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.     

Activation of bovine neutrophils by recombinant bovine tumor necrosis factor-alpha

The in vitro effect of bovine recombinant tumor necrosis factor-alpha (rbTNF-alpha) on bovine neutrophil function and the possibility that rbTNF-alpha and recombinant bovine interferon-gamma (rbIFN-gamma) act synergistically were investigated. Treatment of neutrophils with rbTNF-alpha (0.05 micrograms/ml; approximately 50 U/ml) at 37 degrees C for 2.5 h resulted in enhancement of antibody independent neutrophil-mediated cytotoxicity (AINC) and inhibition of random migration and chemotaxis. The same treatment resulted in a slight decrease in iodination and cytochrome C reduction, but did not affect Staphylococcus aureus ingestion, or antibody dependent cell-mediated cytotoxicity. Kinetic and inhibitor studies indicated that the action of rbTNF-alpha was rapid and was independent of protein and RNA synthesis by neutrophils. Evaluation of the synergistic activities of rbTNF-alpha and rbIFN-gamma indicated that treatment of neutrophils with these two cytokines simultaneously resulted in additive enhancement of AINC and inhibition of random migration and chemotaxis. There was no additive effect of the two cytokines on inhibition of iodination or cytochrome C reduction.     

Effects of serum vitamin E levels on skin vitamin E levels in dogs and cats

Skin problems are common in small animal practice. Oxidative stress, or the imbalance between prooxidants and the body's antioxidant defense system, likely plays a role in the development of skin disease. According to this study, increasing amounts of vitamin E in foods for dogs and cats increases serum and cutaneous concentrations of vitamin E. Based on available scientific data, these increases in vitamin E concentration are likely to be beneficial. However, the relationship between increases in serum and skin vitamin E concentrations and the prevention, development, and treatment of skin disease remains to be elucidated by intervention studies.     

维生素E、K对动物基因表达的影响

本文在介绍维生素E和维生素K营养生理功能的基础上,综述了维生素E和维生素K对相关基因表达的调控及其主要机制。     

高效液相色谱法测定维生素E粉含量

用高效液相色谱法测定饲料添加剂维生素E粉中维生素E含量。采用EclipseXDBC18柱(150mm×4.6mm,5μm),以甲醇-水(98∶2,V∶V)为流动相,流速为1.2mL/min,检测波长为285nm,柱温为25℃。在此色谱条件下,维生素E在0.104￣0.602mg/mL浓度范围内呈良好线性关系,r=0.9998,平均回收率为100.3%,RSD为0.72%。本法操作简便易行,系统适用性良好,适用于饲料添加剂维生素E粉中维生素E含量测定。     

Modulation, in vivo and in vitro, of surface expression of CD18 by bovine neutrophils.

A series of experiments was designed to elucidate some of the factors that may influence surface expression of CD18 by bovine neutrophils. Expression of CD18 was determined by immunofluorescence flow cytometry. Neutrophils recovered from the uterus of cows (n = 9) after intrauterine administration of sterile oyster glycogen solution expressed (mean +/- SD) 123 +/- 21% more CD18 than did circulating neutrophils recovered simultaneously from the same cows (P = 0.003). In 8 cows given 20 mg of dexamethasone IM daily for 3 days, expression of CD18 on blood neutrophils was 29.6 +/- 8% less after treatment than before treatment (P = 0.0078). Neutrophils from 12 cows or bulls exposed to phorbol myristate acetate in vitro increased expression of CD18 by 137 +/- 37% (P = 0.0035). Likewise, exposure of neutrophils from 8 cattle to zymosan-activated bovine plasma increased CD18 exposure by 10.6 +/- 3.8% (P = 0.029). These findings indicate that expression of CD18 by bovine neutrophils is a dynamic system, capable of responding to inflammatory stimuli. Inadvertent activation of neutrophils may be responsible for some of the variance in expression observed when examining large groups of cattle for CD18 expression by neutrophils. The ability of bovine neutrophils to respond rapidly to various stimuli by increasing surface expression of CD18 indicates that a pool of intracellular CD18 may be available for inclusion in the plasmalemma, as has been reported for human neutrophils.     

