Effect of in vitro inoculation of bovine respiratory syncytial virus on bovine pulmonary alveolar macrophage function

Viruses may predispose the respiratory tract to the development of secondary bacterial pneumonia by impairing functions of alveolar macrophages. The effects of bovine respiratory syncytial virus (BRSV) on selected functions of bovine pulmonary alveolar macrophages (PAM) were examined in vitro. Alveolar macrophages were obtained from nonsedated cattle, using a polypropylene tube passed intranasally into the lung. The PAM lavaged from the lung were allowed to adhere to glass coverslips or plastic tissue culture plates, and were exposed to BRSV for 2 hours. Control and BRSV-inoculated PAM were compared at intervals over a 72-hour period for their abilities to phagocytize and kill Staphylococcus epidermidis, rosette with and phagocytize antibody-coated sheep RBC (SRBC), phagocytize latex particles, and influence lysosomal enzyme activity. Challenge exposure with BRSV did not affect the ability of PAM to adhere and did not affect cell viability. There were numerical differences between control and BRSV-inoculated cell populations in phagocytosis and killing of S epidermidis, but these were not significant (P greater than 0.05). There was less than 5% difference in the abilities of control and BRSV-challenged PAM to phagocytize latex beads. When Fc-receptor-mediated phagocytosis of antibody-coated SRBC was compared with controls, BRSV-challenged PAM had significantly (P less than 0.05) impaired phagocytic function, which was maximal 72 hours after BRSV inoculation; the phagocytic impairment occurred in spite of normal Fc-receptor function, as determined by rosetting with antibody-coated SRBC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Patterns of alveolar macrophage activation upon attenuated and virulent African swine fever viruses in vitro

The pattern of porcine alveolar macrophage (AM) activation upon classical stimuli of two strains of African swine fever (ASF) viruses, an attenuated ASFV-BA71V and virulent ASFV-Georgia2007 were investigated. In an in vitro experiment ASFV-Georgia2007-infected AM showed M1 polarization pattern different from the one induced by classical stimuli. Altered morphology, appearance of binuclear cells, decreased synthesis of IFN-alpha as well as IFN-epsilon was observed compared with attenuated ASFV-BA71V, and decreased synthesis of IFN-omega compared with intact cells. However, CD68 level did not significantly differ between alveolar macrophage populations infected by ASFV-Georgia2007 and control group, while both LPS/IFN-gamma stimulation and non-pathogenic ASFV-BA71V virus increased the level of CD68 soluble receptor.AM infection with ASFV-Georgia2007 resulted in remarkable DNA proliferation whereas LPS/IFN-gamma and ASFV-BA71V induced less expressed DNA proliferation in activated cells. The higher value of nitric oxide was obvious in the cells infected with ASFV-BA71V, compared to ASFV-Georgia2007 and LPS/IFN-gamma activated cells.In conclusion, pattern of activation of alveolar macrophages induced by ASFV-Georgia2007 virus differs from the one expressed in LPS/IFN-gamma- and ASFV-BA71V-activated cells. ASFV-BA71V and LPS/IFN-gamma share similar antiviral response of porcine AM. Therefore we assume that wild type virulent ASFV can partially down regulate antiviral response of AM and conclude that evolutionary decrease of virulence in ASFV is related to alterations of control of the host cell antiviral response.     

Effects of Mycoplasma hyopneumoniae and Actinobacillus (Haemophilus) pleuropneumoniae infections on alveolar macrophage functions in swine

