Modulation of fish phagocytic cells by N-terminal peptides of proopiomelanocortin (NPP)

N-terminal peptide of proopiomelanocortin (NPP, or pro-gamma-MSH) has shown to exhibit biological activity such as stimulation of adrenal mitogenesis and prolactin release-inhibiting factor activity. Structurally, studies reveal a significant difference between fish NPP from that of tetrapods, as NPPs from carp and salmonid lack gamma-MSH. Thus, fish NPP may exhibit functions different from that of mammals. The activation of phagocytic cells by NPP was analysed using rainbow trout Oncorhynchus mykiss and carp Cyprinus carpio. Rainbow trout and carp macrophages incubated with chum salmon NPP significantly enhanced the production of superoxide anion in comparison with control macrophages (without hormones). Both rainbow trout and carp macrophages had shown increased phagocytosis when stimulated administered with NPP. The above results were complemented by in vivo studies where NPP was administered to rainbow trout and carp. NPP significantly increased superoxide anion production as well as phagocytosis in macrophages. These results show that NPP in lower vertebrates activates the function of the phagocytic cells.     

Identification of carp proopiomelanocortin-related peptides and their effects on phagocytes

We report the immunomodulating effects of proopiomelanocortin (POMC)-related peptides on phagocytic cells in carp. The complete amino acid sequences of two carp POMCs (I and II) were deduced from the nucleotide sequences after cDNA cloning. Both POMCs consist of 194 amino acids (91% sequence identity) including identical alpha-melanotropin (MSH) and beta-endorphin (EP). All hormonal peptides derived from two POMCs were identified by mass spectrometry after separation by high-performance liquid chromatography of an acid-acetone extract from a single pituitary. These peptides were alpha-MSH, N-Des-Ac-alpha-MSH, di-Ac-alpha-MSH, beta-MSH I, beta-MSH-II, N-Ac-beta-EP(1-29), corticotropin-like intermediate lobe peptide I and II and N-terminal peptide of POMC I and II. The immunomodulating effects of synthetic MSHs and EPs on phagocytic cells from carp head kidney were examined. Di-Ac-alpha-MSH, beta-MSH I, N-Ac-beta-EP(1-29) and beta-EP(1-29) increased the production of superoxide anion at 0.1-100 ng ml-1 for these MSHs and 1-100 ng ml-1 for EPs in RPMI 1640 medium.     

Effects of estradiol, progesterone and testosterone on the function of carp, Cyprinus carpio, phagocytes in vitro

The modulation of phagocytic cells by beta-estradiol, 11-ketotestosterone and progesterone was analyzed in common carp Cyprinus carpio. Carp kidney leukocytes were cultured in RPMI 1640 medium containing 0.1, 1, 10, 100 or 1000 nM concentration of each hormone. The production of superoxide anion, nitric oxide (NO) and phagocytosis were measured in vitro. Similar concentrations of cortisol were used as control. Phagocytic activities of carp macrophages was suppressed by treatment with beta-estradiol, progesterone and 11-ketotestosterone. The production of NO in carp macrophages was suppressed by progesterone and 11-ketotestosterone. However, carp macrophages incubated with beta-estradiol, progesterone and 11-ketotestosterone did not show any difference in the production of superoxide anion in comparison with control macrophages in the absence of hormones. Carp macrophages treated with cortisol suppressed phagocytosis and the production of nitric oxide and superoxide anion.     

Effect of gonadotropin-releasing hormone on phagocytic leucocytes of rainbow trout

To clarify the role of gonadotropin-releasing hormone (GnRH) in the fish immune system, in vitro effect of GnRH was examined in phagocytic leucocytes of rainbow trout (Oncorhynchus mykiss). Gene expression of GnRH-receptor was detected by RT-PCR in leucocytes from head kidney. Administration of sGnRH increased proliferation and mRNA levels of a proinflammatory cytokine, tumor necrosis factor (TNF)-α, in trout leucocytes. Superoxide production in zymosan-stimulated phagocytic leucocytes was also increased by sGnRH in a dose-related manner from 0.01 to 100 nM. There was no significant effect of sGnRH on mRNA levels of growth hormone (GH) expressed in trout phagocytic leucocytes. Immunoneutralization of GH by addition of anti-salmon GH serum into the medium could not block the stimulatory effect of sGnRH on superoxide production. These results indicate that GnRH stimulates phagocytosis in fish leucocytes through a GnRH-receptor-dependent pathway, and that the effect of GnRH is not mediated through paracrine GH in leucocytes.     

