Arachidonic acid metabolites produced by bovine alveolar macrophages

Bovine alveolar macrophages, obtained by bronchoalveolar lavage, were labeled with tritiated arachidonic acid. The cells were stimulated with calcium ionophore A23187, and the radiolabeled arachidonic acid metabolites that were released were identified by reverse-phase high-performance liquid chromatography. Leukotriene B4 and 5-hydroxyeicosatetraenoic acid were consistently observed. The lipoxygenase inhibitor, nordihydroguaiaretic acid, blocked production of these metabolites. The cyclooxygenase products, prostaglandin F2 alpha and thromboxane B2, were observed infrequently in comparison with leukotriene B4 and 5-hydroxyeicosatetraenoic acid.     

Evaluation of cytokine production by equine alveolar macrophages exposed to lipopolysaccharide, Aspergillus fumigatus, and a suspension of hay dust

OBJECTIVE: To evaluate cytokine production by equine alveolar macrophages after exposure to lipopolysaccharide (LPS), Aspergillus fumigatus, and hay dust, and determine the effect of clenbuterol on the cytokine response. ANIMALS: 6 horses. PROCEDURE: Alveolar macrophages were exposed to PBS solution (negative control), LPS, hyphae and conidia of Aspergillus fumigatus (AF), or a suspension of hay dust (HDS) and incubated for 24 hours at 37 degrees C. Concentrations of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta were measured in the supernatant. The procedure was repeated with cells that were concurrently incubated with 0.5 microM clenbuterol. RESULTS: Exposure to HDS and AF significantly increased production of TNF-alpha by equine alveolar macrophages. The increase in TNF-alpha produced in response to HDS and AF was 5 and 7 times as great, respectively, as the increase measured in response to LPS. The concentration of IL-1beta in the supernatant was significantly increased after exposure of cells to AF. Clenbuterol was effective at inhibiting TNF-alpha production by cells exposed to LPS, HDS, or AF. CONCLUSIONS AND CLINICAL RELEVANCE: Increased production of TNF-alpha and IL-1 indicated that the pro-inflammatory cytokines produced by alveolar macrophages in response to allergens may play a role in recurrent airway obstruction (RAO) in horses. Equine alveolar macrophages are not only a primary pulmonary defense mechanism but may also influence the pathogenesis of equine RAO. The beta2-adrenoceptor agonist clenbuterol, a drug that is commonly used for treatment of equine RAO, promotes immediate bronchodilation and may also contribute to downward modulation of the inflammatory response.     

Separation by carrier-free electrophoresis of subpopulations of bovine alveolar macrophages

Carrier-free electrophoresis was used to separate subpopulations of bovine alveolar macrophages according to their electrical surface charges. The electrophoretic mobility of the macrophages changed significantly as a function of the time of cultivation in vitro. After 20 h, three subgroups were identified which differed in their specific contents of lysosomal hydrolases, alpha-naphthyl acetate esterase, and alkaline phosphodiesterase. By comparing the findings with results from the literature it was concluded that the subgroups correspond to different stages of maturation and that the fraction moving fastest may be the most mature fraction. The results were not influenced by phagocytosis and the presence or absence of Ca2+.     

Phagocytosis of latex beads by alveolar macrophages from mice exposed to cigarette smoke.

Cigarette smoking is known to alter the numerical presence and function of alveolar macrophages. It has been speculated that these cigarette-smoke-induced alterations contribute to the depressed pulmonary defence mechanism commonly demonstrated in smokers. Studies of the phagocytic and bactericidal activities of alveolar macrophages from smokers and non-smokers have yielded conflicting results. The purpose of this study was to investigate the phagocytic capacity of alveolar macrophages from mice exposed to cigarette smoke in relation to the ability to ingest inert particles (latex beads). Measurements were made before (basal values), immediately after, and 1, 12 or 24 h after exposure. Significant decreases were observed in the number of latex beads ingested by 100 macrophages (phagocytic index) and in the phagocytic efficiency for ingesting latex (mean number of latex beads ingested per activated macrophages) immediately after and 1 h after exposure, and in the number of activated macrophages (those with phagocytic activity) immediately after exposure.     

Reduced endotoxin-induced production of tumor necrosis factor activity by equine peritoneal macrophages exposed to the dual inhibitor of arachidonic acid metabolism, SK & F 86002.

