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.     

Shedding of sCD14 by bovine neutrophils following activation with bacterial lipopolysaccharide results in down-regulation of IL-8

CD14, the leukocyte co-receptor for lipopolysaccharide (LPS), is important in the response of bovine polymorphonuclear neutrophil leukocytes (PMN) to Gram-negative bacteria. In other species, the expression of CD14 on the surface of PMN was shown to increase after exposure to inflammatory stimuli. These newly expressed molecules may originate from either an intracellular pool or through new gene expression. We sought to characterize bovine PMN cell surface expression and shedding of CD14 molecules, and CD14's effect on secretion of the chemoattractants IL-8 and IL-1beta by PMN. Bovine PMN were incubated in RPMI for 20 h at 37 degrees C with LPS (1, 10, 100 microg/mL). IL-8 release increased with treatment of 1 microg/mL LPS, but decreased 41.5 and 95% at the 10 and 100 microg/mL concentrations of LPS, respectively. In contrast, shedding of CD14 from the surface of PMN only increased at the highest concentration of LPS (100 microg/mL). Secretion of IL-1beta was similar regardless of the LPS concentration used to stimulate PMN. The effect of PMN concentration (1 x 10(7), 2.5 x 10(7), 5 x 10(7), and 10 x 10(7)/mL) on CD14 cell surface expression and shedding of IL-8 and IL-1beta were also determined. Shedding of CD14 by PMN increased with increasing concentration of PMN after exposure to 0.1 and 10 microg/mL of LPS, while secretion of IL-8 decreased. IL-1beta increased at the highest concentration of PMN. The use of real time polymerase chain reaction showed that CD14 mRNA expression was not different between control and LPS-stimulated cells, indicating that the sCD14 came from either membrane bound CD14 or a preformed pool. Our results demonstrate that release of CD14 from PMN suppresses secretion of IL-8, and may be an important regulatory mechanism for controlling excessive migration of PMN into the bovine mammary gland.     

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.     

The production and characterization of anti-bovine CD14 monoclonal antibodies

To characterize further the chemical and biological properties of bovine soluble (bos) CD14, a panel of ten murine monoclonal antibodies (mAb) reactive with recombinant (r) bosCD14 were produced. A sandwich ELISA, using murine mAb and rabbit polyclonal antibodies reactive with rbosCD14 was developed. All the mAb were reactive by ELISA with baculovirus-derived rbosCD14 and they recognized rbosCD14 (40 kDa) by western blot analysis. The mAb also identified by western blot sCD14 (53 and 58 kDa) in milk and blood and sCD14 (47 kDa) in a lysate of macrophages obtained from involuted bovine mammary gland secretions. Analysis by ELISA of whey samples after intramammary injection of lipopolysaccharide (LPS) (10 micro g) revealed increased sCD14 levels between 8 to 48 h after injection. Flow cytometric analysis showed that the mAb bound to macrophages isolated from involuted mammary gland secretions and mouse macrophages but not to swine or horse monocytes. Addition of anti-rbosCD14 mAb to monocytes stimulated with LPS reduced in vitro production of TNF-alpha. The anti-rbosCD14 antibodies generated in this study will be useful in studying CD14, an accessory molecule that contributes to host innate recognition of bacterial cell wall components in mammary secretions produced during mastitis.     

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 the second-generation synthetic lipid A analogue E5564 on responses to endotoxin in [corrected] equine whole blood and monocytes

OBJECTIVE: To evaluate proinflammatory effects of the second-generation synthetic lipid A analogue E5564 on equine whole blood and isolated monocytes and to determine the ability of E5564 to prevent LPS (lipopolysaccharide)-induced procoagulant activity (PCA); tumor necrosis factor (TNF)-alpha production; and mRNA expression of TNF-alpha, interleukin (IL)-1beta, IL-6, and IL-10 by equine monocytes. SAMPLE POPULATION: Venous blood samples obtained from 19 healthy horses. PROCEDURES: Whole blood and monocytes were incubated with Escherichia coli O111:B4 LPS, E5564, or E5564 plus E coli O111:B4 LPS. Whole blood and cell supernatants were assayed for TNF-alpha, and cell lysates were assayed to determine PCA. Expression of mRNA for TNF-alpha, IL-1beta, IL-6, and IL-10 by monocytes was determined by use of real-time quantitative PCR assay. RESULTS: Minimal proinflammatory effects were detected in whole blood and monocytes. In addition, E5564 inhibited LPS-induced PCA and TNF-alpha production in a concentration-dependent manner. Furthermore, E5564 significantly inhibited LPS-induced mRNA expression of TNF-alpha, IL-1beta, and IL-10 and decreased LPS-induced expression of IL-6. CONCLUSIONS AND CLINICAL RELEVANCE: The second-generation synthetic lipid A analogue E5564 lacked agonist activity in equine whole blood and monocytes and was a potent antagonist of enteric LPS. Therefore, E5564 appeared to be the first lipid A analogue that has potential as an effective therapeutic agent in horses with endotoxemia.     

