Copper induces oxidative stress with triggered NF-κB pathway leading to inflammatory responses in immune organs of chicken.

Copper (Cu) is a necessary trace mineral due to its biological activity. Excessive Cu can induce inflammatory response in humans and animals, but the underlying mechanism is still unknown. Here, 240 broilers were used to study the effects of excessive Cu on oxidative stress and NF-κB-mediated inflammatory responses in immune organs. Chickens were fed with diet containing different concentrations of Cu (11, 110, 220, and 330 mg of Cu/kg dry matter). The experiment lasted for 49 days. Spleen, thymus, and bursa of Fabricius (BF) on day 49 were collected for histopathological observation and assessment of oxidative stress status. Additionally, the mRNA and protein levels of NF-κB and inflammatory cytokines were also analyzed. The results indicated that excess Cu could increase the number and area of splenic corpuscle as well as the ratio of cortex and medulla in thymus and BF. Furthermore, excessive Cu intake could decrease activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px); but increase contents of malondialdehyde (MDA), TNF-α, IL-1, IL-1ß; up-regulate mRNA levels of TNF-α, IFN-γ, IL-1, IL-1ß, IL-2, iNOS, COX-2, NF-κB and protein levels of TNF-α, IFN-γ, NF-κB, p-NF-κB in immune organs. In conclusion, excessive Cu could cause pathologic changes and induce oxidative stress with triggered NF-κB pathway, and might further regulate the inflammatory response in immune organs of chicken.

Effects of IBDV infection on expression of ghrelin and ghrelin-related genes in chicken.

Ghrelin is a peptide hormone that plays a modulatory role in the immune system. Studies have demonstrated that mammal ghrelin level is influenced by pathological status. However, it has not been reported whether chicken ghrelin level changes during pathogen infection. This study was designed to investigate changes of ghrelin levels in chickens infected with infectious bursal disease virus (IBDV) and to explore the relationship between ghrelin changes and bursal damage, and inflammatory cells infiltration induced by IBDV. The results showed that (1) plasma ghrelin concentration increased after IBDV infection. It reached a peak at 10443.6 ± 2612.9 pg/mL on 2 dpi, which was about 100-fold as high as that of the control. Then it decreased sharply on 3 dpi, which was only 31.7% as that of 2 dpi, and remained stable until 5 dpi. Meanwhile, ghrelin and ghrelin-related gene, ghrelin-o-acyltransferase (GOAT), and growth hormone secretagogue receptor (GHSR) mRNA expression levels in bursa were also increased after IBDV infection, and reached the peak on 2 dpi at 149, 28.8, and 117.2-fold higher than that of the control, respectively. Then they decreased and remained at a higher status. Correlation analysis showed that plasma ghrelin concentration and ghrelin, GOAT, and GHSR mRNA expressions in bursa were strongly associated with IBDV VP2 mRNA expression in bursa. (2) The damage of bursa was the most severe on 5 dpi with a histopathological score of 12. It had no direct correlation with plasma ghrelin level and ghrelin, GOAT, and GHSR mRNA expressions in bursa. However, the number of inflammatory cells infiltrating into bursa, which was the highest on 2 and 3 dpi, showed significant a positive correlation with the ghrelin and GHSR mRNA expression. Presumably chicken ghrelin may function as an anti-inflammatory factor. In conclusion, IBDV infection upregulates the expression of ghrelin and ghrelin-related gene in chickens, and chicken ghrelin may play an important regulatory role during pathogen infection.

[Tryptase of mast cells in immune organs of developing chicken embryos].

OBJECTIVE: To observe the form, distribution and emerging time of the mast cells (MCs) and tryptase positive cells (TPCs) in the immune organs of different days old chicken embryos. METHODS: The thymus, bursa of Fabricius and spleen of chicken embryos at different development time were taken and fixed in Carnoy's solution. The alcian blue/safranine O (AB/SO) and streptavidin-biotin complex (SABC) immunohistochemistry were used to observe the MCs and TPCs. RESULTS: MCs were first present in the thymus, bursa and spleen of day 13-14 embryos. The number of MCs increased along with the development days. On day 16, TPCs were first found in the immune organs. The form and distribution of TPCs were similar to MCs, but the emerging time of TPCs was later than that of MCs. CONCLUSION: Tryptase appears in the MCs in the immune organs of day 16 or later chicken embryos, and it is mainly located in the MCs of the connective tissues.

