Bacterial lipopolysaccharide stimulates bovine neutrophil production of TNF-alpha, IL-1beta, IL-12 and IFN-gamma

After intramammary infection, polymorphonuclear neutrophil leukocytes (PMN) are the first cells recruited into the mammary gland. Rapid recruitment of and bacterial phagocytosis and killing by PMN are the most effective defenses against establishment of bacterial infection. In addition to their phagocytic and bactericidal properties, PMN may play a key supportive role through secretion of cytokines during the innate immune response. We sought to determine whether bovine PMN produce cytokines in response to stimulation by lipopolysaccharide (LPS). To investigate the effects of LPS on the expression of cytokines secreted by bovine PMN, we measured the expression of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-12, and interferon (IFN)-gamma by ELISA after stimulation with different concentrations of LPS, and secretion of IL-8 after co-stimulation with LPS and either TNF-alpha or IL-1beta. Bovine PMN were shown to secrete TNF-alpha , IL-1beta, IL-12, IL-8 and IFN-gamma in response to LPS. Co-incubation of PMN with LPS and TNF-alpha increased secretion of IL-8 when compared to LPS alone. It was concluded that LPS stimulation up-regulates the secretion of cytokines by bovine PMN, and that co-incubation of LPS with TNF-alpha had an additive effect on the secretion of IL-8. These data show that bovine PMN, in addition to their phagocytic and bactericidal properties, may play a supportive role in the innate immune response to infection by Gram-negative bacteria through their ability to produce immuno-regulating cytokines.     

褪黑素对脂多糖刺激的滩羊骨骼肌卫星细胞炎性因子表达的影响

试验旨在探究褪黑素（MLT）对脂多糖（LPS）诱导的滩羊骨骼肌卫星细胞炎性反应的影响。选取滩羊妊娠30日龄胎儿为研究材料,使用Ⅳ型胶原酶分离获得滩羊骨骼肌卫星细胞并进行体外培养,添加相应的诱导试剂对滩羊骨骼肌卫星细胞进行诱导分化,利用免疫荧光检测骨骼肌卫星细胞表面标记物CD29、CD44、CD73及Vimentin的表达;在无胎牛血清的培养基中添加不同浓度（0、5、8和10 μg/mL）的LPS培养骨骼肌卫星细胞,利用实时荧光定量PCR （qRT-PCR）检测白细胞介素-6（IL-6）、IL-8和肿瘤坏死因子-α（TNF-α）等炎性相关因子mRNA水平变化,筛选最佳的LPS处理浓度;在无胎牛血清的培养基中添加不同浓度（0.1和0.5 μmol/L） MLT与最佳浓度LPS共培养骨骼肌卫星细胞,利用实时荧光定量PCR检测IL-6、IL-8和干扰素-γ（IFN-γ）等炎性相关因子mRNA水平变化。免疫荧光结果显示,滩羊骨骼肌卫星细胞表面标志物CD29、CD44、CD73及Vimentin表达呈阳性,且具有诱导成肌、成脂和成骨分化的特性。实时荧光定量PCR结果显示,与对照组相比,不同浓度的LPS处理均可显著增加细胞炎性相关因子基因的表达（P<0.05）,并且8 μg/mL LPS处理时炎性相关因子mRNA表达最强;当MLT与LPS共培养骨骼肌卫星细胞时,与LPS单独处理相比,0.5 μmol/L MLT与LPS共同处理组中IL-6 mRNA表达水平显著降低（P<0.05）。综上表明,MLT具有缓解LPS刺激的滩羊骨骼肌卫星细胞炎性反应的作用。本试验结果为进一步开展MLT缓解LPS诱导的骨骼肌卫星细胞炎性反应的调控机制研究奠定了基础。     

Effect of apoptosis on phagocytosis, respiratory burst and CD18 adhesion receptor expression of bovine neutrophils

