Differential expression of inducible nitric oxide synthase is associated with differential Toll-like receptor-4 expression in chicken macrophages from different genetic backgrounds.

The purpose of this study was to examine iNOS gene expression and activity in macrophages from different chicken genetic lines against various bacterial LPS. Furthermore, the possible involvement of surface LPS receptors as candidates for differential iNOS gene induction in these genetic lines of chicken was also examined. Sephadex-elicited abdominal macrophages (1 x 10(6)) as well as iNOS hyper-responder macrophages from a transformed chicken macrophage cell line, MQ-NCSU, were exposed to 5 microg/ml LPS from E. coli, Shigella flexneri, Serratia marcensces, and Salmonella typhimurium. Nitrite levels were quantitated in the culture supernatant fractions of macrophages after 24h by the Griess method. The results showed that macrophages from K-strain (B(15)B(15)) (range from two separate trials: 31-89 microM) and MQ-NCSU (22-81 microM) were high responders whereas macrophages from both GB1 (B(13)B(13)) (15-38 microM) and GB2 (B(6)B(6)) (7-15 microM) chickens were low responders against all LPSs used. Northern blot analysis revealed that K-strain macrophages expressed higher intensity of 4.5Kb iNOS mRNA (iNOS/beta-actin ratio) than macrophages from GB2 regardless of the LPS source. To elucidate possible molecular mechanism(s) involved in iNOS gene expression in these two strains of chickens, the constitutive expression of LPS-related macrophage cell surface receptors, CD14, Toll-like receptor-2 (TLR2), and Toll-like receptor-4 (TLR4), was examined via flow cytometry using anti-human CD14, TLR2 and TLR4 antibodies. CD14 surface expression and intensity was not different between macrophages from K-strain or GB2 chickens. In contrast, while the overall percentage of TLR4-positive macrophages was the same (K-strain, trial 1=92%, trial 2=62%; GB2, trial 1=91%, trial 2=64%), the mean fluorescence intensity (MFI), an indicator of receptor number, was significantly higher (P=0.05) in K-strain macrophages (MFI: trial 1=145; trial 2=131) than GB2 macrophages (MFI: trial 1=101; trial 2=98). Furthermore, TLR2 (a previously thought candidate as LPS signaling molecule) positive cell numbers were higher in K-strain than the GB2 macrophages in one of the two trials with no difference in the intensity of TLR2 expression in either trial. These findings suggest that the observed differences in iNOS expression and activity among the K-strain (hyper-responder) and GB2 (hypo-responder) chickens are, at least in part, due to differential expression of TLR4 (an LPS signaling molecule), leading to more intense LPS-mediated activation of K-macrophages.     

Effects of antimicrobial peptides derived from the beetle Allomyrina dichotoma defensin on mouse peritoneal macrophages stimulated with lipopolysaccharide

We previously reported that synthetic peptides, RLYLRIGRR-NH2 (peptide A) and RLRLRIGRR-NH2 (peptide B), derived from the beetle Allomyrina dichotoma defensin, showed antimicrobial activity against both Gram-positive and negative bacteria and suppressed lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha (TNF-alpha) mRNA expression in a murine macrophage cell line. In this study, inhibitory effects of these peptides in LPS-induced mouse peritoneal macrophage activation were investigated. The supplement of peptide A to macrophages cultured with LPS resulted in a significant decrease in nitric oxide and TNF-alpha production. Furthermore, NF-kappaB activation was also blocked by addition of peptide A. These results indicated that peptide A blocked macrophage activation induced by LPS.     