Suppression in function of phagocytic cells in common carp Cyprinus carpio L. injected with estradiol,progesterone or 11-ketotestosterone

The modulation of phagocytic cells by beta-estradiol, 11-ketotestosterone, progesterone and cortisol was analysed in common carp (Cyprinus carpio L.). Carp were injected intraperitoneally (1 and 5 microg/kg fish) with each steroid and the activity of kidney macrophages was recorded 1 and 3 days post-injection. Bovine serum albumin-injected fish served as controls. Phagocytosis, superoxide anion and nitric oxide production were suppressed in all sex steroid treatments, and the suppression was in a dose-dependent manner. Cortisol treatment also inhibited the immune parameters to an extent similar to each of the steroid hormone. These results show that sex steroids can modulate the function of the phagocytic cells.     

Hormonal control of sulfate uptake by branchial cartilage of coho salmon: role of IGF-I.

The direct hormonal control of sulfate uptake by cartilage matrix of coho salmon was examined by exposing branchial cartilage to 1 microCi.ml-1 35SO4 for 48 hours at 15 degrees C in a defined medium. Sulfate uptake occurred primarily in cartilage (rather than bone) and the amount of specific uptake was similar in epibranchial and ceratobranchial cartilages. Intact and hypophysectomized coho salmon starved for 22 days had equivalent in vitro sulfate uptake, which in both cases were 30% of the uptake seen in branchial cartilage of fed, intact controls. In branchial cartilage from starved coho salmon, in vitro exposure to recombinant bovine insulin-like growth factor I (rbIGF-I) at 1, 10, 100, and 1,000 ng.ml-1 caused a dose-dependent increase in sulfate uptake, with a maximum 3-fold increase over control at 1,000 ng.ml-1 rbIGF-I. Coho salmon insulin (1, 10, 100, and 1,000 ng.ml-1) resulted in a maximum 30% increase in sulfate uptake at the highest dose. Growth hormone and triiodo-L-thyronine had no direct effect on in vitro sulfate uptake. The results indicate that IGF-I has direct effects on coho salmon cartilage and may be an important regulator of growth in salmon and other teleosts.     

Distribution of carotenoids in the eggs from four species of salmonids.

1. The distribution of carotenoids in unfertilized ovulated eggs from chum salmon, kokanee (natural and cultured), masu salmon and cultured rainbow trout was examined from the comparative biochemical point of view. 2. The carotenoid contents in the eggs from cultured salmons such as kokanee and rainbow trout were low, whereas those from natural salmons were high. 3. The carotenoids were distributed in both chylomicra particles with high levels of triglyceride and lipovitellin. The carotenoids in the eggs of cultured kokanee and masu salmon were mostly distributed in lipovitellin, whereas those of the other salmons were equally contained in both chylomicra particles and lipovitellin. 4. Comparison of carotenoid distribution in the eggs from immature and mature chum salmon indicated that the carotenoids were bound to lipovitellin, as well as to chylomicra particles, during vitellogenesis.     

In vitro effect of diacetoxyscirpenol and deoxynivalenol on microbicidal activity of murine peritoneal macrophages

Diacetoxyscirpenol and deoxynivalenol, two trichothecene mycotoxins shown previously to exert immunosuppressive effects on the immune system were examined for their in vitro effects on some functions of murine peritoneal macrophages. The cells were pre-incubated for 4 hr with the mycotoxin concentrations of 0.1 ng/ml–1 g/ml. At concentrations that did not affect the cell viability (Specific Lactate Dehydrogenase test), diacetoxyscirpenol and deoxynivalenol suppress microbicidal activity of phagocytic cells. The diacetoxyscirpenol concentrations, which reduce phagocytosis (2ng/ml), microbicidal activity (1 ng/ml), superoxide anion production (1 ng/ml) and phagosome-lysosome fusion (0.1 ng/ml), indicate that the inhibition of killing mechanism arise from both oxidative and non-oxidative pathways. Phagocytosis, microbicidal activity and superoxide anion production are inhibited by deoxynivalenol at 1 ng/ml whereas phagosome-lysosome fusion is reduce above 100 ng/ml. These results suggest that microbicidal activity inhibition by deoxynivalenol did not depend on non-oxidative pathway (phagosome-lysosome fusion) impairment.     