Alveolar macrophages were collected at necropsy from pigs inoculated with Mycoplasma hyopneumoniae or Actinobacillus pleuropneumoniae or both and were tested for phagocytic capabilities, using in vitro techniques. Macrophages from noninoculated littermates were used as controls. Alveolar macrophages from pigs inoculated with either M hyopneumoniae or A pleuropneumoniae had significantly (P less than 0.05 to P less than 0.0025) higher phagocytic capacity than that of noninoculated controls. Macrophages from A pleuropneumoniae-inoculated pigs were comparatively more stimulated than were those from M hyopneumoniae-inoculated pigs. Pigs inoculated with M hyopneumoniae and then challenge-exposed with A pleuropneumoniae 2 and 4 weeks later had greatly reduced phagocytosis. Infection with M hyopneumoniae or A pleuropneumoniae caused stimulation of alveolar macrophage functions, and M hyopneumoniae infections may have suppressed phagocytic responses when pigs were challenge-exposed with a secondary pathogen (A pleuropneumoniae). This potential suppression may represent a prediposition of the host by M hyopneumoniae to secondary bacterial infections.     

Phagocytic functions of pulmonary alveolar macrophages in genetically selected lean and obese swine and the effects of exogenous linolenic acid upon cell function

Alveolar macrophages from genetically selected obese and lean swine were compared for in vitro phagocytic capabilities, using Fc (gamma)- and C3-mediated phagocytosis. Cells from obese pigs were significantly more effective at Fc (gamma)-mediated phagocytosis than those from lean pigs, both for percentage of total cells phagocytosing (P less than 0.044) and for the average number of opsonized sheep erythrocytes (SRBC) ingested per phagocyte (P less than 0.045). A seasonal interaction was noted for average number of SRBC ingested per phagocyte: the relative difference in macrophage responses between obese and lean groups became significantly more pronounced during winter and spring months (P less than 0.080). Macrophages from obese pigs also exhibited higher phagocytic activities at C3-mediated phagocytosis than did cells from lean pigs, but these differences were significant only for average number of SRBC ingested per phagocyte (P less than 0.080). Exogenous linolenic acid was added to selected cultures undergoing Fc (gamma)-mediated phagocytosis. Addition of the fatty acid frequently caused enhanced phagocytosis. Macrophages from obese pigs were stimulated by fatty acid treatment more frequently than cells from lean pigs (P less than 0.05). Relatively greater enhancement was also seen in cells from obese pigs, when compared with those from lean swine (P less than 0.025). These results suggest that genetically transferred factors are of primary importance in alveolar macrophage phagocytic responses and that linolenic acid can induce increased phagocytic activity by porcine alveolar macrophages in vitro.     

Alteration of neutrophil function in BCG-treated and non-treated swine after exposure to Salmonella typhimurium.

Salmonella typhimurium infection in swine causes an enterocolitis followed by a persistent carrier state, but little is known about the mechanisms that allow this organism to colonize and persist in host tissues. Neutrophils provide a first line of defense against invading pathogens such as Salmonella typhimurium. The purpose of this study was to evaluate porcine neutrophil function after in vivo exposure to Salmonella and to determine if the immunomodulator, bacillus Calmette Guerin (BCG), exerts any effect on neutrophil function or on the colonization and persistence of S. typhimurium in the pig. Compared to negative controls, neutrophils from pigs exposed to S. typhimurium exhibited significantly decreased iodination, cytochrome-C reduction, antibody-dependent cell-mediated cytotoxicity, random migration, and chemotaxis (P less than or equal to 0.05). Neutrophil bactericidal activity against S. typhimurium was significantly enhanced. Most of the significant differences were noted in the first two days after exposure to Salmonella. Often the functional alterations were biphasic, peaking again 7-10 days after exposure. BCG alone significantly depressed random migration and cytochrome-C reduction in unstimulated neutrophils. The clinical course, colonization pattern, and persistence of Salmonella were similar between pigs receiving BCG and untreated pigs. These data suggest that S. typhimurium infection causes a depression in oxidative metabolism and motility, yet an increase in overall bactericidal activity against S. typhimurium in circulating porcine neutrophils. It also appears that BCG treatment, as reported here, does not enhance resistance of pigs to S. typhimurium colonization or reduce the number of persistent organisms in the porcine ileum.     

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.     

