Rainbow Trout Leucocytes Research Articles

Viral haemorrhagic septicaemia virus induces vig -2, a new interferon-responsive gene in rainbow trout

An mRNA differential display methodology was used to study the rainbow trout response to viral infection. A new transcript (vig -2) induced by viral haemorrhagic septicaemia virus (VHSV) in rainbow trout leucocytes was identified from the head-kidney. vig -2 was also induced in vivo during experimental infection and following DNA immunisation with a plasmid containing a gene encoding the viral glycoprotein. Viral induction of vig -2 was blocked by cycloheximide (CHX), indicating its dependency on a newly synthesised intermediate protein. This intermediate protein is most probably related to interferon because treatment of cells with a conditioned medium displaying an interferon-like activity resulted in a strong vig -2 expression, which was not blocked by CHX treatment. The cDNA sequence of the vig -2 transcript displays several mRNA destabilisation motifs and two signals characteristic of immediate–early gene expression. Curiously,vig -2 has no evident encoding potential except for a small 51 amino acid putative polypeptide with no clear similarity to any sequence available in the databanks. Therefore, the complete vig -2 genomic sequence was determined from a lambda phage clone retrieved from a genomic DNA library of rainbow trout. The genomic organisation of vig -2 shows five exons delimited with typical splice acceptor and donor sites. A promoter with a canonical ISRE, confirming that vig -2 is an interferon-responsive gene, is also present 115nt upstream of the first exon.

Seasonality of trout leukocytic sensitivity to toxicity: implications for immunotoxic biomonitoring

Aflatoxin B 1 (AFB 1), a widespread food-borne mycotoxin and polycyclic aromatic hydrocarbon, is associated with hepatic carcinogenesis and immunomodulation in a wide variety of vertebrates. Although AFB 1 has been found to be a potent immunomodulator in endotherms, little in the way of immunological assessment has been published in ectothermic species. We found that rainbow trout leukocytes can demonstrate up to a 1000-fold greater sensitivity to the immunosuppression effects of AFB 1 in vitro than is observed with murine leukocytes. This suppression is manifested as a decrease in lymphocytic proliferation and immunoglobulin secretion in response to the polyclonal activator, Escherichia coli lipopolysaccharide. However, this sensitivity appears to be seasonal, exhibiting an acute phase during the July–December period of the year, with the sensitivity falling to lower, more murine-like levels from January to June. These levels of suppression were observed at AFB 1 concentrations which were not toxic to the leukocytes. This sensitivity appears to occur subsequent to a generalized winter-associated period of decreased immune reactivity that has been observed in a number of ectothermic species. It is not unusual to observe a variety of physiologically seasonal effects in ecotherms. Alterations in photoperiod have been noted to have a profound effect on levels of the hormones testosterone, estradiol, and cortisol, all of which been found to alter immune cell function as well as the biotransformation of xenobiotics. This has been specifically demonstrated in the rainbow trout with respect to the impacts of glucocorticoids, estradiol, as well as the process of sexual maturation on P450 activity. If these in vitro observations are indicative of whole animal effects, the dependence of immunotoxicological assessments on seasonality may have tremendous implications for the use of immunoassays for the purposes of biomonitoring in the field. Our results strongly suggest that xenobiotics may have immunosuppressive capabilities that vary over a 1000-fold in their impact depending upon the time of year. Thus, a lack of thorough screening could lead to erroneous conclusions as to the effects of xenobiotics on native fish populations. [Support: NIEHS ES05783.]

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

The dose dependency and kinetics of lipopolysaccharide (LPS) induced interleukin-1β (IL-1β) mRNA expression in rainbow trout leucocytes has been studied. Northern blot analysis revealed weak hybridisation of a trout IL-1β probe to RNA from head kidney leucocytes stimulated with 0.1 μg LPS/ml, with a large increase in IL-1β transcript level seen between 0.1 and 1.5 μg LPS/ml. Using 5 μg 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-1β expression, with low temperatures inhibiting transcription. Indeed, an eight-fold increase in IL-1β transcript level was seen between cells cultured at 4°C and 22°C. Preincubation with cortisol was also shown to inhibit LPS-induced IL-1β expression.

Growth of rainbow trout hemopoietic cells in methylcellulose and methods of monitoring their proliferative response in this matrix.