Detection of Renibacterium salmoninarum antigen in migrating adult chum salmon (Oncorhynchus keta) in Japan.

Renibacterium salmoninarum antigen was detected in the kidney of migrating chum salmon (Oncorhynchus keta) using the indirect dot blot assay and indirect fluorescent antibody test. The adult chum salmon had migrated into a bay in which cultured coho salmon infected with R. salmoninarum were present. Antigen was detected in 5% of the chum salmon although they did not have clinical signs of bacterial kidney disease (BKD). This report describes the first case of R. salmoninarum antigen detection among wild chum salmon populations in eastern Asia.     

Activation of rainbow trout, Oncorhynchus mykiss, phagocytic cells by administration of bovine lactoferrin

The chemiluminescent response of kidney phagocytic cells was significantly increased by the oral administration of bovine lactoferrin to rainbow trout, Oncorhynchus mykiss. The phagocytic activity of kidney cells was also increased by administration of bovine lactoferrin to the fish. Activation of kidney cells was also observed in vitro with cells cultured in the presence of bovine lactoferrin.     

全鱼基因的构建及其在鲫鱼体内的整合与转录

利用PCR技术删除大麻哈鱼生长激素基因的启动序列,通过基因重组构建出全鱼基因（鲤鱼MT启动子－大麻哈鱼生长激素基因）;以融合全鱼基因为外源基因,通过显微注射方法将其线性片段导入鲫鱼受精卵内,研究其整合与转录效率。结果表明,全鱼基因在鲫鱼基因组中的整合率为36．4％（16／44）,对转基因阳性鱼的RNA样本进行Northern印迹杂交检测,转录率为25％（1／4）。因此,该全鱼基因可以作为转基因鱼研究和应用的外源基因。     

Enhancement of innate immunity in rainbow trout (Oncorhynchus mykiss Walbaum) associated with dietary intake of carotenoids from natural products

The effects of orally administered carotenoids from natural sources on the non-specific defense mechanisms of rainbow trout were evaluated in a nine-week feeding trial. Fish were fed four diets containing either beta-carotene or astaxanthin at 100 and 200 mg kg-1 from the marine algae Dunaliella salina and red yeast Phaffia rhodozyma, respectively, and a control diet containing no supplemented carotenoids. Specific growth rate and feed:gain ratio were not affected by dietary carotenoid supplementation. Among the humoral factors, serum alternative complement activity increased significantly in all carotenoid supplemented groups when compared to the control. On the other hand, serum lysozyme activity increased in the Dunaliella group but not in the Phaffia group, whereas plasma total immunoglobulin levels were not altered by the feeding treatments. As for the cellular responses, the superoxide anion production from the head kidney remained unchanged while the phagocytic rate and index in all supplemented groups were significantly higher than those of the control. These findings demonstrate that dietary carotenoids from both D. salina and P. rhodozyma can modulate some of the innate defense mechanisms in rainbow trout.     

Susceptibility of rainbow trout Oncorhynchus mykiss,Atlantic salmon Salmo salar and coho salmon Oncorhynchus kisutch to experimental infection with sea lice Lepeophtheirus salmonis

Physiological, immunological and biochemical parameters of blood and mucus, as well as skin histology, were compared in 3 salmonid species (rainbow trout Oncorhynchus mykiss, Atlantic salmon Salmo salar and coho salmon O. kisutch) following experimental infection with sea lice Lepeophtheirus salmonis. The 3 salmonid species were cohabited in order to standardize initial infection conditions. Lice density was significantly reduced on coho salmon within 7 to 14 d, while lice persisted in higher numbers on rainbow trout and Atlantic salmon. Lice matured more slowly on coho salmon than on the other 2 species, and maturation was slightly slower on rainbow trout than on Atlantic salmon. Head kidney macrophages from infected Atlantic salmon had diminished respiratory burst and phagocytic capacity at 14 and 21 d post-infection (dpi), while infected rainbow trout macrophages had reduced respiratory burst and phagocytic capacities at 21 dpi, compared to controls. The slower development of lice, coupled with delayed suppression of immune parameters, suggests that rainbow trout are slightly more resistant to lice than Atlantic salmon. Infected rainbow trout and Atlantic salmon showed increases in mucus lysozyme activities at 1 dpi, which decreased over the rest of the study. Mucus lysozyme activities of infected rainbow trout, however, remained higher than controls over the entire period. Coho salmon lysozyme activities did not increase in infected fish until 21 dpi. Mucus alkaline phosphatase levels were also higher in infected Atlantic salmon compared to controls at 3 and 21 dpi. Low molecular weight (LMW) proteases increased in infected rainbow trout and Atlantic salmon between 14 and 21 dpi. Histological analysis of the outer epithelium revealed mucus cell hypertrophy in rainbow trout and Atlantic salmon following infection. Plasma cortisol, glucose, electrolyte and protein concentrations and hematocrit all remained within physiological limits for each species, with no differences occurring between infected and control fish. Our results demonstrate that significant differences in mucus biochemistry and numbers of L. salmonis occur between these species.     