Polymorphonuclear neutrophil leukocytes (PMN) play an important role in intramammary defense against infections by Escherichia coli. During mastitis, PMN are confronted with various inflammatory mediators that can modulate their function. In severely diseased cows, increased concentrations of lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-alpha (TNF-alpha) are detected in plasma. Binding of LPS to membrane bound CD14 molecules on monocytes cause release of inflammatory mediators such as TNF-alpha. Because apoptosis of PMN promotes resolution of inflammation and because the LPS and TNF-alpha response in milk and blood is related to the severity of E. coli mastitis, the effect on apoptosis of bovine PMN of increased concentrations LPS and TNF-alpha was studied together with the functionality of apoptotic PMN.Bovine PMN apoptosis, as determined with annexin-V, was induced with high concentrations of either LPS (1000 and 10,000ng/mL) or TNF-alpha (10,000ng/mL) in whole blood following a 6h incubation at 37 degrees C. The apoptosis inducing effect of LPS on PMN was not inhibited following coculture with either anti-bovine TNF-alpha or anti-ovine CD14 monoclonal antibodies. When compared to controls, apoptotic PMN had a similar level of CD18 expression but lacked phagocytic and respiratory burst activity. This is the first study reporting the effects of apoptosis on bovine PMN function. These functional impairments in apoptotic PMN could be important in contributing to the establishment of intramammary infection. Well functioning PMN could finally determine the severity of mastitis following an invasion of bacteria in the mammary gland.     

Effects of fish oil supplementation on the performance and the immunological, adrenal, and somatotropic responses of weaned pigs after an Escherichia coli lipopolysaccharide challenge

Seventy-two crossbred pigs (7.58 +/- 0.30 kg BW) weaned at 28 +/- 3 d of age were used to investigate the effects of fish oil supplementation on pig performance and on immunological, adrenal, and somatotropic responses following an Escherichia coli lipopolysaccharide (LPS) challenge in a 2 x 2 factorial design. The main factors consisted of diet (7% corn oil [CO] or 7% fish oil [FO]) and immunological challenge (LPS or saline). On d 14 and 21, pigs were injected intraperitoneally with either 200 microg/kg BW of LPS or an equivalent amount of sterile saline. Blood samples were collected 3 h after injection for analysis of interleukin-1beta (IL-1beta), prostaglandin E2 (PGE2), cortisol, growth hormone (GH), and insulin-like growth factor (IGF)-I. On d 2 after LPS challenge, peripheral blood lymphocyte proliferation (PBLP) was determined. Lipopolysaccharide challenge decreased ADG (487 vs. 586 g; P < 0.05) and ADFI (as-fed, 776 vs. 920 g; P < 0.05) from d 14 to 21 and ADG (587 vs. 652 g; P < 0.10) from d 21 to 28. Fish oil improved ADG (554 vs. 520 g; P < 0.10) and ADFI (891 vs. 805 g; P < 0.10) from d 14 to 21. On d 14, LPS challenge x diet interactions were observed for IL-1beta (P < 0.10), PGE2 (P < 0.001), and cortisol (P < 0.05) such that these measurements responded to the LPS challenge to a lesser extent (IL-1beta: 93 vs. 114 pg/mL, P < 0.05; PGE2: 536 vs. 1,285 pg/mL, P < 0.001; cortisol: 143 vs. 206 ng/mL, P < 0.05) in pigs receiving the FO diet than in pigs fed the CO diet. In contrast, among LPS-treated pigs, pigs fed the FO diet had higher IGF-I (155 vs. 101 ng/mL; P < 0.10) than those fed the CO diet. On d 21 among LPS-treated pigs, pigs fed FO had lower IL-1beta (70 vs. 84 pg/mL; P < 0.10) and cortisol (153 vs. 205 ng/mL; P < 0.05) than those fed CO. Pigs fed FO had lower PGE2 (331 vs. 444 pg/mL; P < 0.05) and higher IGF-I (202 vs. 171 ng/mL; P < 0.10) compared with those fed CO. Lipopolysaccharide challenge decreased GH (0.27 vs. 0.33 ng/mL; P < 0.05) on d 14, whereas it had no effect on GH on d 21. During both LPS challenge periods, the challenge increased PBLP when these cells were incubated with 8 (1.46 vs. 1.32; P < 0.10) or 16 microg/mL (1.46 vs. 1.30; P < 0.05) of concanavalin A. Fish oil had no effect on PBLP. These results suggest that FO alters the release of proinflammatory cytokines, which might lead to improved pig performance during an immunological challenge.     