Actinobacillus pleuropneumoniae culture supernatants interfere with killing of Pasteurella multocida by swine pulmonary alveolar macrophages.

The effect of Actinobacillus pleuropneumoniae culture supernatant on swine pulmonary alveolar macrophage (PAM) functions was studied. The A. pleuropneumoniae culture supernatant was toxic to PAMs when tested by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and lactate dehydrogenase (LDH) release assays. Biological activity of the supernatant was ascribed to cytotoxins. Both the LDH and MTT assays were used for measurement of crude A. pleuropneumoniae cytotoxin concentration with good reproducibility. A preparation containing 6,800 toxic units/mL (determined by MTT assay) was used for subsequent experiments. The objective was to study the effect of crude cytotoxin on the ability of swine PAMs to kill Pasteurella multocida. Phagocytosis of opsonized P. multocida type A by PAMs was not efficient. Only 8% of incubated organisms were ingested by noncytotoxin-treated PAMs after 30 min phagocytosis. The bactericidal effect of noncytotoxin-treated PAMs only last for 60 min, after which, the rate of growth of surviving P. multocida exceeded the rate of bacterial killing by PAMs. Complete elimination of P. multocida by PAMs was not observed in this study. A total loss of ability to kill P. multocida by PAMs was seen when the PAMs were pretreated with a high concentration (340 toxic units/mL) of A. pleuropneumoniae cytotoxin. If the PAMs were pretreated with a low concentration (3.4 toxic units/mL) of cytotoxin, a significant reduction in the killing of P. multocida was still observed. The reductions in phagocytosis, phagosome-lysosome fusion (demonstrated using yeast particles of Candida albicans), and oxidative burst (demonstrated by nitro blue tetrazolium reduction (NBT) assay) may have contributed to the impaired killing of P. multocida by PAMs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of porcine reproductive and respiratory syndrome virus (PRRSV) on alveolar lung macrophage survival and function

Porcine reproductive and respiratory syndrome virus (PRRSV) recently emerged as an important cause of reproductive disorders and pneumonia in domestic pigs throughout the world. Acute cytocidal replication of PRRSV in alveolar lung macrophages causes the acute pneumonia; however, it remains largely unresolved whether there may also be a predisposition to longer-term local immunodeficiency in the PRRSV-convalescent lung. We applied various flow cytometric techniques to study the interplay between PRRSV replication and macrophage viability/function in pure cultures of porcine alveolar lung macrophages. Monitored by flow cytometric detection of intracellular PRRSV nucleocapsid protein, acute (24 h post infection) PRRSV replication did not impede the ability of alveolar macrophages to ingest fluorescently labelled Escherichia coli. At 48 h post infection, PRRSV-induced cytotoxicity (quantitated by flow analysis of cell size and membrane integrity) led to 40% reduction in the total number of phagocytozing cells. However, viable/uninfected macrophages in PRRSV-infected cultures exhibited normal phagocytic ability at 48 h, indicating that no soluble phagocytosis-suppressive mediators were induced by PRRSV infection in this system. In short, in our minimal system containing only a single cell type, phagocytosis-suppressive effects of PRRSV infection were detected, that acted at the culture level by reducing the total number of alveolar lung macrophages.     

PRRSV和PCV2共感染对猪肺泡巨噬细胞CD80-CD86mRNA转录的影响

用猪生殖与呼吸综合征病毒（PRRSV）与猪圆环病毒2型（PCV2）共感染40日龄健康大白仔猪，利用实时荧光定量PCR技术对共感染仔猪肺泡巨噬细胞（PAM）共刺激分子CD80一CD86的mRNA转录水平进行了定量分析。结果表明，在感染后第3d和第7dCD80与CD86mRNA转录显著下调（P〈0．05），感染后第14dCD86mRNA转录水平仍低于未感染对照组。尽管CD80mRNA转录水平在第14d和第28d高于对照组，CD86mRNA转录水平在第28d高于对照组，两者在第42d均高于对照组，但无显著差异。证实，PRRSV和PCV2共感染可导致猪肺泡巨噬细胞的共刺激分子CD80-CD86基因转录在感染早期明显受到抑制，PAM的抗原呈递能力受到影响。     