A technique for the clonal culture of rainbow trout leukocytes in a methylcellulose matrix can be used to identify growth factors and other substances affecting cell proliferation and development in fish. Methylcellulose supports colony formation by rainbow trout leukocytes isolated from the major hemopoietic organ, the pronephros. The addition of rainbow trout serum dramatically increased the number of colonies formed, scored by counting colonies. As an alternative measure of cell proliferation, 3H-thymidine incorporation by cells can be easily measured in methylcellulose cultures. This method requires only small amounts of test substances, is rapid, and is superior for assessing the growth-stimulating ability of some substances, such as bacterial lipopolysaccharide, which stimulated growth but not the formation of discreet colonies by rainbow trout cells.

Cloning and sequence analysis of rainbow trout LMP 2 cDNA and differential expression of the mRNA

A Rainbow trout Oncorhynchus mykiss (L.) clone encoding the low molecular weight proteasome LMP 2 has been cloned and sequenced from rainbow trout leukocyte cells that had been stimulated with the mitogen phytohemagglutinin (PHA). The cDNA was 1254bp and had an open reading frame of 648bp encoding 217 amino acids. The sequence has a high degree of identity with other published LMP 2 sequences varying from 84·4% identity with medaka (Oryzias latipes) to 61·4% identity with Xenopus laevis. Northern blot analysis showed the LMP 2 mRNA to be expressed in a wide range of tissues, with two transcripts in gill, spleen and anterior kidney RNA. Up regulation of the LMP 2 mRNA transcript was shown in cells that were maintained in primary culture following stimulation with the PHA mitogen for 24h. A 3·7-fold increase in mRNA was observed between control and stimulated cells.

Polyunsaturated fatty acids enhance the heat induced stress response in rainbow trout ( Oncorhynchus mykiss) leukocytes

The heat shock response has been studied extensively, yet the molecular signals that trigger the response remain elusive. The dogma of the heat shock response contends that denatured proteins initiate the response, but evidence is accumulating to point to a more complex system in which at least more than one signal is involved in this process. Thermal stress initiates changes in cellular phospholipid membrane physical state, which when acted upon by phospholipases may release lipid mediators that could serve as triggering signals during the heat shock response. We have examined the heat shock response in freshly isolated leukocytes from the pronephros of rainbow trout ( Oncorhynchus mykiss). In this study, we show that leukocytes isolated from rainbow trout acclimated to 5 or 19°C express elevated levels of heat shock protein 70 (hsp70) mRNA when heat shocked at 5°C above their respective acclimation temperature and supplementation with exogenous docosahexaenoic acid or arachidonic acid followed by heat shock enhanced levels of hsp70 mRNA. The time course for docosahexaenoic acid induced enhancement of hsp70 mRNA was accelerated compared with heat shock alone, and staurosporine inhibited the docosahexaenoic acid induced increase of hsp70 mRNA. We also provide evidence that phospholipase A 2 is involved in the heat shock response.

Subcellular localization of enzyme activities involved in the metabolism of platelet-activating factor in rainbow trout leukocytes

The subcellular distribution of an alkyllyso-GPC: acetyl-CoA acetyltransferase (EC 2.3.1.67) and transacylase, two important enzyme activities involved in the remodeling pathway for the biosynthesis of platelet-activating factor (1- O-alkyl-2-acetyl- sn-glycero-3-phosphocholine, PAF) have been examined in leukocytes isolated from the pronephros of the rainbow trout, Oncorhynchus mykiss. Contrary to mammalian systems, in which the acetyltransferase is localized to intracellular membranes, the subcellular distribution of an acetyltransferase activity in rainbow trout leukocytes was localized to the plasma membrane. Analysis of the acetyltransferase products by thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC) confirmed synthesis of two subclasses of PAF, 1- O-alkyl-2-acetyl- sn-glycero-3-phosphocholine and 1-acyl-2-acetyl- sn-glycero-3-phosphocholine. The transacylase activity in this study was detected in membrane fractions in two domains of the intermediate density region which also contained the NADH dehydrogenase activity, a marker enzyme for the endoplasmic reticulum. Acylation of lysoPAF (1- O-alkyl-2-lyso- sn-glycero-3-phosphocholine) exhibited approximately 95% specificity for ω-3 fatty acids. Acylation patterns were not significantly different in either domain of the endoplasmic reticulum. A model is proposed herein for the metabolism of PAF in rainbow trout leukocytes.

Vig-1, a new fish gene induced by the rhabdovirus glycoprotein, has a virus-induced homologue in humans and shares conserved motifs with the MoaA family.

