results, showing that IL‐11 is widely distributed and modulated by infection and other cytokines, suggest that fish IL‐11 is an active player in the cytokine network and the host immune response to infection.

Eicosanoids and their role in immune modulation in fish—a brief overview

Abstract Eicosanoids have been demonstrated to play a central role in immune regulation in mammals brought about by their direct effects on cells such as macrophages and lymphocytes or by their indirect effects via cytokines. Studies have shown that fish mononuclear phagocytes, granulocytes and thrombocytes synthesize and release both cyclooxygenase- and lipoxygenase-derived products such as prostaglandin E2, leukotriene B4 and lipoxin A4. Whether lymphocytes have the ability to generate leukotrienes and lipoxins is still unclear but they do appear to have 12-lipoxygenase activity that leads to the generation of 12-hydroxy fatty acid derivatives. As in mammals, leukotriene and lipoxin biosynthesis requires the presence of a 5-lipoxygenase activating protein-like molecule that is sensitive to the action of the specific inhibitor, MK-886. The prostaglandin-generating ability of trout macrophages can be altered by incubation with lipopolysaccharide suggesting the possible presence of an inducible cyclooxygenase activity. Prostaglandins have been found to suppress the mitogen-induced proliferation of trout leucocytes and the generation of humoral antibody and plasma cells both in vivo and in vitro. The lipoxygenase products, leukotriene B4 and lipoxin A4 have more variable effects ranging from inhibition to stimulation depending on the assay system employed. Overall, there is clear evidence that eicosanoids play a role in immune regulation in fish in a similar way to that reported in mammals.

Identification of a Novel IL-1 Cytokine Family Member in Teleost Fish

A novel IL-1 family member (nIL-1F) has been discovered in fish, adding a further member to this cytokine family. The unique gene organization of nIL-1F, together with its location in the genome and low homology to known family members, suggests that this molecule is not homologous to known IL-1F. Nevertheless, it contains a predicted C-terminal β-trefoil structure, an IL-1F signature region within the final exon, a potential IL-1 converting enzyme cut site, and its expression level is clearly increased following infection, or stimulation of macrophages with LPS or IL-1β. A thrombin cut site is also present and may have functional relevance. The C-terminal recombinant protein antagonized the effects of rainbow trout rIL-1β on inflammatory gene expression in a trout macrophage cell line, suggesting it is an IL-1β antagonist. Modeling studies confirmed that nIL-1F has the potential to bind to the trout IL-1RI receptor protein, and may be a novel IL-1 receptor antagonist.

Molecular and Functional Characterization of IL-15 in Rainbow Trout Oncorhynchus mykiss: A Potent Inducer of IFN-γ Expression in Spleen Leukocytes

IL-15 is a member of the common γ-chain family of cytokines that possess a heterogeneous repertoire of activities on various cells of the immune system. We report here the first functional characterization of a fish IL-15 in rainbow trout. The trout IL-15 gene is 6-kb long and contains six exons and five introns that transcribe into a 1.2-kb mRNA containing seven out-of-frame AUG initiation codons and translate into a 193-aa peptide. Potential sites for transcriptional activators and repressors have been identified in the trout IL-15 gene. Like IL-15 from other species, trout IL-15 is closely linked to an INPP4B gene, but there is also a BCL10 gene located between the IL-15 and INPP4B genes. Three alternative splicing variants of the trout IL-15 gene have also been identified and their expression in vivo was studied. Trout IL-15 expression is present in all the tissues and cell lines studied. Recombinant trout IFN-γ selectively increased IL-15 expression but had little effect on other cytokines such as IL-1β and IL-11. Recombinant trout IL-15 preferentially stimulated splenic leukocytes from healthy fish, where it induced a large increase in IFN-γ expression, with little, if any, effect on IL-1β expression. This effect was quite long-lived, and was still apparent 24 h poststimulation. Although the exact cell types being affected have still to be determined, it is clear that once produced IL-15 will have a profound affect on the ability of the fish immune system to activate antimicrobial defenses and genes induced themselves by IFN-γ.