Effects of Mannheimia haemolytica leukotoxin on apoptosis and oncosis of bovine neutrophils

OBJECTIVE: To investigate the concentration-dependent effects of Mannheimia haemolytica (formerly Pasteurella haemolytica) leukotoxin (LKT) on apoptosis and oncosis in bovine neutrophils and to examine the role of calcium ions (Ca2+) in LKT-induced apoptosis. SAMPLE POPULATION: Neutrophils isolated from blood samples obtained from healthy calves. PROCEDURE: Neutrophil suspensions were exposed to lytic or sublytic dilutions of LKT and then examined by use of transmission electron microscopy (TEM) or gel electrophoresis. Contribution of extracellular Ca2+ to LKT-induced apoptosis was investigated by incubating neutrophils with LKT or control solutions in buffer containing 1 mM CaCl2 or in Ca2+-free buffer containing 1 mM ethylene glycol-bis (b-aminoethyl ether)-N,N-tetraacetic acid (EGTA) prior to diphenyl amine analysis. RESULTS: Examination by TEM revealed that bovine neutrophils exposed to lytic dilutions of LKT had changes consistent with oncosis, whereas neutrophils exposed to sublytic dilutions of LKT and staurosporin, an inducer of apoptosis, had changes consistent with apoptosis. Effects of sublytic dilutions of LKT on apoptosis were confirmed by gel electrophoresis. Replacement of extracellular Ca2+ with EGTA, a Ca2+ chelator, reduced apoptosis attributable to the calcium ionophore A23187, but it did not have significant effects on apoptosis induced by LKT or staurosporin. CONCLUSIONS AND CLINICAL RELEVANCE: The ability of LKT to cause apoptosis instead of oncosis is concentration-dependent, suggesting that both processes of cell death contribute to an ineffective host-defense response, depending on the LKT concentration in pneumonic lesions. Furthermore, although Ca2+ promotes A23187-induced apoptosis, it is apparently not an essential second messenger for LKT-induced apoptosis.     

Effects of Moraxella bovis and culture filtrates on 51Cr-labeled bovine neutrophils

The cytotoxicity of Moraxella bovis whole cells and culture filtrates was studied, using 51Cr-labeled bovine and human blood neutrophils. The cytotoxicity of living M bovis was directly related to the concentration of bacteria in the neutrophil cultures, and was maximal at an approximate neutrophil to bacteria ratio of 1:10. Cytotoxicity was maximal by 30 minutes after living bacteria were added to the suspension of the 51Cr-labeled neutrophils. Expression of the cytotoxicity was dependent on the presence of Ca2+ in the media, and was independent of the presence of Mg2+. Cytotoxic activity was eliminated by inactivating M bovis in buffers containing formalin or sodium azide. Hemolytic and nonhemolytic isolates of M bovis were examined for cytotoxic activity. All 7 of the hemolytic isolates were cytotoxic for bovine neutrophils, but all 4 of the nonhemolytic isolates were devoid of cytotoxic activity. None of the 11 isolates were cytotoxic for human neutrophils. Sterile filtrates from 6-hour shaker cultures of a hemolytic M bovis isolate were cytotoxic for bovine neutrophils. Cytotoxicity of the filtrate was eliminated by heating, incubation with trypsin, or addition of EDTA to the media. Bacterial homogenates or sterile filtrates prepared from statistically incubated cultures of M bovis were not toxic for bovine neutrophils.     

高效液相色谱法测定维生素E原料含量

用高效液相色谱法测定饲料添加剂维生素E原料中维生素E含量。采用ODSC18柱(150mm×4.6mm,5μm),以甲醇-水(98+2,V+V)为流动相,流速为1.2ml/min,检测波长为285nm,柱温为(30±2)℃,在此色谱条件下,维生素E在0.104～0.828mg/ml浓度范围内呈良好线性关系(r=0.99994),经方法评估函数的评价表明,方法不存在恒定系统误差和相关性系统误差,方法准确性较好,且方法操作简便易行,系统适用性良好,适用于饲料添加剂维生素E原料中维生素E含量测定。     

Effects of dietary vitamin and fatty acid content in E on muscle vitamin E Aohan fine-wool sheep

Background： Increasing the polyunsaturated fatty acid （PUFA） content and decreasing the saturated fatty acid （SFA） content of mutton can help to improve its nutritional value for consumers. Several laboratories have evaluated the effects of vitamin E on the fatty acid （FA） composition of muscle in sheep. However, little information is available on wool sheep, even though wool sheep breeds are an important source of mutton, especially in northern China where sheep are extensively farmed. The present study was designed to address the effects of vitamin E on muscle FA composition in male Aohan fine-wool sheep. Methods： Forty-two male Aohan fine-wool lambs （5 mo old） with similar initial body weight were randomly divided into seven groups and fed diets supplemented with 0 （control group）, 20, 100, 200, 1,000, 2,000, or 2,400 IU/sheep/d vitamin E for 12 mo. Three lambs from each group were slaughtered to measure vitamin E and FA content in the Iongissimus lumborum （LL） and gluteus medius （GM） muscles. Results： Vitamin E concentrations in the LL and GM increased significantly after 12 mo of vitamin E supplementation （P 〈 0.05）. However, this increase did not occur in a dose-dependent manner because the muscle vitamin E concentration was highest in the 200 IU/sheep/d group. Dietary vitamin E supplementation also caused a significant reduction in SFA content and an increase in monounsaturated FA （MUFA） content in the LL and GM （P 〈 0.05）. All six doses of vitamin E significantly increased cis9 tronsl -conjugated linoleic acid （cgtl -CLA） content in the LL compared with the control group （P 〈 0.05）. Conclusions： Dietary supplementation with vitamin E increased muscle vitamin E content and improved the nutritional value of mutton by decreasing SFA content and increasing MUFA and c9tl 1-CLA contents in Aohan fine-wool sheep. These effects were greatest in sheep fed a diet containing 200 IU/sheep/d vitamin E.     