硒化大蒜多糖对小鼠腹腔巨噬细胞功能的影响

【目的】研究硒化大蒜多糖(sGPS)对小鼠腹腔巨噬细胞功能的影响,以期为硒化大蒜多糖的作用挖掘和临床应用提供依据。【方法】依次通过分离、纯化得到大蒜多糖(GPS),并经硝酸-亚硒酸钠硒化修饰得到sGPS₃、GPS₅和sGPS₆。以小鼠腹腔巨噬细胞为研究对象,用6.25、12.5、25、50、100μg/mL sGPS₃、GPS₅、sGPS₆及10μg/mL脂多糖(LPS组)处理小鼠腹腔巨噬细胞48 h,同时设置不加药物的细胞为对照组,用中性红法测定其吞噬功能,CCK-8法测定其増殖能力,筛选出活性最好的硒化大蒜多糖;然后用ELISA法检测活性最好的硒化大蒜多糖对巨噬细胞上清液中一氧化氮(NO)、肿瘤坏死因子-α(TNF-α)、干扰素-γ(IFN-γ)、白介素-1β(IL-1β)、白介素-6(IL-6)、白介素-12(IL-12)含量的影响;再通过小鼠碳廓清实验、小鼠腹腔巨噬细胞吞噬鸡红细胞实验测定空白对照组(生理盐水)及高(2 mg/mL)、中(1 mg/...     

Response of white leghorn chickens of various B haplotypes to infection at hatch with subgroup J avian leukosis virus

White leghorn chickens from seven 15.B congenic lines (genetically similar except for genes linked to the major histocompatibility complex [MHC] B haplotype) and two Line 0.B semicongenic lines were infected at hatch with strain ADOL Hc-1 of subgroup J avian leukosis virus (ALV-J). At 5, 8, 16, and 36 wk of age, chickens were tested for viremia, serum-neutralizing antibody, and cloacal shedding. Chickens were also monitored for development of neoplasia. In the 15.B congenic lines (B*2, B*5, B*12, B*13, B*15, B*19, and B*21) there were no significant differences in the incidence of viremia between B haplotypes. In fact, infection at hatch in all of the 15.B congenic lines induced tolerance to ALV-J because 100% of these chickens were viremic and transient circulating serum-neutralizing antibody was detected in only a few chickens throughout the 36 wk experiment. However, at 16 wk of age more B*15 chickens had antibody and fewer B*15 chickens shed virus than did the 16-wk-old B*2, B*5, or B*13 chickens. Moreover, compared with B*15 chickens, a higher percentage of B*13 chickens consistently shed virus from 8 wk postinfection to termination at 36 wk postinfection. The B haplotype had a transient effect on viral clearance in Line 0.B semicongenics, as more B*13 than B*21 chickens remained viremic through 5 wk of age. Very few (0%-18%) of the Line 0.B semicongenic chickens shed virus. By 36 wk of age, all Line 0 B*13 and B*21 chickens produced serum-neutralizing antibodies and cleared the virus. These results show that following ALV-J infection at hatch the immune response is influenced transiently by the B haplotype and strongly by the line of chicken. Although this study was not designed to study the effect of endogenous virus on ALV-J infection, the data suggest that endogenous virus expression reduced immunity to ALV-J in Line 15I5, compared with Line 0, a line known to lack endogenous virus genes.     

Disassociation of bactericidal and fungistatic activities from the oxidative burst of avian macrophages

Avian peritoneal exudate macrophages, when exposed to phagocytic stimuli, produced an appreciable oxidative burst as measured by production of chemiluminescence, superoxide anion, and hydrogen peroxide. Metabolic inhibitors of the oxidative burst and scavengers of oxygen radicals clearly inhibited macrophage chemiluminescence, but had no significant effect on macrophage bactericidal activity against Escherichia coli or fungistatic activity against Candida tropicalis. Therefore, avian macrophages were capable of oxygen-independent bactericidal and fungistatic activities.     

Genetic control of immunity to Eimeria tenella. Interaction of MHC genes and non-MHC linked genes influences levels of disease susceptibility in chickens

The relative importance of MHC genes and background genes in the genetic control of disease susceptibility and the development of protective immunity to E. tenella infection was investigated in eight different strains of 15I5-B congenic and four inbred chicken strains. RPRL 15I5-B congenic chickens that share a common genetic background but express different B haplotypes demonstrated wide variations in disease susceptibility and the development of acquired resistance to E. tenella infection. Infection of chickens sharing a common B haplotype but expressing different genetic backgrounds showed quite contrasting levels of susceptibility to secondary E. tenella infection. In all chicken strains examined, infected chickens developed high levels of serum and biliary anti-coccidial antibodies regardless of their B haplotypes. Furthermore, no correlation between antibody levels and the phenotypically expressed levels of disease resistance was demonstrated. These findings lend support to the view that interaction of MHC genes and non-MHC genes influences the outcome of host response to E. tenella infection.     