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.     

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.     

An investigation of the role of soluble CD14 in hospitalized,sick horses

The objectives of this study were to (1) evaluate the effects of equine soluble CD14 (sCD14) and monoclonal antibodies (mAb) to equine CD14 on lipopolysaccharide-induced tumor necrosis factor α (TNF-α) secretion from equine peripheral blood mononuclear cells (PBMC); and to (2) determine serum concentrations of sCD14 in a population of horses with gastrointestinal diseases or other illnesses likely to result in endotoxemia. Equine PBMC isolated from 10 healthy horses were incubated with Escherichia coli LPS plus CD14 mAb or sCD14 and assayed for TNF-α activity. Pre-incubation with CD14 mAb did not inhibit LPS-induced TNF-α production, whereas use of sCD14 inhibited LPS-induced TNF-α production in a concentration-dependent manner. Additionally, blood samples from 55 ill and 23 healthy horses were used to determine serum concentrations of sCD14. Concentrations of sCD14 were positively correlated to respiratory rate, duration of clinical signs and band neutrophil count. Although serum sCD14 was significantly increased in the ill horses compared to healthy horses, sCD14 did not correlate with outcome. Results of this study indicate that release of sCD14 is increased in ill horses and that TNF-α production by PBMC is decreased when cells are treated with sCD14.     

The effect of recombinant swine interleukin-4 on swine immune cells and on pro-inflammatory cytokine productions in pigs

The in vitro effect and the in vivo influence of recombinant swine IL-4 (rSwIL-4) were characterized in various swine cells and in nursery pigs on LPS-induced endotoxic shock and pro-inflammatory cytokine productions. In in vitro experiment, the rSwIL-4 induced a proliferation of CD4 positive T cells in mitogen-prestimulated peripheral blood mononuclear cell (PBMC). In addition, the rSwIL-4, which was produced from insect cells, promoted the differentiation of monocytes into immature dendritic cells in combination with granulocyte macrophage-colony stimulating factor (GM-CSF). Furthermore, the rSwIL-4 successfully suppressed the LPS-induced secretion of TNF-alpha, IL-1alpha, IL-6, IL-8, and IL-18 from swine alveolar macrophages when rSwIL-4 was treated at the same time with LPS. In in vivo experiment in nursery pigs, subcutaneous pretreatment of rSwIL-4, which was produced from baculovirus expression system, enhanced the severity of respiratory failure with endotoxic shock, and increased the production of TNF-alpha and IL-18 in response to inoculation with LPS. These results indicate that the rSwIL-4 is biologically active in both in vitro and in vivo treatments. Depending on the administration time, pro-inflammatory cytokine productions by IL-4 can cause either inhibitory or stimulatory regulation.     

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.     

内毒素的信号转导

内毒素即脂多糖介导的细胞信号转导在激活靶细胞产生一系列应答反应，导致机体损伤并出现病理变化中起着关键作用。LPS信号转导涉及到多种血清蛋白和受体，如LBP、sCD14,mCD14，以及CD11/CD18，LBP/CD14系统在LPS信号转导中起着重要作用，但并非LPS信号转导所必需的LPS与受体结合后，细胞内信号转导涉及到酪氨酸激酶与MAPK超家族，通过多个MAPK通路和保守的三级激酶级联反应（MAPKKK-MAPKK-MAPK）将LPS信号转导致胞内，引起细胞应答，同时p38MAPK对LPS介导的细胞反应有重要调节作用，这些均表明，LPS信号转导可能是一个多受体，多通路，可调控的复杂过程。     

Egg white derivatives induce tumor necrosis factor-alpha expression in porcine peripheral blood mononuclear cells