We used mRNA differential display methodology to analyze the shift of transcription profile induced by the fish rhabdovirus, viral hemorrhagic septicemia virus (VHSV), in rainbow trout leukocytes. We identified and characterized a new gene which is directly induced by VHSV. This VHSV-induced gene (vig-1) encodes a 348-amino-acid protein. vig-1 is highly expressed during the experimental disease in lymphoid organs of the infected fish. Intramuscular injection of a plasmid vector expressing the viral glycoprotein results in vig-1 expression, showing that the external virus protein is sufficient for the induction. vig-1 expression is also obtained by a rainbow trout interferon-like factor, indicating that vig-1 can be induced through different pathways. Moreover, vig-1 is homologous to a recently described human cytomegalovirus-induced gene. Accordingly, vig-1 activation may represent a new virus-induced activation pathway highly conserved in vertebrates. The deduced amino acid sequence of vig-1 is significantly related to sequences required for the biosynthesis of metal cofactors. This suggests that the function of vig-1 may be involved in the nonspecific virus-induced synthesis of enzymatic cofactors of the nitric oxide pathway.

Sensitivity of rainbow trout leucocytes to aflatoxin B1

In vitroexposure of rainbow trout (Oncorhynchus mykiss) peripheral blood leucocytes to aflatoxin B1(AFB1) leads to a dramatic suppression of immunological function as indicated by decreased lymphocyte proliferation and immunoglobulin production in response to the mitogen lipopolysaccharide. These leucocytes were up to 1000-fold more sensitive to this carcinogen than were murine leucocytes. Additionally, leucocytes procured from trout during July–December were significantly (P=0·01) more sensitive to AFB1than those obtained during January–June. Immunosuppression was observed with AFB1concentrations that were not toxic as measured by cell viability. This decrease in AFB1sensitivity appears to occur subsequent to a generalised winter-associated period of decreased immune reactivity that has been observed in a number of ectothermic species and in this study.

Lipid Mediator Mechanisms in Fish

Abstract Salmonids synthesize platelet-activating factor (PAF: 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), and much of the interest in the potential use of lipids to mediate physiological events in fish metabolism is focused on this compound. In sharp contrast to mammalian cells, salmonid cells acylate lysoPAF with a high degree of specificity for omega-3 fatty acids. Polyunsaturated fatty acids in the lipids in tissues of rainbow trout Oncorhynchus mykiss are compartmentalized differently than in mammalian tissues. On extracellular challenge by PAF, trout leukocytes exhibit both chemotaxis and respiratory burst responses. During the metabolism of PAF in rainbow trout leukocytes, acylation of lysoPAF appears highly selective for docosahexaenoic acid (C22:6 n-3) and eicosapentaenoic acid (C20:5 n-3) and is localized in the endoplasmic reticulum. The acyl moiety in the sn-2 position of the acylation product 1-O-alkyl-2-acyl-glycero-3-phosphocholine may be removed by hydrolysis to produce lysoPAF in the...

L3 10:45 Monoclonal antibody labeling of rainbow trout leucocytes: Pattern changes in immunized fish

L3 10:45 Monoclonal antibody labeling of rainbow trout leucocytes: Pattern changes in immunized fish

F4 2:30 Seasonal sensitivity of rainbow trout leukocytes to aflatoxin B 1

F4 2:30 Seasonal sensitivity of rainbow trout leukocytes to aflatoxin B 1

The influence of environmental temperature on membrane fluidity, fatty acid composition and lipoxygenase product generation in head kidney leucocytes of the rainbow trout, Oncorhynchus mykiss

The adaptive responses of leucocytes isolated from the head kidney of rainbow trout, Oncorhynchus mykiss, to temperature changes were studied. Membrane fluidity measurements showed that these cells underwent an adaptive response to reduced environmental temperature. This was mediated, at least in part, by changes in the fatty acid profiles of these cells. The unsaturated to saturated fatty acid ratio of total lipid extracted from leucocytes isolated from summer (14.5°C) and winter (6.5°C) temperature-acclimated fish was increased at the lower temperature. This alteration was as a result of reduced saturated fatty acid and increased polyunsaturated fatty acid content of these cells. The eicosanoid generating capacity of leucocytes from winter-and summer-acclimated trout showed a significant reduction in leukotriene B 4 and B s synthesis in the winter, whereas lipoxin A 4 and A s biosynthesis was unaffected. There was no difference in the ratio of 4-to 5-series lipoxins or leukotrienes derived from 20:4(n-6) and 20:5(n-3), respectively, synthesised by head kidney leucocytes between these two periods. Investigations of the time course of the acclimation to a reduction in temperature showed an increase in the membrane fluidity of leucocytes from fish after 5–10 days maintenance at low (4°C) temperature. This correlated with a progressive increase in the unsaturated to saturated fatty acid ratio in the lipids of these cells. This in turn was as a result of a significant decrease in the relative amounts of 16:0 and increases in 18:2, 18: 3 and 20: 2. Overall, this study shows that rainbow trout leucocytes undergo progressive changes in their membrane fluidity and fatty acid composition following short-term and long-term changes in environmental temperature.