Effect of angiotensin II and losartan on the phagocytic activity of peritoneal macrophages from Balb/C mice

Angiotensin II (AII), a product of rennin-angiotensin system, exerts an important role on the function of immune system cells. In this study, the effect of AII on the phagocytic activity of mouse peritoneal macrophages was assessed. Mice peritoneal macrophages were cultured for 48 h and the influence of different concentrations of AII (10(-14) to 10(-7) M) and/or losartan, 10(-16) to 10(-6) M), an AT1 angiotensin receptor antagonist, on phagocytic activity and superoxide anion production was determined. Dimethylthiazoldiphenyltetrazolium bromide reduction and the nucleic acid content were used to assess the cvtotoxicity of losartan. A stimulatory effect on phagocytic activity (P < 0.05) was observed with 10(-13) M and 10(-12 M) AII concentrations. The addition of losartan (up to10(-14) M) to the cell cultures blocked (P < 0.001) the phagocytosis indicating the involvement of AT1 receptors. In contrast, superoxide anion production was not affected by AII or losartan. The existence of AT1 and AT2 receptors in peritoneal macrophages was demonstrated by immunofluorescence microscopy. These results support the hypothesis that AII receptors can modulate murine macrophage activity and phagocytosis, and suggest that AII may have a therapeutic role as an immunomodulatory agent in modifying the host resistance to infection.     

Role of distinct subpopulations of peritoneal macrophages in the regulation of reactive oxygen species release.

It has been reported in vitro that during the respiratory burst of phagocytic cells the superoxide anion production per cell shows a negative relation with the cell density. This process has been described as autoregulation. The aim of this work was to analyze the superoxide anion production in thioglycollate-elicited peritoneal macrophage exudates to evaluate the importance of the peritoneal cavity environment in the autoregulation process. 12-O-tetradecanoylphorbol-13-acetate (PMA) was used to stimulate the respiratory burst and superoxide anion production was measured evaluating the intracellular formazan deposits that precipitate as a result of nitro blue tetrazolium (NBT) reduction. We have demonstrated a negative correlation between superoxide anion production and cell density in the peritoneal cavity in macrophages challenged with PMA. The response of individual cells was analyzed by means of an image analyzer, measuring the amount of formazan per cell and cell-size changes during the process of activation. The results revealed that the decrease in individual cell response as a function of higher cell densities were due to a significant increase in the amount of basal reaction macrophages. Concomitantly, the number of reactive cells remained unchanged irrespective of the cell density of the population. A direct correlation between cell size and superoxide anion production was observed. This phenomenon was demonstrated in SENCAR and Balb/c strains. However, macrophages from SENCAR mice showed greater superoxide anion production than those from Balb/c.The differences between strains could be associated to the increased sensitivity to PMA tumor promotion of SENCAR mice. Based on this property, macrophages from SENCAR mice were stimulated with opsonized zymosan, a particulate stimulus that reflects the interaction macrophage-microorganism during the phagocytic process. This data will contribute to the knowledge of infection control. We conclude that variations in basal reaction cells modulates the macrophage activation response when excess macrophages are recruited to the peritoneum. This is demonstrated using different stimuli, thus suggesting that this response may be applied to a wide variety of stimuli-macrophage interactions. The differences between strains may be associated to the increased sensitivity to PMA tumor promotion of SENCAR mice.     