The purpose of this study was to determine if a structurally novel dual inhibitor of arachidonic acid metabolism, SK & F 86002, would inhibit the endotoxin-induced production of tumor necrosis factor (TNF) activity by equine peritoneal macrophages. Equine peritoneal macrophages were variously pretreated for 0, 0.5 and 2 h with SK & F 86002 at 10(-9) to 10(-4) molar final concentrations or were left untreated. Then, the macrophages were cultured in vitro in the presence of endotoxin (5 ng/mL). Supernatant media were collected after 4 h and stored at -70 degrees C until assayed for TNF activity and immunoreactive thromboxane B2 (iTxB2). Macrophage supernatant TNF activities were estimated by an in vitro cytotoxicity bioassay using the murine fibrosarcoma cell line, WEHI 164 clone 13. Concentrations of iTxB2 were quantitated by radioimmunoassay. Coincubation of macrophages with SK & F 86002 significantly decreased the subsequent supernatant TNF activity. Concentrations of SK & F 86002 from 10(-7) to 10(-4) molar effectively reduced TNF production when added to macrophages 0 and 0.5 h prior to endotoxin. After 2 h of preincubation, SK & F 86002 significantly reduced supernatant TNF activity at 10(-5) and 10(-4) M concentrations. Supernatant concentrations of iTxB2 were reduced when SK & F 86002 was added at 10(-6) to 10(-4) M concentrations, 0 and 0.5 h prior to endotoxin, and at all concentrations (10(-9) to 10(-4)) when preincubated with macrophages for 2 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytotoxic interactions between bovine parainfluenza type 3 virus and bovine alveolar macrophages

This paper describes an investigation of the cytotoxic activity of bovine alveolar macrophages for parainfluenza type 3 (PI-3) virus-infected target cells, using 51Cr release assays. Alveolar macrophages from uninfected calves were shown to be capable of killing PI-3 virus infected cells without the presence of antibody or complement (antibody-independent cell-mediated cytotoxicity). The level of killing was shown to vary from animal to animal with specific lysis values ranging from <5% to 70%. Presence of PI-3 virus antiserum was shown to inhibit, rather than enhance macrophage cytotoxicity in a dose-dependent manner, suggesting that bovine alveolar macrophages do not always exhibit antibody-dependent lysis in all cases. Following intranasal and intratracheal inoculation of calves with PI-3 virus, the level of cytotoxicity by macrophages lavaged from the lungs of the calves increased substantially, and by Day 5 post inoculation, levels of 95% to 98% specific lysis were recorded. After Day 5, the killing ability decreased rapidly to low levels. Cell-free lavage fluids, collected from PI-3 virus infected and control calves at various times throughout the experiment, were incubated with aliquots of an alveolar macrophage population from an uninfected donor calf, which initially showed a low level of killing, and were subsequently added to PI-3 virus infected target cells. The recorded levels of cytotoxicity, mirrored those which were seen with the initial macrophage effector cells from the infected and control animals, suggesting that macrophage cytotoxicity was largely controlled by extracellular factors.     

Oxidative metabolism of the bovine alveolar macrophage

Oxidative respiratory burst activity was examined in lavage-procured bovine pulmonary alveolar macrophages. Nonstimulated alveolar macrophages released a minimal quantity of superoxide anion and had small amounts of glucose flux through the pathways of energy metabolism. Nonstimulated cells metabolized substantial amounts of glucose through the hexose monophosphate shunt. Stimulation with opsonized zymosan particles induced a tenfold increase in the release of superoxide anion and a twofold increase in the flux of glucose through the hexose monophosphate shunt and the pathways of energy metabolism. Preliminary observations also indicated that the magnitude of the burst varied between sets of bronchoalveolar cells obtained from the same calf over time.     

Comparison of bovine IgG1, IgG2 and IgM for ability to promote killing of Mycoplasma bovis by bovine alveolar macrophages and neutrophils

The IgG1, IgG2 and IgM fractions were purified by chromatography from bovine antisera to Mycoplasma bovis. They were assayed for specific antibody and compared for ability to promote killing of M. bovis by bovine peripheral-blood neutrophils and alveolar macrophages. None of the Ig preparations killed the mycoplasma in the absence of the cells. The IgG1 and IgG2 preparations both promoted mycoplasma killing by the macrophages; IgM appeared to have no effect. The IgG2 preparation promoted killing by the neutrophils but neither the IgG1 or IgM fraction appeared effective.     