Oxidative responses in ferret macrophages

Although the basic function of T and B lymphocytes in ferrets has been known for some time, the function of mononuclear phagocytes has not been described in this species. The present study has characterised basic oxidative responses in ferret macrophages, and has investigated the effects of endogenous and exogenous modulators of macrophage function on oxidative capacity in vitro. Macrophages derived from the blood or lungs of ferrets were shown capable of generating the reactive oxygen intermediate (ROI) molecules superoxide and hydrogen peroxide, and secreting a lysosomal enzyme (acid phosphatase), in response to appropriate stimuli. A T cell supernatant (derived from mitogen-stimulated peripheral blood lymphocytes) was able to activate both blood- and lung-derived macrophages for enhanced ROI production, while specific ROI inhibitors (superoxide dismutase and catalase) were able to partially ablate ROI activity. The accumulation of nitrite in culture supernatants, as an indicator for the production of reactive nitrogen intermediates, could not be demonstrated by ferret macrophages derived from either tissue source. In contrast to the enhancing effects of TCS on the oxidative function of blood-derived macrophages, exposure to bacterial LPS caused marked suppression of ROI and lysosomal enzyme production by these cells. Finally, the generation of superoxide anion, following phagocytosis of live or heat-killed Mycobacterium bovis or zymosan, indicated that ROI production in response to phagocytic stimulation was relatively weak in ferret blood-derived macrophages. These results are discussed in relation to the study of immune function in a novel species, and with particular reference to research into tuberculosis (Tb), since ferrets are important wildlife vectors of bovine Tb in New Zealand.     

Influence of turkey herpesvirus vaccination on the B-haplotype effect on Marek's disease resistance in 15.B-congenic chickens.

Eight recently developed 15.B congenic lines of chickens were tested for Marek's disease (MD) resistance by intra-abdominal injection of cell-associated preparations of MD virus of a virulent strain (JM), a very virulent strain (Md5), or Md5 after vaccination with turkey herpesvirus (HVT) strain FC126. Chickens of the 15.N congenic line (B15B21 or B21B21) were very resistant to JM-induced MD, in contrast to chickens homozygous for the B-haplotypes 2, 5, 12, 13, 15, or 19. After Md5 infection, more than 88% of the chickens in all of the congenic lines developed MD. However, when chickens were vaccinated with HVT before being inoculated with Md5, the B5 and B12 homozygotes were more resistant to MD than were the B2, B13, or B19 homozygotes, and B15 and B21 homozygotes had intermediate resistance. B5B5 and B2B5 F2 chicks inoculated with HVT and Md5 had a lower prevalence of MD than B2B2 sibs. These results demonstrate that a protocol involving HVT vaccination of chicks followed by infection with very virulent MD virus will allow the detection of B-haplotypes determining MD resistance, some of which are not detectable in unvaccinated chicks challenged with virulent MD.     

苦瓜提取物对小鼠腹腔巨噬细胞吞噬活性及NO、H2O2分泌的影响

探讨苦瓜提取物对小鼠腹腔巨噬细胞吞噬中性红以及对在脂多糖（LPS）刺激下巨噬细胞分泌产生一氧化氮（NO）和过氧化氢（H2O2）的影响。将分离提取得到的苦瓜总提取物作用于小鼠腹腔巨噬细胞,检测结果表明其能够显著增强小鼠腹腔巨噬细胞吞噬中性红的功能,并显著抑制LPS刺激的巨噬细胞分泌H2O2,而抑制巨噬细胞分泌NO的作用不明显。苦瓜提取物发挥药理作用的有效成分主要分布在水层。结果提示,苦瓜提取物能够增强小鼠腹腔巨噬细胞的吞噬功能,抑制LPS刺激下巨噬细胞过量分泌H2O2。     

Mechanisms of the hypoglycaemic and immunopotentiating effects of Nigella sativa L. oil in streptozotocin-induced diabetic hamsters

