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C J Secombes

Publications and source records attributed to C J Secombes.

101 records · Page 6Linked to original sources
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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 degrees C and stimulated to produce MAF immediately after isolation were capable of secreting MAF down to 6 degrees C (the lowest temperature tested). However, after 48 h at 6 degrees C, these leucocytes show impaired MAF secretion. Acclimation of fish to low temperatures (7 degrees 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 degrees C for 48 h, MAF was produced from these cells. Interestingly, macrophages isolated from fish kept at 7 or 14 degrees C and cultured at low temperatures (6 degrees 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 degrees 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.

Acclimatization↗
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In vitro studies on the regulation of rainbow trout (Oncorhynchus mykiss) macrophage respiratory burst activity.

Modulation of the respiratory burst activity of head kidney macrophages isolated from rainbow trout (Oncorhynchus mykiss) was observed following treatment with several biologically active substances. Macrophage-activating factor (MAF) induced the highest increment if respiratory burst activity relative to treatment with lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF alpha) or beta-glucans from Saccharomyces cerevisiae. Increased responses were more evident when these molecules were combined in pairs. Negative regulation of respiratory burst activity was observed when diMePGE2 was added to the macrophages, with maximal inhibition seen using a concentration of 2.6 microM. Inhibition was also seen using stimulated macrophages, either by co-incubation of stimuli and diMePGE2 or by adding diMePGE2 to previously stimulated cells. The inhibitory effect on macrophages was detectable with 3 h of incubation with diMePGE2 and by 24 h the level of the response was even lower than that from unstimulated (control) macrophages. Of significance was the finding that the inhibitory effect of prostaglandin on macrophage function could be overcome by co-incubation with stimulatory molecules or by pre-treatment with MAF and LPS or MAF and TNF alpha Thus, the regulation of macrophage activation in fish is likely to be as complex as in mammals.

16,16-Dimethylprostaglandin E2↗
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Constitutive and LPS-induced gene expression in a macrophage-like cell line from the rainbow trout (Oncorhynchus mykiss).

The lack of cell lines from mononuclear phagocytes of salmonid fish has impeded the study of immune function at a cellular level in these economically and ecologically important animals. Here, we report on the further characterization of RTS11, a previously described macrophage-like cell line from the rainbow trout spleen, with regard to its expression of a number of immunologically relevant genes. Analysis of gene expression by reverse transcription-polymerase chain reaction, using rainbow-trout-specific primers demonstrated that RTS11 cells express the beta chain of the class II major histocompatibility complex, the cytokines transforming growth factor-beta (TGF-beta) and interleukin-1beta (IL-1beta), and cyclo-oxygenase-2 (COX-2). Inducing the cells with lipopolysaccharide led to increased expression of IL-1beta and COX-2, as determined by Northern blotting. These results together suggest that RTS11 retains many of the characteristics expected of mature macrophages, and should be a valuable tool for further study of the expression and function of these immunomodulatory proteins in fish.

Animals↗
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Generation of primary monocyte-like cultures from rainbow trout head kidney leukocytes.

Trout primary kidney monocyte-like cultures (T-PKM) were generated by incubating head kidney leukocytes in the presence of cell-conditioned medium (CCM). This technique was adapted from procedures that were previously used to cultivate in vitro-derived kidney macrophages (IVDKM) from the goldfish. Flow cytometric analysis of the initial T-PKM cultures, identified three cell sub-populations, but only one of these sub-populations survived extensive cultivation periods (i.e. >8 days) in the presence of CCM. Functionally, reactive oxygen intermediate (ROI) production was detected following stimulation of T-PKM with PMA. However, these cells failed to produce reactive nitrogen intermediates (RNI) in response to immunological stimuli. In contrast, goldfish IVDKM were capable of producing both ROI and RNI. Using the dihydrorhodamine (DHR) assay and flow cytometry, we identified two ROI-producing sub-populations in goldfish IVDKM but only a single ROI-producing sub-population was present after extended cultivation of T-PKM. This T-PKM sub-population was subsequently sorted using the flow cytometer and shown to possess monocyte-like morphology by microscopic and cytometric analysis. Thus, acquisition of antimicrobial functions following cultivation of kidney leukocytes of rainbow trout and goldfish is markedly different, and may be due to the failure of trout monocyte-like cells to undergo a final differentiation step in vitro.

Animals↗
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Effects of transforming growth factor beta 1 on rainbow trout Oncorhynchus mykiss macrophage respiratory burst activity.