Histochemical and histological evaluations of the effects of high incubation temperature on embryonic development of thymus and bursa of Fabricius in broiler chickens.

1. The effects of experimentally induced heat-stress on the embryonic development of bursa of Fabricius and thymus of the chicken were investigated by means of histological and enzyme histochemical methods. 2. In the experiments, 250 fertile eggs of the Ross 308 broiler strain were divided into two groups. The control eggs were maintained under optimal conditions (378 degrees C and 65 +/- 2% relative humidity, RH) during the whole incubation period. Heat stressed eggs were maintained under normal conditions (378 degrees C and 65 +/- 2% RH) until the 10th d of incubation and then exposed continuously (24 h per d) to high temperature (388 degrees C and 65 +/- 2% RH). Blood and tissue samples were taken from 10 animals of each group at d 13, 15, 18 and 21 of incubation and at d 2, 4 and 7 post-hatch. Tissue samples were processed for enzyme histochemical methods in addition to routine histological techniques. 3. The results revealed that egg temperatures were higher than incubator air temperature. Long-term heat-stress (401-406 degrees C egg temperature) retarded development of thymus and bursa of Fabricius. Peripheral blood ACP-ase and ANAE-positive lymphocyte levels of heat-stressed animals were lower than in the controls. 4. These results give some morphological evidence for immunosuppression induced by high temperature exposure during the embryonic development. Temperature distribution and air circulation in incubator should be questioned in the case of lower broiler flock immunity.

Reduced mucosal injury of SPF chickens by mast cell stabilization after infection with very virulent infectious bursal disease virus.

Recent studies here have demonstrated that increased mast cell populations and tryptase activity contribute to lesion formation in regions of immune organs in special-pathogen-free chickens after infection with very virulent infectious bursal disease virus (vvIBDV). Mast cells and their mediators have been implicated in acute inflammatory injury after vvIBDV infection, but their precise role in this process remains elusive. In this study, the role of mast cells in the vvIBDV infection process was examined using ketotifen, a mast cell membrane stabilizer. On days 1, 2, and 3 postinfection, the bursa of Fabricius (BFs) were collected to quantify mast cells, tryptase and histamine contents by cytochemistry, immunohistochemistry and fluorospectrophotometry analyses, respectively. The results showed that the mast cell populations, tryptase expression, and histamine released increased significantly in the BFs (p<0.01) of infected birds compared to controls, and acute inflammatory responses were observed in the former. In contrast, in infected chickens pretreated with ketotifen, mast cells, tryptase, and histamine were markedly decreased (p<0.01) and probably as a result, the BFs remitted significantly. The overall results suggest that mast cells are positively involved in BF injury induced by vvIBDV infection. Inhibition of mast cell degranulation and concurrent mediator release may represent a novel strategy to modulate this process. This study, thus, advances the understanding of the acute inflammatory injury mechanisms triggered by vvIBDV infection and the contribution of mast cell activity in this process.

Effects of aflatoxin B1 on the development of the bursa of Fabricius and blood lymphocyte acid phosphatase of the chicken.