Effects of bovine parainfluenza-3 virus on phagocytosis and phagosome-lysosome fusion of cultured bovine alveolar macrophages

Bovine parainfluenza-3 virus (PI-3V) replicated in cultured bovine alveolar macrophages (BAM) in vitro. Cytopathic changes included spindle cell and giant cell formation, diffuse cell lysis, and cellular detachment progressing to total destruction of the monolayer. Direct immunofluorescent microscopy revealed viral antigen in greater than 90% of BAM on post-inoculation day (PID) 3. Virus titers in culture supernatants increased by a factor of 1.6 X 10(6) TCID50 by PID 6. Candida glabrata was phagocytized by a similar proportion of glass-adherent PI-3V-infected and sham-inoculated control BAM. However, when the number of glass-adherent cells available for assay was taken into account (normalization), the percentage of phagocytic PI-3V-infected BAM was significantly lower than that of the control cells at PID 5 and 7 (P less than 0.01). Phagosome-lysosome fusion assays had a marked reduction of fusion activity in PI-3V-infected BAM as compared with that of control BAM. Proportions of infected cells that showed fusion were approximately 50% of that of the control cells at PID 4 and 6 (P less than 0.05). Normalization of these values reduced the fusion rate of PI-3V-infected BAM to 34% of that of control BAM (P less than 0.01).     

Effect of "in vitro" exposure of bovine alveolar macrophages to different strains of bovine respiratory syncytial virus.

A vaccine strain of respiratory syncytial (RS) virus and an isolate from pneumonic calves (AC2) were inoculated onto cultures of bovine alveolar macrophages recovered by lung lavage, and the functional properties of the cells observed over a period of 10 days. In most cultures no infectious virus was produced although immunofluorescence indicated the presence of virus antigens in some cells. No significant difference was noted between infected and control macrophage cultures in their capacity to phagocytose latex particles (neutral phagocytosis), although the ability to phagocytose complement-coated Candida krusei cells was affected, particularly with the AC2 strain after 6 days. Killing of C. krusei cells was slightly affected by infection of macrophages with the vaccine strain and was dramatically affected by infection with strain AC2. C3b and Fc receptor expression was adversely affected by both virus strains. Production of neutrophil chemotactic factors was increased in cultures infected with both strains, but was greater with AC2, suggesting that some properties of the cells were activated.     

Chemotaxis,phagocytosis, and oxidative metabolism in bovine macrophages exposed to a novel interdigital phlegmon (foot rot) lesion isolate,Porphyromonas levii

OBJECTIVE: To examine the host response toward Porphyromonas levii, by evaluating chemotaxis, phagocytosis, and oxidative burst of bovine macrophages in vitro. SAMPLE POPULATION: Cultured bovine macrophages obtained from monocytes harvested from blood samples of 15 Holstein steers. Porphyromonas levii was isolated from the foot rot lesion of an acutely affected feedlot steer. PROCEDURE: Monocytes were cultured for macrophage differentiation over 7 days. Porphyromonas levii was cultured in strict anaerobic conditions for experimentation. Chemotaxis was evaluated by quantifying macrophage migration toward P. levii in Boyden chambers. Phagocytosis was assessed by quantification of macrophages engulfing P. levii following incubation with or without anti-P. levii serum or purified IgG. Oxidative burst was measured by use of the nitroblue tetrazolium reduction assay. RESULTS: Chemotaxis toward P. levii was not significantly different from control values at any of the tested bacterial concentrations. Phagocytosis of P. levii was approximately 10% at a 10:1 bacterium to macrophage ratio and did not change significantly over time. When higher proportions of P. levii were tested for phagocytosis, the 1,000:1 bacterium to macrophage ratio had a significant increase, compared with the 10:1 test group. Opsonization of P. levii with high-titeranti-P. levii serum or anti-P. levii IgG produced a significant increase in macrophage phagocytosis. Oxidative production significantly increased compared with control in the 1,000:1 test group only. CONCLUSIONS AND CLINICAL RELEVANCE: Porphyromonas levii may evade host detection by decreased chemotaxis, phagocytosis, and oxidative burst by macrophages. Acquired immunity may be beneficial for clearance of P. levii in foot rot lesions in cattle.     