Effects of immunopotentiating agents on alveolar macrophage properties

Infectious respiratory diseases in man and in domestic animals are characterized by the presence of a large number of different microorganisms: viruses, bacterias, mycoplasmas. It is therefore necessary to stimulate non-specific defense mechanisms in the lung and especially alveolar macrophages (AM). These cells, located in the alveolar air-spaces, play a major role in the lung clearance mechanisms and exert antibacterial, antiviral and antitumoral activities. Activation of alveolar macrophages was studied in vitro with lipopolysaccharide (LPS), lymphokines or mycobacterial derivatives (MDP). Rodent alveolar macrophages were rendered cytotoxic by in vitro exposure to LPS, free MDP or liposome-encapsulated MDP derivatives. In vivo, intravenously administered liposomes containing lipophilic MDP derivatives induced cytotoxic alveolar macrophages and protected mice against the development of pulmonary metastases.     

Determination of minimum alveolar concentration of sevoflurane in juvenile swine

Pigs are important animal models in veterinary and medical research and have been widely used in experiments requiring surgical anesthesia. Sevoflurane is an inhalant anesthetic with unique properties that make it an ideal anesthetic for mask induction and anesthesia maintenance. However, there are relatively few studies reporting the anesthetic requirements for sevoflurane in juvenile swine, an age group that is commonly used in research experiments. Therefore the objective of this study was to determine the Minimum Alveolar Concentration (MAC) for sevoflurane in juvenile swine. Sevoflurane anesthesia was induced in six Yorkshire-cross pigs of approximately 9 weeks-of-age and MAC for sevoflurane was determined. The sevoflurane MAC value was determined to be 3.5+/-0.1% which is notably higher than values reported in the literature for pigs. This discrepancy in MAC values may represent changes in anesthetic requirements between different age groups of pigs and differences in the type of stimulus used to determine MAC.     

Detection of the nucleolar activity in alveolar macrophages of swine]

With the help of the toluidine blue-staining-technique different characteristic shapes of alveolar macrophage nucleoli were detected, indicating differences in the rRNA synthesis activity of these cells. After application of a Pasteurella multocida aerosol the percentage of alveolar macrophages with a high rRNA synthesis increased. No correlation between the nucleolar activity and the efficacy of the clearance of aerogenously administered Pasteurella could be measured.     

Bovine alveolar macrophage neurokinin-1 and response to substance P

In this study bovine alveolar macrophage neurokinin-1 (NK-1) and the in vitro response to substance P (SP) exposure were investigated. Bovine alveolar macrophage membrane extracts separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted using anti-NK-1 antiserum demonstrated the presence of an approximately 60kDa band. Phagocytosis of fluorescent bioparticles by SP-exposed macrophages was 39% greater than that of non-exposed macrophages (P=0.0089). Likewise, there was 28% greater TNF production by macrophages following SP exposure compared to non-exposed controls (P=0.116). These results suggest that bovine alveolar macrophages respond to SP at least in part by enhancing phagocytosis and TNF production.     

Evaluation of neutrophil function in female goats

Polymorphonuclear neutrophils (PMN) were isolated from the blood of healthy adult female goats on each of 3 consecutive days. The PMN isolated were evaluated, using random migration, chemotaxis, Staphylococcus aureus ingestion, cytochrome C reduction, iodination, and antibody-dependent cell-mediated cytotoxicity assays. Over the 3 days, mean values for each of the assays ranged as follows: area of random migration, 11.3 to 19.9 mm2; chemotactic index, 6.4 to 11; chemotactic difference, 2.8 to 4.2 mm; S aureus ingestion, 18.3% to 26.1% ingested; cytochrome C reduction, 2.7 to 3.2 nmoles of O2- produced/well; iodination, 19.4 to 25.1 nmoles of NaI/10(7) PMN/h; and antibody-dependent cell-mediated cytotoxicity 59% to 90% 51Cr released. Significant (P less than 0.05) day-to-day variations were found for all assays. Parallel increases and decreases for all test results on a per day basis indicated a common denominator influencing cell functional status rather than variability inherent in the assays themselves. Alterations induced in the cells during the cell isolation procedure were considered a probable cause.     