The aim of this study was to elucidate the mechanisms underlying the hypoglycaemic effect of N. sativa oil (Nigella sativa oil) in streptozotocin (STZ)-induced diabetic hamsters, in terms of hepatic glucose production, and to investigate the possible immunopotentiating effect of N. sativa oil on peritoneal macrophages. Diabetes was induced by intraperitoneal injection of 65 mg/kg body weight of STZ. Treatment with N. sativa oil commenced 6 weeks after induction of diabetes at a dose of 400 mg/kg body weight by gastric gavage. Isolated hepatocytes were collected using collagenase to determine liver glucose production. Phagocytic activity was evaluated by injection of fluorescent latex (2 microm diameter) intraperitoneally, followed 24 h later by collection of peritoneal macrophages. N. sativa oil reduced blood glucose from 391+/-3.0 mg/dl before treatment to 325+/-4.7, 246+/-5.9, 208+/-2.5 and 179+/-3.1 mg/dl after the first, second, third and fourth weeks of treatment, respectively. Hepatic glucose production from gluconeogenic precursors (alanine, glycerol and lactate) was significantly lower in treated hamsters. Treatment with N. sativa oil significantly increased the phagocytic activity and phagocytic index of peritoneal macrophages and lymphocyte count in peripheral blood compared with untreated diabetic hamsters. Our data indicate that the hypoglycaemic effect of N. sativa oil is due to, at least in part, a decrease in hepatic gluconeogenesis, and that the immunopotentiating effect of N. sativa oil is mediated through stimulation of macrophage phagocytic activity either directly or via activation of lymphocytes.     

中药与甲氧苄啶复方对小鼠血液学和免疫学指标的影响

为明确中药与甲氧苄啶复方在动物体内抗感染作用的机制,本试验研究了其对小鼠血液学和免疫学指标的影响。将受试药物按1、5、10 g/kg体重连续腹腔注射15 d,通过Coulter-JT血细胞分析仪对血液中的红细胞、白细胞、中性粒细胞、单核细胞和淋巴细胞进行了计数。采用脏器系数测定法、腹腔巨噬细胞吞噬鸡红细胞法、比浊法和MTT法分别对小鼠免疫器官指数、腹腔巨噬细胞吞噬功能、血清溶菌酶含量、血清溶血素水平和淋巴细胞增殖能力进行测定。结果表明中药与甲氧苄啶复方可显著增加血液中的红细胞数、单核细胞比例等(P＜0.05),显著增加免疫器官指数,增强腹腔巨噬细胞吞噬功能和血清溶菌酶活性,提高血清溶血素水平和淋巴细胞增殖能力(P＜0.05)。因此中药与甲氧苄啶复方对小鼠非特异性免疫和特异性免疫功能均有增强作用。     

A rapid and simple method to obtain canine peripheral blood-derived macrophages

Macrophages play an important role in a variety of situations, including pathogen elimination, inflammation, and tissue repair. However, these cells are not fully studied in dogs, in part, due to the difficulty of efficiently isolating and culturing them in vitro. In this study, we cultured canine peripheral blood mononuclear cells (PBMCs) with 10 ng/ml of phorbol 12-myristate-13-acetate (PMA) for 5 days to obtain macrophages. A high number of round-adherent cells were obtained without the addition of any cytokine. These cells showed active phagocytic activity and a cell surface antigen profile different from dendritic cells. Our method facilitates a high yield of macrophages in a short cultivation period compared with previous studies. This method might be a powerful tool to study macrophage functions in dogs.     

Cultured bovine monocytes exhibit decreased release of superoxide anion and increased levels of lysosomal enzymes but do not secrete detectable lysozyme activity