1. In this study, embryotoxicity and effects of in ovo administration of aflatoxin B1 (AFB1) on the embryonic development of the bursa of Fabricius were determined in fertilised chicken eggs by histological methods and histochemical demonstration of acid phosphatase (ACP-ase) enzyme. Peripheral blood lymphocyte percentages were also estimated. 2. Embryonic stages were determined according to the Hamburger-Hamilton (H-H) scale. Mortality rates increased in AFB1-injected groups in a dose-dependent manner. Embryonic deaths were concentrated at H-H 31 in the 5 ng AFB1/egg group while the deaths were highest at H-H 25 in the 10 and 20 ng AFB1/egg groups. In the 40ng AFB1/egg group, embryonic deaths mostly occurred during the first 70 to 72 h of incubation (H-H 20). 3. Bursal development was quite similar at d 7 of incubation in control and all experimental groups although development had been substantially impaired and retarded at d 10 of incubation in 10, 20 and 40 ng AFB1/egg groups. A gradual decrease of ACP-ase positive lymphocyte numbers was observed with increasing AFB1 doses. The chicks in AFB1-treated groups hatched with poorly developed bursae compared with those of the controls. However, proportions of ACP-ase positive lymphocytes substantially increased at hatch and at 21 d of incubation all groups had similar numbers. 4. The results revealed that breeder diets should be investigated for aflatoxins and specifically AFB1, in cases of low hatchability and flock immunity, because low concentration of AFB1 transferred into the fertilised eggs might be the cause of serious problems.

Identification of cells secreting a thymostimulin-like substance and examination of some histoenzymatic pathways in aging avian primary lymphatic organs: II. Bursa of Fabricius.

The Bursa of Fabricius of 15 day, 1-, 3-, and 6 month-old adult chickens (White Leghorn strain) were studied by histological and histochemical staining, histoenzymatic reactions (LDH, SDH, a-GPDH, NAD, NADPH, Ca++-dependent ATP-ase, pH 8.5) and by anti-thymostimulin immunoreaction. Positive reactions for mucopolysaccharides and enzymatic activities were located in the epithelia of the follicles, i.e. in follicle-associated-epithelium (FAE), inter-follicle-epithelium (IFE) and in different epithelial compartments of cortical and medullary zones. Positive reaction for thymostimulin-like (TS-like) substance was restricted to FAE cells and weakly to the basal lamina of IFE. In 6-month-old chickens, the FAE cells disappeared; the phenomenon of bursal regression was evident, although not all the follicles were involved. In the few still normal follicles, the good reactivity to the enzymes tested suggests that residual physiological activity is still present, even if reduced.

Glucocorticoid production in the chicken bursa and thymus.

Glucocorticoid (GC) hormones play an important role in thymic T cell selection and in the development of autoimmune diseases. Previous studies have shown that the mammalian thymus itself is able to produce GC. In order to assess the importance of these findings in terms of the evolutionary development of the immune system, we investigated the functional presence of steroidogenic enzymes in primary lymphoid organs of chickens, which represent one of the best studied non-mammalian species. To this end, we attempted to demonstrate enzyme activities of the whole set of steroidogenic enzymes for the synthesis of GC in the bursa of Fabricius and the thymus. We isolated steroidogenic organelles from primary lymphoid tissues, incubated these with radioactive (precursor) steroids in vitro and visualized the resulting products by thin-layer chromatography. Our results show that the chicken bursa as well as the chicken thymus possesses all enzymes and cofactors required for GC production. The observation of GC production in an organ responsible for B cell selection and maturation is a further step in uncovering the yet ill-defined mechanism of B cell selection. These results provide the biochemical basis for the in situ hormonal effects, and underline the general importance of GC hormones on T and B lymphocyte development and selection.

Effects of PCB 126 on thymocyte surface marker expression and immune organ development in chicken embryos.