Natural bovine transforming growth factor beta 1 (TGF-beta 1) was found to have both enhancing and suppressing effects on the respiratory burst activity of rainbow trout, Oncorhynchus mykiss, head kidney macrophages. Incubation with TGF-beta 1 alone increased respiratory burst activity using doses from 0.05 to 0.2 ng/mL. However, incubation of activated macrophages with TGF-beta 1 decreased their respiratory burst activity in a dose-dependent manner. Coincubation of macrophages with activating signals (macrophage activating factor containing supernatants and tumor necrosis factor alpha) and TGF-beta 1 inhibited the ability of such signals to increase respiratory burst activity. Similarly, pretreatment of macrophages with TGF-beta 1 prior to treatment with activating signals resulted in a time and dose-dependent decrease in activity relative to cells not treated with TGF-beta 1. These suppressive effects were largest using higher doses of TGF-beta 1 (1 ng/mL). The conservation of TGF-beta s and their possible mode of action are discussed.

Animals↗
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Lysozyme and antiprotease activity in the lesser octopus Eledone cirrhosa (Lam.) (Cephalopoda).

Antiprotease and lysozyme activities were detected in various tissue samples including the haemocytes and haemolymph of Eledone cirrhosa. Injection of live Vibrio anguillarum caused an increase in lysozyme activity in the branchial heart over 48 hours and a decrease in the lysozyme activity of haemocytes over 24 hours. Haemocytes from control PBS injected animals demonstrated increased lysozyme levels 4 hours after injection whereas it decreased after the injection of live bacteria in PBS. The lysozyme activity of the haemolymph was not affected by these procedures. Bacteria injections had no effect on the antiprotease activity of the organ samples but increased the antiprotease activity of the haemocytes compared to controls in the 4 h samples. Haemolymph antiprotease activity decreased at a greater rate following bacteria injection than in control PBS injected animals. Haemocyte numbers/ml increased for both the control and bacteria injected animals with a greater increase demonstrated for the bacteria injected animals in the 4 h sample. Concomittant with the increase in the numbers of circulating haemocytes live V. anguillarum were cleared from the circulation of E. cirrhosa in less than 4 hours.

Animals↗
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Binding of immune complexes to Atlantic salmon peripheral blood leucocytes.

The ability of peripheral blood leucocytes (PBL) of Atlantic salmon to bind immune complexes in an antibody-dependent fashion was investigated. Immune complexes were labelled with fluorescein isothiocyanate and binding of these complexes to isolated PBL was determined by flow cytometry. The data show that a high proportion (up to 65%) of PBL were capable of binding immune complexes, and this binding did not occur when immune serum was replaced with normal serum. The presence of fresh normal serum inhibited or abrogated immune complex-binding of PBL. This is the first report of high levels of immune complex receptors on leucocytes in fish, and the dependence of complex binding on the presence of antibody suggests that these receptors may be similar to Fc receptors which are widely distributed on immunocytes of mammals.

Animals↗
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Expression of an inducible nitric oxide synthase gene in rainbow trout Oncorhynchus mykiss.

Using oligonucleotide primers based on mammalian nitric oxide synthases (NOS), expression of an inducible NOS (iNOS) gene was detected in head kidney and gill tissue of bacterially-challenged rainbow trout. Three overlapping fragments were amplified by RT-PCR and used to construct a contiguous sequence of 1410bp, with high nucleotide homology to iNOS in birds (61%) and mammals (57-59%). The nucleotide sequence translated in one reading frame to produce a partial peptide containing 470 amino acids, with 69-71% amino acid homology with mammalian iNOS, 81% homology with chicken iNOS and 85% homology with a partial (492bp) goldfish iNOS sequence. In vitro stimulation of head kidney macrophages with LPS also induced expression of the trout iNOS RNA, with optimal expression seen using 20-50 microg/ml LPS at 2h to 6h post-stimulation. The evolutionary and functional significance of the trout iNOS sequence are discussed.

Amino Acid Sequence↗
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Genomic organisation of rainbow trout, Oncorhynchus mykiss TGF-beta.

The genomic organisation of Oncorhnchus mykiss TGF-beta has been determined through the generation of contiguous clones by PCR. The O. mykiss TGF-beta gene is approximately 3.4 Kb in length and consists of 7 coding exons with no introns in the 5'-UTR. Whilst this is the same number of exons found in TGF-beta genes of amphibians, birds and mammals, in the O. mykiss gene intron 2 of other vertebrates is absent and an additional intron is present at the 3' end of the molecule, splitting exon 7 of the other known TGF-beta genes into two exons (trout exons 6 and 7). Comparison of exon sizes in the coding region support the suggestion that the Xenopus TGF-beta5 and trout TGF-beta sequences are the forerunners of TGF-beta1. Conservation of exons coding for the mature TGF-beta peptide is relatively high (63-73% identity) but other exons show lower identities (37-58%). Comparison of the TGF-beta intron sequences reveals that in general the O. mykiss introns are considerably shorter than the avian homologs. The impact of the teleost TGF-beta gene organisation on theories of the gene evolution of this cytokine family are discussed.

Amino Acid Sequence↗
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Factors influencing the expression of interleukin-1 beta 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.

Animals↗