脂多糖对体外培养奶牛子宫内膜上皮细胞子宫容受性因子表达的影响

试验旨在通过脂多糖（lipopolysaccharide,LPS）诱导奶牛子宫内膜上皮细胞构建子宫内膜炎体外感染模型,研究子宫内膜炎对奶牛子宫容受性因子大分子转膜黏蛋白-糖蛋白1（MUC-1）、高度保守的分泌型WNT家族亚型糖蛋白（Wnt-7a）、β受体1（IFNAR1）、IFNAR2、Integrin αvβ3 mRNA及蛋白相对表达量的影响,进而阐明子宫内膜炎引起奶牛屡配不孕和繁殖率低下的机制。采用组织块法分离培养奶牛子宫内膜上皮细胞,免疫荧光方法鉴定细胞纯度,不同浓度LPS刺激子宫内膜上皮细胞,在不同时间点用CCK8测定细胞存活率,ELISA检测炎性因子白细胞介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）、IL-6、IL-8的分泌变化,实时荧光定量PCR和Western blotting分别检测子宫内膜感染模型中子宫容受性因子MUC-1、Wnt-7a、IFNAR1、IFNAR2、Integrin αvβ3 mRNA及蛋白的表达变化。结果显示,通过组织块法纯化获得的奶牛子宫上皮细胞数量较多,经免疫荧光角蛋白染色证实纯度较高。采用CCK8测定细胞存活率发现,与对照组相比,浓度为0、5、10、50μg/mL的LPS作用6、12、24、48 h后,细胞活力无显著变化（P>0.05）;浓度为100μg/mL的LPS作用24 h时,细胞存活率显著低于对照组（P<0.05）,细胞培养上清中的IL-1β、TNF-α、IL-6、IL-8含量均显著高于对照组及低浓度组（P<0.05）,引起细胞的炎症反应。与对照组相比,模型组MUC-1 mRNA及蛋白的相对表达量均极显著增加（P<0.01）;Wnt-7a mRNA及蛋白的相对表达量均极显著下降（P<0.01）;Integrin αvβ3、IFNAR1和IFNAR2 mRNA相对表达量均极显著下降（P<0.01）,蛋白相对表达量均显著下降（P<0.05）。结果表明,浓度为100μg/mL的LPS作用24 h可成功构建子宫内膜炎体外感染模型,子宫内膜炎对奶牛子宫容受性因子MUC-1、Wnt-7a、IFNAR1、IFNAR2、Integrin αvβ3 mRNA及蛋白表达的影响可能是引起奶牛屡配不孕和繁殖率低下的重要原因。     

孕酮对LPS诱导小鼠腹腔巨噬细胞表达TNF-α、IL-1β、TLR4、CD14和MD2的影响

先分离培养小鼠腹腔巨噬细胞,经差速贴壁法纯化后,随机分为6组：空白对照组、0.5mg/L脂多糖（LPS）组、10-6 mol/L孕酮（P4）组、LPS＋10-5 mol/L P4组、LPS＋10-6 mol/L P4组、LPS＋10-7 mol/L P4组。各组在处理12、24h分别提取上清液,ELISA法测TNF-α和IL-1β的含量;各组在处理24h分别提取细胞总RNA,用RT-PCR法测TLR4、CD14、MD2mRNA的表达。结果显示,处理12、24h,0.5mg/L LPS组TNF-α和IL-1β的含量均极显著高于对照组（P〈0.01）;10-6 mol/L P4组与对照组差异不显著（P〉0.05）;LPS＋10-5 mol/L P4组极显著低于对照组（P〈0.01）;LPS＋10-6 mol/L P4组显著低于对照组（P〈0.05）;而LPS＋10-7 mol/L P4组TNF-α的表达差异不显著（P〉0.05）,IL-1β的表达差异显著（P〈0.05）。说明P4可降低LPS刺激小鼠腹腔巨噬细胞TNF-α和IL-1β的分泌,且呈剂量依赖关系。LPS单独处理,TLR4和CD14mRNA的表达极显著高于对照组（P〈0.01）;10-6 mol/L P4单独处理与对照组无显著差异（P〉0.05）;分别添加1-5、10-6、10-7 mol/L P4组均极显著降低LPS诱导TLR4和CD14mRNA的表达（P〈0.01）,而MD2mRNA的表达差异不显著（P〉0.05）。说明P4可极显著降低LPS刺激小鼠腹腔巨噬细胞TLR4和CD14mRNA表达,但对MD2mRNA表达影响不显著。结果显示,P4能抑制LPS刺激的小鼠腹腔巨噬细胞TNF-α和IL-1β的分泌,此过程与细胞TLR4和CD14表达下降相关,而与MD2的表达无关。     