Previous studies have shown that chicken (Gallus domesticus) embryos are sensitive to the immunotoxic effects of Ah receptor agonists. These chemicals cause atrophy of the thymus gland and bursa of Fabricius, the sites of T- and B-lymphocyte maturation, respectively. The objectives of this study were (1) to evaluate the effects of 3,3,4,4',5-pentachlorobiphenyl (PCB 126) on thymocyte phenotypes (CD4-CD8-, CD4+CD8+, CD4+CD8-, CD4-CD8+, TCRalphabeta+, and TCRgammadelta) in chicken embryos, and (2) to compare phenotype alterations with masses and cellularity of lymphoid organs. To simulate exposure in wild avian embryos, chicken eggs were injected with PCB 126 (sunflower oil carrier) into the air cell before incubation. Doses ranged from 0.051 to 0.8 ng/g egg with carrier-injected and noninjected control groups. The thymus and bursa were removed, weighed, and homogenized on d 20 of egg incubation (1 d before hatch). Thymocyte phenotypes were quantified by flow cytometry using monoclonal antibodies for CD4, CD8, TCRalphabeta (Vbeta1), and TCRgammadelta. Right thymus mass declined with dose, decreasing significantly between 0.32 and 0.8 ng/g to a size 28% lower than controls. Live lymphoid cell numbers in the right thymus dropped markedly (21% lower than controls) between 0.051 and 0.13 ng/g, with a further decrease (35% lower than controls) at higher doses. There was no significant change in the percentage of thymocytes expressing TCRalphabeta. The total number of TCRalphabeta+ thymocytes decreased with dose as a function of the declines in TCRalphabeta+ percentages and total thymocyte numbers. The percentages of all other measured phenotypes were unaltered by PCB 126. The total number of CD4+CD8+ cells, and to a lesser degree CD4-CD8+ cells, decreased in a dose-dependent manner following the pattern of total live thymocytes. The number of viable lymphoid cells in the bursa decreased to 45% lower than controls at 0.13 ng/g and fell to 76% lower than controls at 0.8 ng/g. Lymphoid atrophy occurred at doses that were 8- to 12-fold lower with full-term incubation as compared to exposure only during later stages of incubation, and the lymphoid atrophy was associated with decreased TCRalphabeta+ thymocytes at higher doses. These immunological effects were observed at concentrations of PCB 126 comparable to those found in Great Lakes herring gull eggs, after correcting for interspecies differences in sensitivity to PCB 126.

Multiparametric assessment of bursal lymphocyte apoptosis.

When bursal lymphocytes are placed in cell culture, they undergo an apoptotic form of cell death that can be inhibited by phorbol esters and protein synthesis inhibitors. The goal of the current study was to evaluate the time course of this process and the inhibition of this process using several different assays to detect apoptosis: (1) terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) of lymphocyte DNA strand breaks with dUTP-FITC; (2) propidium iodide (PI) staining of lymphocyte chromatin; (3) chloromethyl-x-rosamine (CMX-Ros) binding to lymphocyte mitochondria; (4) merocyanine-540 (MC-540) binding to the lymphocyte plasma membrane; (5) flow cytometric analysis of light scatter from lymphocytes; (6) analysis of genomic DNA from lymphocytes by agarose gel electrophoresis; and (7) cellular caspase-3 activity of lymphocytes. When bursal lymphocyte apoptosis was analyzed as a function of time, or inhibited by phorbol esters or cycloheximide, all of these assays corroborated the apoptotic process. However, treatment of lymphocytes with a cytotoxic level of the proteinase inhibitor, n-ethylmaleimide (NEM) resulted in a putative, necrotic form of cell death that revealed discrepancies among the various assays in the detection of apoptotic cells. Specifically, the CMX-Ros and MC-540 assays erroneously detected the necrotic cells as being apoptotic cells following NEM treatment. These findings indicate the need for additional assays and appropriate treatment controls to verify the apoptotic process when using the CMX-Ros and MC-540 assays.

Detection and localization of 3,3',4,4'-tetrachlorobiphenyl-induced P4501A protein in avian primary immune tissues.