In this study we determined the effects of in vitro maturation on the phagocytic activity, lysosomal enzyme content and oxidative response of bovine monocytes. Intracellular levels of the lysosomal enzymes acid phosphatase, beta glucuronidase, and beta glucosaminidase increased as bovine monocytes matured in vitro. However, in marked contrast to the mononuclear phagocytes of other mammalian species, lysozyme activity was undetectable in the culture supernatants and cell lysates of adherent bovine blood monocytes cultured for one to fifteen days. In vitro maturation of bovine monocytes also increased their phagocytic activity as determined by the ingestion of opsonized yeast. A greater percentage of monocyte-derived macrophages that were stimulated with opsonized yeast and phorbol myristic acetate (PMA) reduced nitroblue tetrazolium than did similarly treated monocytes. Monocyte-derived macrophages stimulated with PMA released significantly less superoxide anion than did PMA-stimulated monocytes. Bovine monocytes and macrophages also failed to bind the monoclonal antibody Mac-1, which binds to human and mouse macrophages. Bovine monocytes demonstrated both similarities and differences with other mammalian mononuclear phagocytes, thus making them a useful model for further study of the comparative and developmental biology of mononuclear phagocytes.     

Endotoxin lipopolysaccharide from Escherichia coli and its effects on the phagocytic function of systemic and pulmonary macrophages in turkeys.

The effect of Escherichia coli lipopolysaccharide (LPS) on the competence of pulmonary macrophages and phagocytic cells from the systemic circulation of turkeys was examined using luminol-enhanced zymosan-stimulated chemiluminescence. The results showed a rapid and accelerated oxidative burst in both systemic and pulmonary macrophages in LPS-treated turkeys that was significantly greater than in untreated controls. However, this increased oxidative metabolism induced by LPS was not associated with enhanced intracellular bacterial killing by pulmonary macrophages. Turkeys treated with LPS showed a highly significant decrease in pulmonary bactericidal activity against Staphylococcus aureus challenge, indicating a defect in pulmonary macrophage function induced by LPS.     

Response of B congenic chickens to infection with infectious bursal disease virus.

Six congenic lines of chickens that differ from the parental inbred line RPRL-15I5 for genes in the major histocompatibility (B) complex were used to study the influence of the B haplotypes on the response of chickens to infection with virulent infectious bursal disease virus (IBDV) at 1 day or 4 weeks of age, and on the antibody response to vaccination with live or inactivated oil-emulsion (OE) IBDV vaccines at 7 weeks of age. IBDV-induced immunodepression and lesions in the bursa, spleen, and thymus in chickens infected with virus at 1 day of age were of the same degree of severity, regardless of line of chickens used. The response of blood cells to the mitogens phytohemagglutinin-M and concanavalin A was elevated in chickens infected with IBDV at 1 day of age. In an experiment conducted to study the effect of the B haplotype on IBDV infection in 4-week-old chickens, B congenic line C-12 (B12B12) showed the highest susceptibility to clinical IBD, with mortality of 79%. No detectable difference in the serological response to vaccination with live or OE IBDV vaccines was noted among chickens of various congenic lines. We conclude that the B haplotypes may influence IBDV-induced mortality, but not immunodepression or severity of lesions in lymphoid organs, or the antibody response to live or OE IBDV vaccines.     

Phagocytosis and erythrocyte antibody-rosette formation by three populations of mononuclear phagocytes obtained from dogs treated with glucocorticoids

The effect of systemic administration of glucocorticoids was evaluated on 3 populations of macrophages obtained from healthy dogs. Phagocytic and Fc-receptor activities were determined for mononuclear phagocytes from blood and pulmonary and peritoneal lavage fluids. Samples were collected from 12 dogs before treatment and again on the same dogs after glucocorticoid administration. Thirty or more days were allowed between treatment periods. Twelve hours after combined prednisolone sodium succinate and dexamethasone sodium phosphate administration, the percentage of phagocytosing cells decreased for blood monocytes and increased for pulmonary macrophages. The percentage of pulmonary macrophages positive for erythrocyte antibody-rosette formation (Fc-receptor activity) increased. After 7 days of oral administration of prednisone, the percentage of phagocytosing peritoneal macrophages increased, whereas the percentage of blood monocytes with Fc-receptor activity increased. Results indicate that significant (P less than 0.05) changes in macrophage function occur after systemic administration of the glucocorticoid doses used in this study. Also, the effect of systemic administration of glucocorticoids on mononuclear phagocytes varies, depending on the specific cell location.     