Pasteurella haemolytica lipopolysaccharide-induced arachidonic acid release from and neutrophil adherence to bovine pulmonary artery endothelial cells

Bovine pulmonary artery endothelial cells (BPAEC) were labeled with 3H-arachidonic acid. Exposure of the labeled BPAEC to Pasteurella haemolytica lipopolysaccharide (LPS) resulted in a time- and dose-dependent release of radioactivity. The release was inhibited by 5 mM indomethacin, but inhibition was not caused by less than or equal to 500 microM indomethacin or hydrocortisone, which suggests that the release was caused primarily by a mechanism other than cyclooxygenase or phospholipase A2 metabolism of arachidonic acid. Pasteurella haemolytica LPS also caused increased adherence of bovine neutrophils to BPAEC through independent effects on both cell types. The increased adherence was inhibited by treatment of either cell type with cycloheximide or actinomycin D prior to LPS exposure, indicating that de novo protein synthesis was required in both cell types to promote the LPS-induced adherence. Lipopolysaccharide may be an important factor in neutrophil-mediated effects in pneumonic pasteurellosis by causing increased neutrophil adherence and, thus, the vascular sequestration of neutrophils. Together, these experiments provide additional evidence for the involvement of LPS in pneumonic pasteurellosis. Moreover, they provide evidence of LPS-induced endothelial activation, which could have broad ramifications in the inflammatory and immune responses of pneumonic pasteurellosis.     

Modulation of murine macrophages and T lymphocytes by lysozyme dimer

The effects of lysozyme dimer (2 and 20 microg/kg) administered i.p. once and four times to mice on the phagocytic and killing ability of peritoneal macrophages, interleukin-1 (IL-1) production by murine macrophages stimulated in vitro with lipopolisaccharide of E. coli and expression of thymocyte, splenocyte and mesenteric lymphonode cell CD3+, CD4+ and CD8+ markers were studied. It was found that lysozyme dimer administered once or four times at doses of 2 microg/kg and 20 microg/kg augments the phagocytic and killing activity of peritoneal macrophages. The strongest stimulating effect was noted after four injections of lysozyme dimer at a dose of 20 microg/kg. Moreover, lysozyme dimer is able to modulate the production of IL-1 by murine macrophages stimulated in vitro with LPS. Exposure to four doses of lysozyme dimer (20 microg/kg) enhances the synthesis and release of IL-1, but this drug administered once (2 microg/kg and 20 microg/kg) or four times (2 microg/kg) decreases IL-1 production by peritoneal macrophages. It was also found that administration of lysozyme dimer at a dose of 20 microg/kg, irrespective of the number of doses applied, increases the percentage of CD4+ thymocytes and splenocytes. Moreover, exposure to four doses of lysozyme dimer (2 and 20 microg/kg) increases the percentage of CD4+ and CD8+ mesenteric lymphonode cells.     

Secretion of interleukin-1 by bovine milk macrophages

The relative sensitivity of bovine blood monocytes and macrophages isolated from milk to lipopolysaccharide, with respect to interleukin 1 (IL-1) production, was evaluated. Addition of lipopolysaccharide (0 to 30 microgram/ml) to the culture medium resulted in increases in secreted and intracellular IL-1 activity for monocytes and milk macrophages, with maximal stimulation achieved at 30 micrograms of lipopolysaccharide/ml of medium. At this concentration of lipopolysaccharide, monocytes released 76% of the total IL-1, whereas milk macrophages released only 26% of the total IL-1 produced within the cell. Secretion of a small quantity of IL-1 was a common property of macrophages isolated from healthy and mastitic quarters. We concluded that limited secretion of IL-1 may render the milk macrophages less efficient in promoting lymphocyte activation.     

Endotoxin-induced bovine mastitis: arachidonic acid metabolites in milk and plasma and effect of flunixin meglumine