Differential cytokine gene expression in CD4+ and CD8+ T cell subsets of calves

The distinct patterns of cytokine expression in CD4+ and CD8+ T cells are well understood in mice and humans. However, little information is available about cytokine expression in bovine CD4+ and CD8+ T cells. In this study, mRNA expression of 19 different cytokines was analyzed in CD4+ and CD8+ T cells of calves with or without Concanavalin A (Con A) stimulation. CD4+ and CD8+ T cell populations were enriched to 98% purity by positive selection using magnetic cell sorting (MACS). CD4+ T cells spontaneously expressed the mRNAs of interleukin-1alpha (IL-1alpha), IL-1beta, IL-2, IL-6, IL-7, IL-8, IL-10, IL-18, IFN-gamma, TNF-alpha, TNF-beta and TGF-beta, and augmented the mRNA expression of IL-10, IFN-gamma and TNF-beta after Con A stimulation. The mRNAs of IL-3, IL-4, IL-5, IL-13 and GM-CSF were newly expressed in Con A-stimulated CD4+ T cells. CD8+ T cells displayed spontaneous mRNA expression of IL-6, IL-18, TNF-alpha, TNF-beta and TGF-beta, and newly expressed the mRNA of IL-2, IL-7, interferon-gamma (IFN-gamma) and GM-CSF after Con A stimulation. It was found that CD4+ T cells expressed the mRNA of 17 cytokines except for IL-12 and IL-15, while CD8+ T cells expressed only the mRNA of 9 cytokines after Con A stimulation. The profile of cytokine mRNA expression was substantially different in the CD4+ and CD8+ T cells of calves, indicating that CD4+ T cells can be distinguished from CD8+ T cells by the cytokine gene expression of IL-1alpha, IL-1beta, IL-3, IL-4, IL-5, IL-8, IL-10 and IL-13. Differential cytokine expression between CD4+ and CD8+ T cells serve to interpret an individual function of T cell subsets in the immune system of calves.     

Differential gene regulation of interleukin-1 ligands and receptors in bovine peripheral blood neutrophils and mononuclear cells in response to E. coli lipopolysaccharide (LPS)

The proinflammatory cytokine, interleukin-1, plays a prominent role in the inflammatory reactions that characterize numerous diseases. In this study, we examined the gene expression for bovine IL-1 ligands and receptors by bovine peripheral blood mononuclear cells (MNCs) and neutrophils (PMNs) in response to E. coli lipopolysaccharide (LPS) in vitro. Gene expression of mRNA for IL-1alpha, IL-1beta, IL-1 receptor antagonist (IL-1ra), type 1 IL-1 receptor, type 2 IL-1 receptor, and IL-1 beta converting enzyme (ICE), were measured by a semi-quantitative RT-PCR technique. LPS had little effect on type 1 IL-1R expression in MNC, whereas, it strongly up-regulated type 1 IL-1R expression in PMNs. Co-incubation of PMNs with LPS and bovine recombinant IL-1beta had little additional effect on type 1 IL-1R expression. Incubation of MNCs with LPS resulted in up-regulation of IL-1beta, IL-1ra, and type 2 IL-1R, no change in IL-1alpha, and a decrease in ICE gene expression. Incubation of PMNs with LPS up-regulated IL-1beta gene expression, whereas, IL-1alpha, IL-1ra, type 2 IL-1R and ICE were unchanged. This study provides evidence for differential regulation of gene products of the bovine IL-1 family by peripheral blood mononuclear cells (MNC) and neutrophils (PMNs) in response to E. coli LPS.     

In vitro regulation of Mac-1 expression on bovine polymorphonuclear leukocytes by endotoxin and tumor necrosis factor-α at different stages of lactation

The purpose of this in vitro study is to clarify some of the underlying mechanisms leading to the decreased migratory capacity of polymorphonuclear leukocytes (PMN) during mastitis in dairy cows soon after calving. Surface expression of Mac-1 (CD11b, CR3) on PMN and of CD14 on monocytes was measured in early- (EL), peak- (PL), and midlactation (ML) by flow cytometric analysis. In addition, we evaluated the effect of lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-alpha on CD11b surface expression in PMN at different stages of lactation in a whole blood model. During EL, while resting monocytes expressed diminished levels of CD14, the basal expression of CD11b on PMN was not significantly altered. The relative increase of CD11b on PMN after incubation with LPS or TNF-alpha did not significantly differ among EL, PL, or ML at any of the concentrations tested. The current findings do not support an important role for basal CD11b levels nor for a defective mobilization of CD11b by LPS and TNF-alpha in the reduced migratory capacity of PMN during EL.     