Sequence and expression analysis of two T helper master transcription factors, T-bet and GATA3, in rainbow trout Oncorhynchus mykiss and analysis of their expression during bacterial and parasitic infection

The polarization of naïve CD4+ T cells to T helper (Th)1 or Th2 cells is specified by two master transcription factors, T-bet and GATA3, and is an essential feature of mammalian adaptive immune responses to pathogens and the development of long-lasting immunity. We report here the cloning of rainbow trout Oncorhynchus mykiss T-bet and GATA3, to allow the future evaluation of the existence of Th1 and Th2 cells in salmonid fish. The trout T-bet translation shares high amino acid identities to other fish T-bet molecules (71-72%) but low identities to mammalian T-bet genes (41-42%), although the middle T-box DNA binding domain is highly conserved among all the T-bet proteins from fish and mammals. The trout GATA3 has high amino acid sequence identities (73-88%) to all known vertebrate molecules, with two highly conserved zinc finger motifs. The identity of the trout T-bet and GATA3 molecules was confirmed by phylogenetic tree analysis. A comparable expression level of T-bet and GATA3 was seen in the spleen, head kidney and muscle in healthy trout, but a higher expression level of GATA3 was seen in the gills, brain, skin and intestine relative to that of T-bet. T-bet and GATA3 expression was modulated by different stimulants. The T cell stimulant PHA up-regulated the expression of both T-bet and GATA3 in splenocytes, suggesting that they may be mainly expressed by activated T cells. The expression of T-bet and GATA3 in the spleen was increased by acute stress, but their expression was inhibited by bacterial (Yersinia ruckeri) infection. In a parasitic infection model, Tetracapsuloides bryosalmonae infection induced a biased gene expression profile where a large increase in the expression of T-bet, IFN-gamma and IL-2 was seen, suggesting that a Th1-like response is likely induced by this disease. A better understanding of pathogen modulated expression of T-bet and GATA3, and the potential underlying host immune responses elicited as a consequence of their expression, may allow novel future control measures against disease in fish to be developed.

Eicosanoid generating capacities of different tissues from the rainbow trout,Oncorhynchus mykiss

The eicosanoid generating potential of the brain, gills, skin, ovary, muscle, eye, liver, spleen, heart, and alimentary canal in the rainbow trout,Oncorhynchus mykiss, was examined. All the organs/tissues examined synthesized the 12-lipoxygenase products, 12-hydroxyeicosatetraenoic acid (12-HETE), and 12-hydroxyeicosapentaenoic acid (12-HEPE), implying the widespread nature of this enzyme in trout. Both prostaglandin E and LTC were also found in variable amounts in the organs, with the greatest amount of PGE found in the gill. Leukotriene (LT) B4 and LTB5 were found in supernatants from calcium ionophore-challenged brain, skin, ovary, liver, spleen, and heart, but the lipoxins A4 and A5 were only present in brain, ovary, and spleen in relatively small amounts. As lipoxins have previously been shown to be synthesized by macrophages in rainbow trout [Pettittet al., J. Biol. Chem. 266, 8720–8726 (1991)], and related cells (microglial cells) are found in the brain of mammals, the localization of macrophage-like cells in trout brain was investigated immunocytochemically. Monoclonal antibodies specific for trout leucocytes failed to identifity any microglial-like cells in sections of the brain, although microvessels containing immuno-positive reaction products were observed. A number of distinct lipoxygenase products were found in supernatants of ionophore-challenged gill, including 14-hydroxydocosahexaenoic acid, 12-HETE, and 12-HEPE, and a large number of dihydroxy fatty acid derivatives with conjugated triene chromophores. One of these products was tentatively identified as 8(R),15(S)-dihydroxyeicosatetraenoic acid, a dual 12- and 15-lipoxygenase product, but apparently no LTB4 was generated by this tissue.