Salmon growth hormone is transported into the circulation of rainbow trout,Oncorhynchus mykiss,after intestinal administration

Summary Transport of recombinant salmon growth hormone (rsGH) into the circulation of rainbow trout following intragastric or rectal administration was investigated. Changes in plasma GH levels were analyzed by radioimmunoassay specific to chum salmon GH. Intragastric administration of rsGH by oral intubation resulted in elevation of GH immunoreactivity in plasma after 11h. The plasma GH increased maximally after 15h, and then declined rapidly to reach a normal level after 19h. On the other hand, oral intubation of rsGH to carp, which has no stomach, caused elevation of plasma GH levels after 1 h which lasted for 21 h. In sharp contrast, rectal administration of rsGH to rainbow trout significantly elevated plasma GH levels after 15 min, which reached a maximum after 30 min. The rsGH was labeled with fluorescein isothiocyanate (FITC) to distinguish exogenous from endogenous GH, and administered into rainbow trout rectally. Subsequent analysis of plasma samples on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed two fluorescent bands at the same molecular weights as those of the monomeric and dimeric rsGHs. Intragastric and rectal administration of rsGH into juvenile trout resulted in significant increases in length and body weight compared to the control fish. This study strongly suggests that the rsGH is transported into the circulation of salmonids via the lower part of the intestine and takes part in growth stimulation.Abbreviations DCA deoxycholic acid - FITC fluorescein isothiocyanate - GH growth hormone - HPLC high-performance liquid chromatography - HRP horseradish peroxidase - RIA radioimmunoassay - rsGH recombinant salmon growth hormone - sGH chum salmon growth hormone - SEM standard error of mean - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TFA trifluoroacetic acid     

Mitochondrial cytochrome b sequence variation in Korean salmonids

The complete 1141 bp mitochondrial cytochrome b sequences were determined for lenok Brachymystax lenok , cherry salmon Oncorhynchus masou masou , Ishikawa's cherry salmon O. m. ishikawai , chum salmon O. keta , rainbow trout O. mykiss , and an albino mutant of rainbow trout. Common substitutions detected in these species were transitional mutations. There were no significant differences in the intraspecific variation of the cytochrome b genes. Interspecific divergences were greater than intraspecific variation. The level of variation ranged from 8·026–15·686%. The cherry salmon was closer to chum salmon than to rainbow trout, and the lenok was the most distantly related species.     

In vitro effects of steroid hormones on IgM-secreting cells and IgM secretion in common carp (Cyprinus carpio)

The effects of steroid hormones on in vitro IgM-secreting cells (IgMSC) and IgM secretion by lymphocytes of the lymphoid organs in common carp, Cyprinus carpio were examined by ELISPOT and ELISA assay, respectively. Cells isolated from peripheral blood (PB), spleen and head kidney were cultured for 12, 24 and 48 h either in the absence or in the presence of steroid hormones, i.e. cortisol, testosterone, 11-ketotestosterone (11-KT), and estradiol-17beta (E(2)) at doses of 1, 10 and 100 ng/ml. Cortisol reduced the IgMSC numbers and IgM secretion by cells from all organs. In addition, cortisol induced apoptosis in lymphocytes from all organs. High dose of testosterone showed tissue-specific functions; it reduced the number of IgMSC and amount of IgM secretion by cells from spleen and head kidney, but not in PB, though IgM secretion was suppressed. However, no effects of sex steroids were observed in this study. The results show that sex-specific steroid hormones may have no immunosuppressive effects in common carp.     

Histone-like proteins from Atlantic cod milt: stimulatory effect on Atlantic salmon leucocytes in vivo and in vitro

This study was carried out to reveal some characteristics of cationic proteins from Atlantic cod (Gadus morhua) milt chromatin and to investigate their ability to activate Atlantic salmon (Salmo salar) macrophages. Cationic proteins extracted from cod milt chromatin were fractionated on a cation exchange chromatography column. SDS-PAGE and amino acid analyses of the resulting fractions indicated that these proteins are similar to calf thymus histones. Two cationic protein fractions were used to stimulate leucocytes from Atlantic salmon in vitro and in vivo. Increased production of superoxide, measured as reduction of nitroblue tetrazolium (NBT), was used as indication of macrophage activation. Both fractions induced elevated superoxide anion production in the macrophages after 3 and 6 days of in vitro stimulation. Intraperitoneal injection of the cationic protein fractions in Atlantic salmon (100 mg kg(-1)) four days prior to slaughtering stimulated superoxide production when assayed after one and two days of cell cultivation. In macrophages from fish slaughtered two days after injection, activation could first be seen after two days of cell cultivation.