P4501A can be detected in thymic and bursal microsomes from chickens pretreated with 3,3',4,4'-tetrachlorobiphenyl (TCB) using a polyclonal antibody against purified P4501A from 3-methylcholanthrene (3-MC)-induced chicken embryo liver. A dose-response for induction by TCB of P4501A protein was detected by Western blotting in both bursal and thymic microsomes. Ethoxyresorufin-O-deethylase (EROD), a specific catalytic activity of P4501A, was also induced in a dose-response fashion. More TCB-induced P4501A was detected in thymus than bursa by both methods. No EROD was detected in bursal or thymic microsomes from untreated chickens, although P4501A protein was detected at very low levels in thymic microsomes from untreated chickens. P4501A was detected by immunohistochemistry in scattered patches of non-lymphocytic cells residing in medullary regions of the TCB-induced thymus but was not detected in lymphocytes. This result supports previous work demonstrating that TCB-inducible EROD is much higher in the supporting tissue cell fractions than in lymphocyte fractions of the primary immune tissues. Although EROD was induced by TCB in the late stage embryo after 20 h exposure, no effect of TCB on the cell cycle in thymic or bursal lymphocytes was observed over the same period. The same TCB exposure resulted in bursal but not thymic cellular depletion. Thymic and bursal supporting tissue cells may be primary sites of immunosuppression within these organs by P4501A inducers or substrates whether immunosuppression occurs subsequent to metabolism or through interaction with Ah receptors.

The effect of single administration of aflatoxin B1 and ACTH on the intensity of acid phosphatase reaction in bursa Fabricii and in periellipsoidal lymphatic tissue of the spleen of ducklings.

The experiments were carried out with Peking breed female ducklings which on their second day of life were administered into the crop a single dose of 1.5 micrograms of aflatoxin B1. On the 14th and 15th day from administration of aflatoxin B1 the ducklings were intravenously injected a single dose of ACTH at 1 i.u. per 100 g bodyweight. Disturbances in the ducklings, initiated by aflatoxin and then increased by ACTH, manifested themselves by a change in the intensity of acid phosphatase reaction in the lymphatic organs tested. The occurrence of differentiated APh reaction (but intensified differently as compared to that in control ducklings) within bursa Fabricii and in periellipsoidal lymphatic tissue of the spleen was the expression of unevenly changed reactivity of respective tissues in those organs due to the action of aflatoxin B1. These results seem to present an interesting starting point for further multidirectional experiments, first of all in the range of immunological reactivity in birds.

Acid phosphatase in lymphoid tissues of developing chick embryos.

Activity of acid phosphatase (ACPase) in bursa and thymus has been determined in developing chick embryos of either normal or treated with bovine serum albumin (BSA). From the study of light microscopical histochemistry, ACPase activity could be detected in cytoplasm. These ACPase activities were detected in both the follicular medulla and cortex in bursa. In thymus, moderate ACPase activities were also obtained in both the cortex and medulla. ACPase activity in the tissue homogenate was increased in the bursa but the thymic ones showed constant level during the incubation days examined in normal embryos. ACPase distribution observed at the distinct developing stage indicates that ACPase is a useful parameter for growth and development of chicken lymphoid tissues.

Modification of ornithine decarboxylase activity by adrenergic stimulation in cultured chicken spleen cells.

1. In vivo, adrenergic agonists promote an increase of ornithine decarboxylase activity (ODC) in chicken spleen, as opposed to a decrease in thymus and bursa of Fabricius. The increase is not due to the cell fraction separated on Lymphoprep, i.e. the spleen cells, but it could be due to the macrophages. 2. With spleen cells in culture, a marked increase of ODC activity is observed during the first 3 hr, followed by a decrease. 3. cAMP drastically decreases after 10 min in culture. 4. Adrenergic agonists promote a decrease of activity, both alpha and beta receptors being involved in these modifications. TPA promotes partial desensitization. 5. Selenite, which in vivo has the same effect as epinephrine, enhances ODC activity in culture. Propranolol partially counteracts this effect, while prazosin has a synergistic effect. TPA partially desensitizes spleen cells to selenite.

Distribution of esterase activity at the level of the epithelium of the diffusely infiltrated area (DIA) and of the cloaca in the Gallus domesticus: an ultrastructural study.