Arachidonic acid metabolites (AAM) were measured in milk and plasma during the course of acute endotoxin-induced mastitis in 12 lactating cows. Mastitis was induced by intramammary challenge exposure with 10 micrograms of Escherichia coli (026:B6) endotoxin. Endotoxin was injected into the teat cistern via the teat canal of a single randomly selected rear quarter of each cow. Concentrations of prostaglandin (PG) F2 alpha and thromboxane (Tx) B2 in fat-free unextracted milk and of 15-keto-13,14-dihydro-PGF2 alpha in plasma were measured by radioimmunoassay. Total production of AAM in milk was determined by measuring quarter milk production. The AAM were compared in 6 cows administered flunixin meglumine (1.1 mg/kg of body weight) and in 6 cows administered saline solution. Concentrations of TxB2 in milk were significantly (P less than 0.001) increased during the early course of acute mastitis in endotoxin-treated quarters of cows not administered flunixin meglumine. Peak concentrations of TxB2 in milk occurred at 8 hours after endotoxin inoculation. Flunixin meglumine treatment produced significant (P less than 0.05) reductions in milk TxB2 and plasma 15-keto-13,14-dihydro-PGF2 alpha concentrations. Concentrations of PGF2 alpha in milk and total PGF2 alpha and TxB2 production per quarter per milking were not significantly influenced by endotoxin challenge or by flunixin meglumine treatment.     

利用被孢霉发酵生产油脂和花生四烯酸

本文研究了被孢霉液体发酵过程中菌体形态及油脂积累、油脂成分的变化。结果表明:1)在摇瓶培养时其菌体形态与接种方式和接种量有关。采用孢子接种,并使孢子量不低于4.0×105个/mL时,可以得到菌体的最佳生长形态即短而多分枝状。2)其油脂积累发生在菌体快速生长期,培养第5天达到最高,在随后的静止期油脂被利用,且花生四烯酸的积累与油脂积累同步。油脂中的脂肪酸含量随培养时间而变化,棕榈酸、油酸的变化规律基本一致,花生四烯酸的变化规律与之相反。     

Chemiluminescence of bovine alveolar macrophages as an indicator of developing immunity in calves vaccinated with aromatic-dependent Salmonella

Chemiluminescence of bovine alveolar macrophages was used to study the development of opsonins in calves vaccinated parenterally with live aromatic-dependent strains of either S. dublin or S. typhimurium. These calves responded by producing Salmonella-specific opsonins detected by increased chemiluminescent responses, and were able to survive oral challenge with live virulent organisms of either serotype. Non-vaccinated calves of the same age lacked Salmonella-specific opsonins and were not able to survive challenge. Thus it was concluded that the ability to produce opsonins is among the immunological responses that are associated with protection against salmonellosis in calves. Antigenic similarities between S. dublin and S. typhimurium were shown by the ability of either organism to absorb significant amounts of opsonic capacity from the sera of calves vaccinated with either of the two vaccines. These antigenic similarities are thought to explain in part the ability of either vaccine to protect against challenge with either the homologous or heterologous Salmonella serotype.     

Generation of neutrophil chemoattractants by phagocytosing bovine mammary macrophages

Macrophages from bovine mammary gland were cultured in vitro and the growth medium collected at intervals. Using an in vitro system in which neutrophils migrated under agarose, both chemotactic and chemokinetic activity for bovine neutrophils was detected in supernatants of macrophage cultures to which heat killed preopsonised Staphylococcus aureus had been added. The suspensions of killed bacteria were not themselves chemotactic for neutrophils and no chemotactic activity was present either in supernatants from unstimulated macrophage cultures or in sonicated macrophages. The chemotaxin(s) was generated within two hours of the addition of staphylococci to the cultures and was largely stable to heating at 56 degrees C for 45 minutes, although its activity was reduced by boiling for 15 minutes. Traces of proteolytic activity were also detected in some supernatants. Substantial proteolytic activity was found in lysates of neutrophils. Unlike chemotaxis, proteolytic activity was suppressed by addition of milk from early lactation and containing high natural levels of protease inhibitors. Proteolytic activity was destroyed by boiling for 15 minutes.     

Pasteurella haemolytica A1 purified capsular polysaccharide does not stimulate interleukin-1 and tumor necrosis factor release by bovine monocytes and alveolar macrophages

Purified capsular polysaccharide (CPS) stimulated significant release of interleukin-1 (IL-1) activity from bovine blood monocytes but not alveolar macrophages in vitro. The ability of CPS to induce IL-1 release was resistant to boiling and inhibited by the addition of polymyxin beta. Thus, it is likely that the IL-1 release stimulated by CPS resulted from the small amount of contaminating lipopolysaccharide (LPS) that was present (an estimated 5 pg LPS per microgram CPS) rather than to a direct effect of CPS. Tumor necrosis factor activity was not detected in the culture supernatants of bovine monocytes incubated with purified CPS for 1-18 h in vitro.