Effects of dietary vitamin E on muscle vitamin E and fatty acid content in Aohan fine-wool sheep

Background

Increasing the polyunsaturated fatty acid (PUFA) content and decreasing the saturated fatty acid (SFA) content of mutton can help to improve its nutritional value for consumers. Several laboratories have evaluated the effects of vitamin E on the fatty acid (FA) composition of muscle in sheep. However, little information is available on wool sheep, even though wool sheep breeds are an important source of mutton, especially in northern China where sheep are extensively farmed. The present study was designed to address the effects of vitamin E on muscle FA composition in male Aohan fine-wool sheep.

Methods

Forty-two male Aohan fine-wool lambs (5 mo old) with similar initial body weight were randomly divided into seven groups and fed diets supplemented with 0 (control group), 20, 100, 200, 1,000, 2,000, or 2,400 IU/sheep/d vitamin E for 12 mo. Three lambs from each group were slaughtered to measure vitamin E and FA content in the longissimus lumborum (LL) and gluteus medius (GM) muscles.

Results

Vitamin E concentrations in the LL and GM increased significantly after 12 mo of vitamin E supplementation (P < 0.05). However, this increase did not occur in a dose-dependent manner because the muscle vitamin E concentration was highest in the 200 IU/sheep/d group. Dietary vitamin E supplementation also caused a significant reduction in SFA content and an increase in monounsaturated FA (MUFA) content in the LL and GM (P < 0.05). All six doses of vitamin E significantly increased cis9 trans11-conjugated linoleic acid (c9t11-CLA) content in the LL compared with the control group (P < 0.05).

Conclusions

Dietary supplementation with vitamin E increased muscle vitamin E content and improved the nutritional value of mutton by decreasing SFA content and increasing MUFA and c9t11-CLA contents in Aohan fine-wool sheep. These effects were greatest in sheep fed a diet containing 200 IU/sheep/d vitamin E.     

Influence of conditioned media from bovine cotyledon tissue cultures on function of bovine neutrophils.

Bovine fetal placental (cotyledon) tissue obtained from pregnant cows on days 255, 265, and 275 of gestation, as well as immediately after parturition (n = 5) was incubated in media for 48 hours, and the incubation media were collected. Neutrophils from 4 ovariectomized nonpregnant cows were incubated for 2 hours with conditioned media from placental tissue cultures or medium (control). Immediately after incubation, the neutrophils were subjected to the following leukocyte function assays: chemotaxis against zymosan-activated serum, chemotaxis against undiluted conditioned media (only neutrophils that were incubated in medium only), random migration, ingestion of 125I-iododeoxyuridine Staphylococcus aureus (125I-IdUR-S aureus), iodination of proteins, cytochrome C reduction, and antibody-independent and -dependent cell-mediated cytotoxicity. Conditioned media from cultured cotyledon tissue was chemoattractant for bovine neutrophils, and increased chemotactic response of neutrophils against zymosan-activated serum by 13%. The following neutrophil functions were decreased: random migration by 25%, iodination of proteins by 44%, cytochrome C reduction by 13%, and antibody-dependent cell-mediated cytotoxicity by 5%. Ingestion of 125I-IdUR-S aureus and antibody-independent cell-mediated cytotoxicity were not influenced by coincubation of neutrophils and conditioned media. Time of gestation did not alter the effects of conditioned media on neutrophil function. It was concluded that chemotactic properties of cotyledon tissue extracts, as has been reported earlier, may be attributable to substances released by fetal placental tissue. Those substances might also locally or systemically influence the oxygen-dependent antimicrobial system of neutrophils, thereby causing an increased susceptibility to bacterial infections in the peripartum period.     

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.     