Esterase activity studies on the areas of lymphoid infiltration in the bursa of Fabricius (diffusely infiltrated area) and the dorsal wall of the cloaca showed that the epithelial cells exhibit varying degrees of diffuse esterase activity; it was possible to demonstrate the presence of spot-like esterase positivity in star-shaped cells in the epithelium itself. These cells can be found at various levels from the proximal to the distal region and have also been observed in the connective tissue of the tunica propria; as a result, the hypothesis has been advanced that they are connective tissue.

Selenium and polyamine metabolism: different effect of selenite on liver and bursa of Fabricius ornithine decarboxylase activity.

1. When injected i.p., sodium selenite promoted a marked increase of rat liver ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) activities; when administered with the diet for 6 weeks, a less marked increase in liver ODC was observed, whereas SAMDC was not significantly changed. 2. Protein synthesis was involved in the observed modifications. The rate of ODC inactivation was also changed. 3. ODC increase was accompanied by an enhanced putrescine concentration in liver. 4. A marked increase of ODC, accompanied by an enhancement of putrescine, was promoted by selenite (i.p.) also in chicken liver, together with an enhancement of glutathione concentration. Spermidine acetyltransferase (SAT) was also increased. 5. In the bursa of Fabricius, SAT activity was also increased, whereas ODC was decreased. However the expected modifications in polyamine concentration were not observed. 6. Decrease of ODC activity in the bursa was not due to an antizyme. 7. In vitro, selenite concentrations known to inhibit cell proliferation (greater than 1 microgram/ml) inhibited both ODC and SAT activities; at lower concentration, SAT activity was enhanced.

Endonucleolytic activity that cleaves immunoglobulin recombination sequences.

An endonucleolytic activity has been identified in nuclear extracts of chick embryo bursa and mouse fetal liver cells. The activity introduces a double-strand cut in the vicinity of the recombination site of immunoglobulin joining gene segments. The cleavage occurs at the dinucleotide pair TG-AC. This activity is a good candidate for the putative endonuclease involved in recombination of the immunoglobulin variable, diversity, and joining regions. It is distinct from the endonuclease activities previously reported by others.

DNA ligases as markers of lymphoid cell maturation and heterogeneity in the chicken.

The activities of 8 S and 6 S DNA ligases have been studied in the chicken lymphoid cells of blood, spleen and bursa of Fabricius at different stages of development, from late embryonic life to about 3 months after hatching. These cells have been sorted with the fluorescence-activated cell sorter FACS II on the basis of size and T or B antigenicity (immunofluorescence). The light 6 S DNA ligase has been previously demonstrated to be associated to a late stage of differentiation of thymocytes. In the bursa, a unique form of 8 S DNA ligase is found during the whole period of observation. This form of enzyme remains in the B cells of the spleen until 3 weeks after hatching, but is never present in the blood B cells. As far as T cells are concerned, the light DNA ligase is present in the blood from 18-day embryonic life on. In the spleen T cells, on the contrary, this enzyme appears only 3 weeks after hatching. Before this stage, splenic T cells are devoid of any form of DNA ligase activity. These findings show biochemical differences in T and B lymphocytes colonizing the periphery, blood and spleen, and suggest, at least for the T cells at early stages, a heterogeneity in the degree of differentiation.

Isolation and characterization of endonuclease J: a sequence-specific endonuclease cleaving immunoglobulin genes.

An endonuclease activity which cleaves close to the recombination sites of the immunoglobulin JK segments was found in extracts of chicken bursa of Fabricius and characterized after partial purification. The enzyme preparation also cleaved a VK segment at its 3' end. A similar activity was found in mouse liver, mouse myelomas and Hela cells. The enzyme designated as endonuclease J introduces double-stranded cleavages preferentially at sequences containing G clusters of pBR322 as well as the JK segments. However, not all the G clusters were cleaved by endonuclease J, suggesting that the enzyme recognizes additional sequences. Deletion of the conserved nonamer (GGTTTTTGT) located immediately 5' to the JK4 segment drastically reduced the cleavage activity of its immediate downstream G cluster. Although biological function of endonuclease J is not clear at this stage, the possibilities of its involvement in the immunoglobulin gene recombination and general recombination were discussed.