Porcine PBMC derived phagocytic activity in peripheral blood polymorphonuclear cells (PMN) induced by egg white derivatives (EWD) treatment was analyzed at the protein and mRNA level. EWD alone failed to induce phagocytic activity of PMN measured by flow cytometry. But PMN phagocytosis was enhanced by culture supernatant from PBMC treated with EWD, human (h)rTNF-alpha and porcine (p)rIL-1beta, respectively. To identify this phagocytic inducing factor, the culture supernatant was partially purified by gel filtration. Only fraction 8 revealed the enhanced PMN phagocytic activity. This fraction also had a high cross-reactivity with anti-prTNF-alpha polyclonal (p)Ab but not with anti-prIL-1beta pAb, as measured by ELISA, indicating that the culture supernatant from PBMC treated with EWD was independent from IL-1beta. The enhanced PMN phagocytic activity of fraction 8 was also inhibited by anti-prTNF-alpha pAb. Both fraction 8 and hrTNF-alpha produced a single protein band between 16 and 18kDa upon analysis by sodium-dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting using anti-prTNF-alpha pAb, suggesting that the promoter of PMN phagocytosis is TNF-alpha, a 16-18 kDa protein produced by EWD-stimulated PBMC. Porcine TNF-alpha mRNA expression in porcine PBMC analyzed by RT-PCR was also increased by addition of EWD. This study strongly suggests that the immunoenhancing effect of EWD on the phagocytic response of porcine PMN is mediated through TNF-alpha produced by PBMC stimulated with EWD. In addition, the expression of porcine TNF-alpha on PBMC is also increased when stimulated with EWD.     

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。     

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.     

白头翁汤通过抑制TLR4-ERK1/2信号通路缓解LPS诱导的微血管内皮细胞炎性反应

近年来,中药复方抗炎机制的研究不断深入,白头翁汤复方（Pulsatilla decoction,PD）作为经典的清热解毒中药方剂常用于预防和治疗细菌性腹泻。然而其抗炎机制和靶细胞研究仍然不明确,本课题以大鼠肠黏膜微血管内皮细胞（RIMVECs）为模式细胞,旨在研究白头翁汤对LPS诱导的RIMVECs炎症反应的调控作用。利用LPS刺激RIMVECs,通过荧光定量PCR（RT-PCR）、蛋白免疫印迹（Western blot）的方法检测白头翁汤对LPS刺激后RIMVECs的炎性信号通路TLR4-ERK1/2信号通路关键蛋白TLR4、TRAF6、ERK的mRNA及蛋白表达。进一步采用酶联免疫吸附方法（ELISA）检测白头翁汤对LPS刺激后的炎性因子IL-6、IL-8、IL-1β、TNF-α的分泌情况。结果表明：白头翁汤可以显著降低LPS诱导的TLR4、TRAF6、ERK的mRNA水平及蛋白表达并降低了LPS诱导的细胞下游炎性因子的分泌。白头翁汤通过抑制TLR4-ERK1/2信号通路缓解LPS所诱导的RIMVECs炎性反应,发挥抗炎作用。     

Role of inflammatory mediators in priming, activation, and deformability of bovine neutrophils

OBJECTIVE: To determine the capacity of inflammatory mediators tumor necrosis factor-alpha (TNF-alpha), interleukin-8 (IL-8), platelet-activating factor (PAF), lipopolysaccharide (LPS), and leukotoxin to prime, activate, or alter deformability of adult bovine neutrophils. SAMPLE POPULATION: Blood collected from 5 healthy adult Holstein cows. PROCEDURE: Isolated neutrophils or whole blood was incubated with TNF-alpha, IL-8, PAF, LPS, or leukotoxin, and neutrophil chemiluminescence, degranulation, deformability, shape change, CD11b expression, and size distribution was measured. RESULTS: Incubation with TNF-alpha, IL-8, PAF, and LPS primed neutrophils for oxygen radical release but caused minimal oxygen radical release by themselves. None of the inflammatory mediators induced degranulation. Incubation with TNF-alpha and PAF resulted in a decrease in neutrophil deformability and induced shape change in neutrophils. Incubation with PAF consistently resulted in an increase in neutrophil size as measured by use of flow cytometry. Only IL-8 caused an increase in expression of CD11b by neutrophils. CONCLUSIONS AND CLINICAL RELEVANCE: Inflammatory mediators tested had minimal effects on neutrophil oxygen radical production or degranulation but did prime neutrophils for oxygen radical production. Incubation with PAF and TNF-alpha caused a decrease in neutrophil deformability and altered neutrophil shape and size. Results of our study indicate that PAF- and TNF-alpha-induced changes in neutrophil deformability and size may cause integrin- and selectin-independent trapping of neutrophils in the lungs of cattle with pneumonic pasteurellosis.     

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。