In vitro activation of rainbow trout macrophages stimulates inhibition of Renibacterium salmoninarum growth concomitant with augmented generation of respiratory burst products

DAO Diseases of Aquatic Organisms Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials DAO 25:175-183 (1996) - doi:10.3354/dao025175 In vitro activation of rainbow trout macrophages stimulates inhibition of Renibacterium salmoninarum growth concomitant with augmented generation of respiratory burst products Hardie LJ, Ellis AE, Secombes CJ Rainbow trout head kidney macrophages pretreated for 48 h with a supernatant which contained macrophage activating factor (MAF) obtained from Concanavalin A/phorbol myristate acetate stimulated rainbow trout leucocytes express an enhanced ability to inhibit Renibacterium salmoninarum growth in vitro compared with untreated controls. Enhanced 'killing' capacity of these macrophages was detectable after 24 h exposure to R. salmoninarum in vitro. Addition of NG-methyl-L-arginine in vitro did not diminish bacterial growth inhibition by MAF-activated macrophages, suggesting that nitric oxide radicals were not involved in this event. However, MAF-treated macrophages expressed enhanced respiratory burst activity compared with control macrophages after a 3 d exposure to R. salmoninarum in vitro. In addition, it was demonstrated that R. salmoninarum was susceptible to hydrogen peroxide (H2O2) in vitro using a cell-free assay. Introduction of catalase to the in vitro macrophage assay abrogated the 'killing' activity, suggesting that H2O2 release from activated macrophages was involved in the inhibition of R. salmoninarum growth. The results are discussed in relation to other intracellular pathogens and the possible importance of cell-mediated immunity for protection against bacterial kidney disease. Renibacterium salmoninarum . Macrophage activation . Respiratory burst . Growth inhibition Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in DAO Vol. 25, No. 3. Publication date: June 06, 1996 Print ISSN:0177-5103; Online ISSN:1616-1580 Copyright © 1996 Inter-Research.

Characterization of an Androgen Receptor in Salmonid Lymphocytes: Possible Link to Androgen-Induced Immunosuppression

Cytosol of rainbow trout (Oncorhynchus mykiss) leukocytes demonstrated specific and saturable binding of [3H]testosterone (Kd = 0.99 ± 0.17 nM and Bmax = 30.4 ± 4.9 fmol/mg protein based on a total of 6 determinations from three different cytosolic pools). Specific binding of [3H]testosterone was high in leukocytes and other tissues with known androgen binding affinity (plasma, skin, and liver) and low in other tissues (heart, muscle, and red blood cells). Specific binding of [3H]testosterone was displaced by testosterone and dihydrotestosterone. Androstenedione displaced 50% of specifically bound [3H]testosterone between, 10- and 100-fold excess, while 17α-methyltestosterone. 11-ketotestosterone, and progesterone displaced 50% of specifically bound [3H]testosterone between 100- and 500-fold excess. Cortisol, 17β-estradiol, 17α,20β-dihydroxyprogesterone, the synthetic androgen mibolerone, and the synthetic estrogen ethenylestradiol did not displace [3H]testosterone binding, even at 500-fold excess. Treatment of cytosol with proteolytic enzyme significantly reduced the specific binding of [3H]testosterone. HPLC analysis determined that [3H]testosterone was not metabolized during assay incubation with cytosol. These data strongly suggest that androgen receptors exists in salmonid leukocytes and support the hypothesis that these receptors play a role in androgen induced immunosuppression.

Immunocytochemical analysis of a monoclonal antibody specific for rainbow trout ( Oncorhynchus mykiss) granulocytes and thrombocytes

A monoclonal antibody against rainbow trout peripheral blood leucocytes was selected for its lack of reactivity with rainbow trout immunoglobulin. Its reactivity with leucocytes from peripheral blood, head kidney and spleen was analysed by flow cytometry and electron microscopy, and compared with that of monoclonal antibodies directed against rainbow trout immunoglobulin, which reacted with B cells, B lymphoblasts and plasma cells. The antibody reacted with 5–20% of the peripheral blood leucocytes, 8–9% of head kidney leucocytes and 5–7% of spleen leucocytes. Electron microscopical immunocytochemistry revealed that the antibody reacted strongly with granulocytes and weakly with thrombocytes, and not with erythrocytes, lymphocytes, monocytes or macrophages. The antibody has possible applications in the identification and isolation of rainbow trout leucocytes, either alone or in combination with other monoclonal antibodies.