Effects of continuous low-dose infusion of lipopolysaccharide on expression of E-selectin and intercellular adhesion molecule-1 messenger RNA and neutrophil accumulation in specific organs in dogs

OBJECTIVE: To determine the effects of continuous low-dose infusion of lipopolysaccharide (LPS) on the expression of E-selectin and intercellular adhesion molecule-1 (ICAM-1) mRNA and neutrophil accumulation in the lungs, liver, spleen, small intestine, and pancreas in dogs. ANIMALS: 11 healthy adult Beagles. PROCEDURE: Dogs received a continuous infusion of a low dose (10 microg/kg/h, i.v.) of LPS (Escherichia coli 055:B5) or saline (0.9% NaCI) solution (20 mL/kg/h, i.v.) for 8 hours. Activity levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interleukin-6 (1L-6) and the number of WBCs in circulation were examined before and 1, 2, 4, and 8 hours after the onset of LPS infusion. Expression of E-selectin and ICAM-1 mRNA and the number of neutrophils in each tissue were examined. RESULTS: After the onset of LPS infusion, serum TNF-alpha and IL-1beta activities transiently increased. Thereafter, IL-6 activity increased, and high IL-6 activity was maintained throughout the experiment. In dogs in the LPS group, expression of E-selectin mRNA increased only in the lungs, and expression of ICAM-1 mRNA increased in the lungs and liver; the number of neutrophils in the tissue increased in the lungs and liver. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that expression of E-selectin and ICAM-1 mRNA increased during sepsis, particularly in the lungs and liver, and that this increase was associated with neutrophil accumulation. Hence, inhibiting the activation of endothelial cells in the lung and liver may decrease organ damage caused by accumulated neutrophils and help regulate multiple-organ dysfunction.     

Recombinant bovine soluble CD14 reduces severity of experimental Escherichia coli mastitis in mice

Endotoxin, or lipopolysaccharide (LPS), is responsible for pathogenesis of infections induced by Gram-negative bacteria, such as E. coli. The cellular response to LPS is modulated by interactions among LPS, LPS-binding protein (LBP) and CD14. Accumulated evidence shows that the soluble form of CD14 (sCD14) competes with membrane-bound CD14 (mCD14) for LPS and plays a pivotal role in regulating bacterial infection and septic shock caused by Gram-negative bacteria. Recombinant bovine sCD14 (rbosCD14) was produced by transfected insect sf/9 cells and its biological function was evaluated in mice. Eighty-one 8-week old BALB/cj female mice were randomly assigned to two groups, and injected intraperitoneally with either LPS (8 microg/g of body weight, n = 41) or LPS plus rbosCD14 (6.8 microg/g of body weight, n = 40). Survival rate at 24 h after injection for mice injected with either LPS or LPS plus rbosCD14 was 30 and 72%, respectively (P < 0.01). At 48 h survival rate was 7 and 37%, respectively (P < 0.01). To investigate the protective effect of rbosCD14 on experimentally induced mastitis in mice, two abdominal contralateral mammary glands of 7 lactating BALB/cj mice were injected through the teat canal with 10-20 colony-forming units (CFU) of Escherichia coli. One gland simultaneously received rbosCD14 (6 microg) and the other saline. At 24 h after challenge, glands that received rbosCD14 had less swelling and hemorrhaging, significantly lower bacterial counts (P < 0.05) and lower concentrations of TNF-alpha (P < 0.05). Results indicate that rbosCD14 is biologically functional and reduces mortality in mice from endotoxin shock and severity of intramammary infection by E. coli.     