Serum haemolytic complement activities in 11 different MHC (B) typed chicken lines

To study the relation between serum complement levels and the chicken MHC (B) complex, complement haemolytic activity was measured in sera from hens from seven pure-bred B-typed White and one Brown Leghorn lines, and three ISA-Warren lines that had been divergently selected for antibody responses to sheep red blood cells (SRBC). Significant differences occurred in the serum haemolytic complement activities, both belonging to the classic (CPW) and the alternative (APW) pathways, among the 11 different haplotyped chicken lines. Hens with high CPW and high APW titres predominantly displayed the B2 or B21 haplotypes. Chickens with low CPW and APW were found in B14 and B15 haplotypes. Haplotype B14 appears to be different in complement levels when present into the pure-bred lines or into the ISA-Warren line selected for low antibody responses to SRBC. Otherwise, the presence of B21 in ISA-Warren line selected for high antibody responses to SRBC does not differ with the B21 in the inbred lines (except in the NL-line for CPW values). In general the haplotypes B2 and B21 are found in chicken lines with enhanced disease resistance, and the B15 haplotype has been connected with enhanced disease susceptibility. Our results suggest that levels of haemolytic complement activity, either from the classical or from the alternative pathways, may underlie part of the immunocompetence ascribed to the MHC (B) complex in chickens.     

The effects of direct and microsomal activated aflatoxin B1 on chicken peritoneal macrophages in vitro.

Sephadex-elicited peritoneal exudate cells were cultured on glass coverslips in order to determine the effects of aflatoxin B1 (AFB1) on chicken macrophages. Adherent macrophage monolayers were exposed for 1 h to 5, 10, and 20 micrograms ml-1 of AFB1, directly or to 0.01, 0.1, 0.5, 1, and 5 micrograms ml-1 of AFB1 in the presence of a chicken microsomal mixed function oxidase system (MFO). After exposure, the macrophage cultures were washed and allowed to recover for 2 h in fresh culture medium. Parameters measured at 2 h post recovery period were the substrate adherence potential, morphological alterations, phagocytic ability, and number of sheep red blood cells (SRBC) internalized per phagocytic macrophage. Direct in vitro exposure to AFB1 resulted in a dose-dependent decrease in macrophage adherence potential, and an increase in cell damage as determined by nuclear disintegration and cytoplasmic blebbing, but no detrimental effects were observed on percent phagocytic cells or the number of internalized SRBC. However, significant reductions in adherence potential, increased morphological alterations, and reduced phagocytosis and internalization of SRBC were observed when MFOs were added to cultures treated with much lower doses of AFB1. Addition of piperonyl butoxide (a P-450 inhibitor) abrogated AFB1-MFO induced alterations. This study suggests that microsomal activated AFB1 causes significant alterations in chicken macrophage functions.     

Resistance and susceptibility to Marek's disease: nitric oxide synthase/arginase activity balance

The metabolic NO pathway, catalyzed by the enzyme NO synthase in macrophages, is a key defense element against viruses and tumors. However, arginase is an other enzyme able to metabolize the substrate L-arginine, and the two enzymes are alternatively regulated by Th1 and Th2 cytokines in murine macrophages. Marek's disease is characterized by strong immunosuppression and development of T-cell lymphomas in chickens. Inoculation of the very virulent strain of MDV RB-1B induced strong and long-lasting arginase macrophage-dependent activity, which was inhibited by L-norvaline in vitro, but induced low NO production in monocytes and splenocytes from highly susceptible B(13)/B(13) chickens. By contrast, in B(21)/B(21) chickens genetically resistant to tumor development, RB-1B induced a weak and transient increase in arginase activity and strong NO production. The vaccinal HVT strain did not induce any arginase activity in monocytes or splenocytes. Moreover, vaccination with HVT prevented tumor appearance after RB-1B challenge and increase in arginase activity, but favored NO production in susceptible chickens. Differential expression of NO synthase and arginase was modulated in chicken macrophages, with IFN-gamma and LPS being strong inducers of both, depending on the type of macrophage, and TGF-beta 1 and PGE(2) stimulating only arginase activity. This increase in arginase activity in macrophages from chickens inoculated with Marek's disease virus might thus be due to a direct effect of the virus on macrophages, possibly through viral products, or to indirect effects on the cytokine balance.