Effects of dietary fatty acids on eicosanoid-generating capacity, fatty acid composition and chemotactic activity of rainbow trout ( Oncorhynchus mykiss) leucocytes

Rainbow trout, Oncorhynchus mykiss, were maintained on isocalorific diets in which either sunflower, menhaden or Fosol oils were used as the dietary source of fatty acids. At intervals over a period of 6 months, head kidney leucocytes were isolated and used for the analysis of their fatty acid composition and eicosanoid-generating capacity. Major changes in fatty acid composition were apparent within 4 weeks on the diets, with fish fed sunflower oil diets showing a 2.1-fold increase in total n − 6 fatty acids and a 2.3-fold decrease in n − 3 fatty acids, compared with the original basal levels. By week 8 the fatty acid composition changes were greater in the sunflower-fed fish, but thereafter remained relatively stable to the end of the experiment at week 24. Leucocytes from the fish maintained for > 8 weeks on the sunflower oil containing diet produced significantly lower percentages of 5-series lipoxygenase products derived from eicosapentaenoic acid including 12-hydroxyeicosapentaenoic acid, leukotriene B 5 and lipoxin A 5 compared with those cells from fish fed either menhaden or Fosol based diets. Unlike the fatty acid composition, differences in lipoxygenase product profiles between the dietary groups increased throughout the experiment and by week 24 the arachidonic acid/eicosapentaenoic acid derived product ratios were approx. 14:1 in the sunflower oil-fed fish compared with approx. 1:1.5 in the menhaden oil-fed fish. A functional consequence of these differing ratios was seen in the ability of supernatants containing these products to cause the in vitro locomotion of trout neutrophils. Supematants from sunflower oil-fed fish were less chemo-attractive than supernatants from menhaden or Fosol oil-fed fish.

Effect of temperature on macrophage activation and the production of macrophage activating factor by rainbow trout ( Oncorhynchus mykiss) leucocytes

Production of macrophage activating factor (MAF) by rainbow trout leucocytes has been shown to be temperature dependent in vivo and in vitro. Cells from fish held at 14°C and stimulated to produce MAF immediately after isolation were capable of secreting MAF down to 6°C (the lowest temperature tested). However, after 48 h at 6°C, these leucocytes show impaired MAF secretion. Acclimation of fish to low temperatures (7°C) did not recover the inhibitory effects of low in vitro temperatures on MAF production, but if these leucocytes were preincubated at 10 or 18°C for 48 h, MAF was produced from these cells. Interestingly, macrophages isolated from fish kept at 7 or 14°C and cultured at low temperatures (6°C) were responsive to MAF-containing supernatants, and showed a higher relative increase in respiratory burst activity compared with their counterparts cultured at 10 and 18°C. Such observations clearly demonstrate that a major impairment of bactericidal activity at low temperatures resides within the specific immune compartment of fish. The implications for fish health are discussed.

Individual Variations of Natural Killer Activity of Rainbow Trout Leucocytes against IPN Virus-Infected and Uninfected RTG-2 Cells.

Natural killer (NK) activity of head kidney leucocytes of rainbow trout (Oncorhynchus mykiss) against IPN virus-infected and uninfected RTG-2 cells was examined by 51Cr release assay. Fish leucocytes were classified into four groups by cytotoxic activities : Type I, cytotoxic activity against uninfected RTG-2 cells was higher than that against infected cells; Type II, cytotoxic activity against infected cells was higher than that against uninfected cells; Type III, cytotoxic activity was low against both infected and uninfected cells; Type IV, cytotoxic activity was high against both cells. These results indicate that there are individual variations in NK activity of rainbow trout against allogenic and viral antigens.

A comparative study of T and B lymphocytes in rainbow trout (Oncorhynchus mykiss) following their separation by nylon wool adherence and lectin agglutination techniques

A nylon wool separation technique was employed to separate rainbow trout leucocytes into adherent and non-adherent populations. The non-adherent population showed a greater response to concanavalin A (ConA) and a lesser response to lipopolysaccharide (LPS) than did the adherent population in the spleen, kidney and peripheral blood. The great majority (>90%) of thymocytes were in the non-adherent population. The non-adherent population from the spleen, kidney and peripheral blood showed signicantly (P>0.05) higher numbers of acid phosphatase-positive lymphocytes than the adherent population, but there was no significant difference in the pattern of immunochemical staining using a mouse anti-trout IgM monoclonal antibody.