HO-1在巨噬细胞中的抗炎和抗氧化作用

【目的】旨在揭示血红素加氧酶1(HO-1)在巨噬细胞中的抗炎和抗氧化作用。【方法】利用不同浓度脂多糖(LPS,0、3、5、10、15、20、25μg/mL)、HO-1(0、0.02、0.04、0.06、0.08、0.10μg/mL)及锌原卟啉(zinc protoporphyrin, ZPP;0、5、10、15、20、30 ng/mL)分别处理巨噬细胞(RAW264.7),12 h后通过CCK8法检测RAW264.7细胞活力,计算LPS、HO-1和ZPP处理RAW264.7细胞的最佳浓度。将RAW264.7细胞随机分为对照组(CT)、LPS组(LPS)、LPS+HO-1组(LH)、HO-1组(HO-1)、LPS+HO-1+ZPP组(LHZ),每组3个重复。CT组细胞用含10%胎中血清的DMEM培养基培养;LPS组细胞用LPS处理;LH组细胞用LPS和HO-1共处理;HO-1组细胞用HO-1处理;LHZ组细胞用LPS、HO-1和ZPP共处理,各组细胞的处理时间均为12 h,收集细胞和上清。采用ELISA法检测上清液中白细胞介素-6(IL-6)、IL-8、肿瘤坏死因子α(TNF-α)、活性...     

大肠埃希菌和金黄色葡萄球菌对奶牛睾丸间质细胞炎症反应和睾酮分泌的影响

探讨不同数量大肠埃希菌(E.coli)和金黄色葡萄球菌(S.aureus)对奶牛睾丸间质细胞(LCs)炎性因子、类固醇合成快速调节蛋白(StAR)以及睾酮(T)分泌的影响。通过体外培养LCs,选取生长状态良好的第3代LCs进行试验。根据E.coli和S.aureus作用数量,试验各分为6组,即用不同数量的E.coli(0、10~4、10~5、10~6、10~7、10~8 CFU/mL)和不同数量的S.aureus(0、10~4、10~5、10~6、10~7、10~8 CFU/mL)感染LCs。通过实时荧光定量PCR检测相关炎性因子和StAR mRNA表达的变化;用牛睾酮(T)酶联免疫分析(ELISA)试剂盒检测睾酮分泌水平的变化。结果表明,E.coli数量为10~8 CFU/mL和S.aureus数量为10~7 CFU/mL时,IL-6和IL-1βmRNA的表达量最高,与其他组相比较增加极显著(P<0.01);E.coli数量为10~4、10~5、10~6、10~7、10~8 CFU/mL时感染LCs后,StAR mRNA表达量显著低于对照组(P<0.05);S.aureus浓度为10~6、10~7和10~8 CFU/mL时,StAR mRNA的表达量显著低于对照组(P<0.05);分别用E.coli和S.aureus感染LCs 12 h,睾酮分泌量与对照组相比无显著变化(P>0.05)。结果表明,LCs被E.coli和S.aureus感染12 h,能引起LCs的炎症反应,同时抑制StAR mRNA的表达,但对睾酮的分泌无显著影响。     

The effect of silver nanoparticles on splenocyte activity and selected cytokine levels in the mouse serum at early stage of experimental endotoxemia

The objective of this study was to determine the effect of a nonionic silver nanocolloid administered orally for 7 or 14 days at three concentration levels (25 ppm, 2.5 ppm, and 0.25 ppm) on the phagocytic activity and mitogenic response of splenocytes and selected cytokine serum levels (IL-1beta, IL-6, IL-10, IL-12 p70, TNF-alpha) in NMRI mice at the early stage of experimental endotoxemia induced with single 30 microg/mouse dose of bacterial LPS. Regardless of the period of administration, silver nanoparticles enhanced the production of proinflammatory cytokines and anti-inflammatory cytokine IL-10, and they inhibited IL-12 p70 levels in response to LPS challenge. The studied nanoparticles' effect on splenocyte activity was determined by the period of administration. After 7 days of use, silver nanoparticles enhanced the phagocytic activity, and doses of 2.5 ppm stimulated the mitogenic response of splenocytes. After 14 days of administration, silver nanoparticles lowered the phagocytic activity regardless of the dose applied. Although the results obtained are ambiguous, they suggest that silver nanoparticles administered via the alimentary tract are more likely to increase an inflammatory response of an organism than offer protection after LPS challenge.     