维生素A、E对獭兔繁殖性能的影响

将 2 8只繁殖獭兔随机均分 2组 ,基础日粮相同 ,但试验组在配种前 3天妊娠第 7天于日粮中添加VA8mg/kg、VE10 0mg/kg。结果与对照组相比 :试验组产活仔数提高了 2 0 2 3 %(P <0 0 5 ) ,育成率提高了 6 5 8%(P <0 0 5 ) ,增重速度提高 7 11%(P <0 0 5 )。     

Pharmacologic enhancement or suppression of phagocytosis by bovine neutrophils

Sixty-three drugs, belonging to 10 chemical classes, were tested in vitro to determine effects on phagocytosis of 32P-labeled Staphylococcus aureus by neutrophils isolated from milk. Within each class, the number of antibiotics tested were: nonsteroidal anti-inflammatory drugs (NSAID; 8), peptolids (2), aminoglycosides (8), tetracyclines and fusidic acid (4), beta-lactam antibiotics (25), secretolytic agents (2), macrolides (5), polypeptides (2), and antibacterial quinolones (8). Percentage of phagocytosis was determined after incubating (2 hours at 37 C) 12.5 x 10(6) viable neutrophils, 200 x 10(6) 32P-labeled S aureus with antibiotics and 5% skimmed milk. Concentrations of antibiotics tested were 1,000, 500, and 10 micrograms/ml of incubation media. When compared with nonantibiotic controls at the highest drug concentration, the NSAID acetylsalicylic acid and centrophenoxine increased phagocytosis 23.2 and 8.8%, respectively, and benzydamine, indomethacin, phenylbutazone, ibuprofen, and acetominophen decreased phagocytosis 22.8, 14.2, 9.8, 27.0, and 18.2%, respectively. The peptolids novobiocin and pristinamycin decreased phagocytosis 24.5 and 22.0%, respectively. The aminoglycosides tobramycin, amikacin, and gentamicin decreased phagocytosis 21.1, 15.4, and 19.2%, respectively. For the tetracyclines and fusidic acid, minocycline and doxycycline decreased phagocytosis 39.8 and 54.2%, respectively. The beta-lactam antibiotics carfecillin, cephapirin sodium, and cephacetrile sodium decreased phagocytosis 11.2, 12.8, and 23.8%, respectively. The secretolytic agent, bromhexin, increased phagocytosis 10.8%. These data indicate that the potential for enhanced phagocytosis exists through use of some NSAID, and for depressed phagocytosis through use of aminoglycosides, peptolids, tetracyclines, and beta-lactams, as well as certain other NSAID.     

Proteomic survey of bovine neutrophils

Mastitis is a major economic concern for the dairy industry. Conditions such as parturition cause a transient immunosuppression that leads to increased incidence of mastitis. One facet of periparturient immunosuppression is a functional impairment of the blood and milk neutrophils in dairy cows. To better understand the biology of the bovine neutrophil we report the first proteomic analysis of the bovine neutrophil. We have identified over 250 proteins using one-dimensional electrophoresis followed by reverse-phase chromatography in line with electrospray tandem mass spectrometry. A large number of metabolic proteins were identified, including most of the enzymes required for generation of NADPH and ATP. In addition, many proteins were identified that participate in cell mobility and phagocytosis. All the bovine members of the cathelicidin family were identified, as well as other proteins with immunological functions. Proteins important for cell signaling, vesicular transport, control of apoptosis and other functions were identified giving an overview of the bovine neutrophil proteome.     

Activation-induced mobilization of secretory vesicles in bovine neutrophils.

OBJECTIVE: To characterize mobilization of secretory granules in bovine neutrophils. SAMPLE POPULATION: Neutrophils obtained from four 6- to 18-month-old Holstein cattle. PROCEDURE: Mobilization of secretory granules in bovine neutrophils was determined by measuring changes in cell-surface alkaline phosphatase activity on cells treated with various inflammatory mediators. Subcellular distribution of the alkaline phosphatase activity was determined by analysis of bovine neutrophil homogenates fractionated on density gradients. RESULTS: Alkaline phosphatase-containing secretory granules of bovine neutrophils were readily mobilized by a number of inflammatory agents, including platelet-activating factor, interleukin-8, tumor necrosis factor-alpha, lipopolysaccharide, leukotriene B4, and zymosan-activated plasma. In contrast, N-formyl-methionyl-leucyl-phenylalanine did not have a significant effect. Phorbol myristate acetate induced a biphasic response with up-regulation of cell-surface alkaline phosphatase at low doses and a return to baseline or even a reduction in cell-surface alkaline phosphatase at higher doses (> or = 10 ng/ml). Subcellular fractionation of bovine neutrophil homogenates revealed that alkaline phosphatase activity resided in light-density membrane vesicles (ie, location of secretory granules), which were distinct from specific, azurophil, and large granules. CONCLUSIONS AND CLINICAL RELEVANCE: Bovine neutrophils respond to various inflammatory mediators by mobilizing alkaline phosphatase-containing secretory granules. This suggests that the process is an important early step in the host-defense response of bovine neutrophils.