A novel minicollagen gene links cnidarians and myxozoans

Myxozoans are enigmatic endoparasitic organisms sharing morphological features with bilateria, protists and cnidarians. This, coupled with their highly divergent gene sequences, has greatly obscured their phylogenetic affinities. Here we report the sequencing and characterization of a minicollagen homologue (designated Tb-Ncol-1) in the myxozoan Tetracapsuloides bryosalmonae. Minicollagens are phylum-specific genes encoding cnidarian nematocyst proteins. Sequence analysis revealed a cysteine-rich domain (CRD) architecture and genomic organization similar to group 1 minicollagens. Homology modelling predicted similar three-dimensional structures to Hydra CRDs despite deviations from the canonical pattern of group 1 minicollagens. The discovery of this minicollagen gene strongly supports myxozoans as cnidarians that have radiated as endoparasites of freshwater, marine and terrestrial hosts. It also reveals novel protein sequence variation of relevance to understanding the evolution of nematocyst complexity, and indicates a molecular/morphological link between myxozoan polar capsules and cnidarian nematocysts. Our study is the first to illustrate the power of using genes related to a taxon-specific novelty for phylogenetic inference within the Metazoa, and it exemplifies how the evolutionary relationships of other metazoans characterized by extreme sequence divergence could be similarly resolved.

Fish Suppressors of Cytokine Signaling (SOCS): Gene Discovery, Modulation of Expression and Function

The intracellular suppressors of cytokine signaling (SOCS) family members, including CISH and SOCS1 to 7 in mammals, are important regulators of cytokine signaling pathways. So far, the orthologues of all the eight mammalian SOCS members have been identified in fish, with several of them having multiple copies. Whilst fish CISH, SOCS3, and SOCS5 paralogues are possibly the result of the fish-specific whole genome duplication event, gene duplication or lineage-specific genome duplication may also contribute to some paralogues, as with the three trout SOCS2s and three zebrafish SOCS5s. Fish SOCS genes are broadly expressed and also show species-specific expression patterns. They can be upregulated by cytokines, such as IFN-γ, TNF-α, IL-1β, IL-6, and IL-21, by immune stimulants such as LPS, poly I:C, and PMA, as well as by viral, bacterial, and parasitic infections in member- and species-dependent manners. Initial functional studies demonstrate conserved mechanisms of fish SOCS action via JAK/STAT pathways.

The expression of immune-regulatory genes in rainbow trout, Oncorhynchus mykiss , during a natural outbreak of proliferative kidney disease (PKD)

Proliferative kidney disease (PKD) is a parasitic infection of salmonid fish characterized by an apparently abnormal immune response to the presence of the myxozoan parasite, Tetracapsuloides bryosalmonae. In order to examine the nature of the immune response at the molecular level, the expression of a range of immune regulatory genes, including cytokines and cyclooxygenase (COX)-2 was examined in naive unexposed fish and in naive fish exposed to parasite-infected water at three points during the course of a natural outbreak of PKD. Since fish with advanced PKD pathology generally exhibit increased susceptibility to secondary infections which is typical of stress/cortisol-mediated immune suppression, a further aim of this work was to examine in vitro the influence of the glucocorticoid cortisol on the bacterial lipopolysaccharide (LPS)-induced expression of the trout cytokine genes studied. Two weeks after the initial sampling, naive exposed fish showed a specific profile of up-regulated tumor necrosis factor (TNF)-alpha2, COX-2 and, to a lesser extent, transforming growth factor (TGF)-beta1 expression. As the disease pathology increased, TNF-alpha2 and COX-2 expression returned to normal levels. Stress levels of cortisol suppressed the LPS inducibility of pro-inflammatory cytokine genes, although TGF-beta1 and TNF-alpha2 appeared to be refractory. These data demonstrate that specific immune responses at the molecular level are affected during PKD infection, with the cortisol suppression of cytokine expression in vitro providing a possible link to PKD-mediated cytokine down-regulation and immune suppression.