Recombinant bovine soluble CD14 sensitizes the mammary gland to lipopolysaccharide

Standard therapies including administration of potent antibiotics, aggressive fluid resuscitation and metabolic support have not been successful in relieving symptoms and reducing mortality associated with acute coliform mastitis. It is important to understand the pathophysiological response of the mammary gland to coliform infections when designing preventive or therapeutic regimens for controlling coliform mastitis. Our laboratory has previously shown that macrophages and polymorphonuclear neutrophils in milk express CD14 on their cell surface. In this study, we found that soluble CD14 (sCD14) is present in milk whey as a 46kDa protein reacted with anti-ovine CD14 antibody. Additional functional studies found that: (1) under serum-free condition, complexes of LPS-recombinant bovine soluble CD14 (rbosCD14) induced activation of mammary ductal epithelial cells (as measured by changes in interleukin-8 (IL-8) mRNA level by competitive RT-PCR) at low concentrations of LPS after 6 or 24h incubation (1-1000ng/ml), whereas LPS alone did not induce activation of mammary ductal epithelial cells at the same concentrations, and (2) intramammary injection of low concentrations of LPS did not increase concentration of leukocytes in milk. In contrast, LPS-rbosCD14 complex containing the same concentration of LPS increased the concentration of leukocytes in the injected mammary gland at 12 and 24h post-injection. These results indicate that rbosCD14 sensitizes mammary epithelial cells to low concentrations of LPS in vitro and in vivo. Endogenous sCD14 in milk may be important in initiating host responses to Gram-negative bacterial infections.     

Effect of leukotoxin of Mannheimia haemolytica and LPS of E. coli on secretory response of bovine neutrophils in vitro

To evaluate the role of leukotoxin (LKT) of Mannheimia haemolytica and lipopolysaccharide (LPS) of E. coli 055:B5 in pathogenesis of bovine respiratory disease (BRD) we investigated their in vitro effects on cultured bovine neutrophils. Functional parameters of neutrophils including degranulation, generation of superoxide, and nitric oxide were distorted in response to both toxins. The most essential reaction of neutrophils was found in respect to release of elastase after addition of LKT as well as LPS at concentration of 300 microg/ml. Moreover, we observed an increased release of myeloperoxidase (MPO) and alkaline phosphatase (ALK-P) from polymorphonuclear cells (PMN) after addition of LKT and LPS. We also found enhanced superoxide generation by bovine neutrophils after exposure to different concentrations of LKT and LPS. In cultures of PMN treated with LKT, concentration of nitrite increased with growing concentrations of LKT. Lower values of nitrite were obtained in cultures exposed to LPS. Partial lysis of PMN, determined by LDH (lactate dehydrogenase) leakage, started at concentration of 300 microg/ml for both toxins, meanwhile LKT concentration above 300 microg/ml was lethal. Our study has revealed that neutrophils in response to both toxins exaggerate release of analysed substances, which participate in worsening the course of the disease and play a role in lung injury during BRD. Toxins introduced to the cultural medium stimulate release of studied constituents from neutrophils by combined activation and lysis of neutrophils.     

Characterisation of ovine alveolar macrophages: regulation of surface antigen expression and cytokine production.

Regulation of ovine alveolar macrophage function by recombinant interferon gamma (rIFN gamma) and lipopolysaccharide (LPS) was investigated. Ten units per millilitre of rIFN gamma increased surface expression of MHC class I and class II (DR alpha, DP alpha, and DQ alpha) molecules but not other surface antigens examined. The upregulation of MHC class II expression was specifically blocked by rIFN gamma specific monoclonal antibodies and determination of a dose/response curve established that the minimum concentration of rIFN gamma required for increased class II expression was 0.1 U ml-1 and for increased class I expression, 1 U ml-1. Northern blot analysis indicated that rIFN gamma mediated increases in surface MHC class I and class II expression were due to increased levels of specific mRNA. Using Northern blot analysis and homologous human cDNA probes we failed to detect mRNA encoding the cytokines IL-1 alpha, IL-1 beta, and TNF alpha in RNA extracted from freshly isolated macrophages or macrophages cultured in medium alone. Exposure of macrophages to LPS increased production of all three cytokines although kinetics of upregulation varied. TNF alpha mRNA was induced to maximal levels within 1 h, declining thereafter. IL-1 alpha mRNA was detected at 1 h post stimulation with a maximal level at 5 h, but none at 24 h. In contrast, IL-1 beta mRNA was not detected until 5 h after stimulation with a low level remaining at 24 h. Dose response analysis indicated that LPS concentrations of 100 pg ml-1 induced detectable levels of TNF alpha mRNA while levels as low as 10 pg ml-1 induced secretion of bioactive IL-1. Analysis of the kinetics of secretion of bioactive IL-1 from LPS stimulated macrophages indicated that levels peaked at 24 h post stimulation.