Assessment of neutrophil function in canine cancer patients undergoing chemotherapy and correlation with neutrophil numbers

Decreased neutrophil function following administration of chemotherapy has been reported in dogs with lymphoma. The first objective of our study was to determine if neutrophil oxidative burst and phagocytic activity are affected by chemotherapy 7 to 10 days following initiation of treatment in dogs with lymphoma and non-lymphoma malignancies. The second objective was to determine if there is a correlation between neutrophil numbers and neutrophil function before or after initiation of chemotherapy. Flow cytometric assessment of neutrophil oxidative burst and phagocytosis following stimulation with Escherichia coli was performed in 9 dogs diagnosed with lymphoma and 17 non-lymphoma tumor-bearing dogs pre- and post-chemotherapy, as well as 14 tumor-free control dogs. Spearman rank correlation was performed to determine if blood neutrophil numbers and neutrophil function were significantly correlated. Lymphoma patients showed significantly reduced percentage neutrophil oxidative burst post-chemotherapy compared to healthy controls as well as compared to pre-chemotherapy values (P = 0.0022 and P = 0.0020, respectively). Lymphoma patients also exhibited significantly reduced neutrophil phagocytosis activity post-chemotherapy compared to controls and pre-chemotherapy values (P = 0.0016 and P = 0.014, respectively). Dogs with non-lymphoma malignancies also showed a significant decrease in both percentage oxidative burst and phagocytosis post-chemotherapy compared to pre-chemotherapy values (P = 0.00040 and P = 0.029, respectively). Neutrophil numbers and function were not significantly correlated. The results of the study suggest that chemotherapeutic treatment decreases neutrophil oxidative burst and phagocytic activity 7 to 10 days post-treatment in dogs with various malignancies. Furthermore, neutrophil numbers cannot be used to predict neutrophil function.     

PRRSV与PCV2体外共感染对猪肺泡巨噬细胞免疫学功能的影响

制备猪肺泡巨噬细胞,分别接种PCV2、PRRSV、PCV2 PRRSV(先接种PCV2,12h后接种PRRSV)、PRRSV PCV2(先接种PRRSV,12h后接种PCV2)、PRRSV/PCV2(同时接种)和对照组。接种后不同时间观察细胞病变(CPE),并用real-time PCR和IFA方法检测PRRSV和PCV2滴度,INF-α、INF-γ、TNF-α、IL-8和IL-10的mRNA。结果为:①PRRSV能在PAM中增殖,有CPE;PCV2在PAM中感染率较低,无CPE。②PRRSV对PCV2增殖无明显影响。PCV2先于或同时与PRRSV感染对PRRSV的复制具有抑制作用,而PCV2后于PPRSV感染,可促进PRRSV增殖。③PCV2单独感染PAM后能促进INF-α、INF-γ、IL-8和IL-10的大量表达,对TNF-α的表达量影响不大。④PCV2与PRRSV混合感染,尤其是PCV2后于PRRSV感染后,TNF-α、IL-8和IL-10的表达量与单感染组相比明显增加,而INF-γ的表达量明显受到抑制。实验结果表明,PCV2感染时间对PRRSV的增殖影响不同,PRRSV感染后如果再感染PCV2,可以明显促进PRRSV病毒增殖;两种病毒共同刺激了TNF-α、IL-8和IL-10的大量表达,抑制了抗病毒因子INF-γ的表达,从而可能抑制体液免疫和细胞免疫,加重了病理学免疫应答。