Isolation and chemiluminescent properties of ferret (Mustela putorius furo) polymorphonuclear cells

Ferret polymorphonuclear cells (PMNs) and peripheral blood mononuclear cells (PBMCs) were separated from whole blood by density gradient centrifugation. Using a 50% Percoll solution (density=1.066), PMNs and PBMCs were successfully isolated after centrifugation; the purities of the PMNs and PBMCs were 94.2% and 95.6%, respectively. To evaluate the function of isolated ferret PMNs, we measured the superoxide generation with a MCLA-dependent chemiluminescence assay. The isolated ferret PMNs responded to phorbol 12-myristate 13-acetate (PMA) with kinetics similar to that of human PMNs. The ferret PMNs did not respond to N-formyl-Met-Leu-Phe (fMLF), unlike human PMNs, which rapidly responded. Thus, authors established a method for the rapid separation of highly purified populations of functional PMNs from the whole blood of ferrets.     

Characteristics of the response of ovine granulocytes (PMNs) to zymosan-activated serum (ZAS) and to recombinant human interleukin-8 (IL-8)

The chemotactic activity of zymosan-activated serum (ZAS) and of two concentrations of recombinant human IL-8 (IL-8(25), 25 ng/ml; IL-8(50), 50 ng/ml) for ovine polymorphonuclear granulocytes (PMNs) was tested in a modified Boyden chamber. Thick cellulose acetate filters and the leading front method were used to quantify the movements of the cells. Both ZAS and IL-8(25) exerted a chemotactic effect on ovine PMNs (P < 0.01): IL-8(50) induced a more homogeneous response (P < 0.001). To verify the characteristics of the responsiveness to the chemokines after short-term (st) or long-term (lt) repeated samplings, chemotaxis was investigated 1 (T1st), 2 (T2st), 24 (T3st) and 48 h (T4st) after the basal sampling (T0st) and 15 days (T1lt) after the basal sampling (T0lt). No differences in chemotaxis were found in long-term repeated samplings. In contrast an increase in the responsiveness to IL-8(25) and to IL-8(50) (P < 0.05) was detected at T2st in comparison with T0st. Furthermore, the significance of the distance run by activated PMNs compared with the controls, increased from T0st to T2st, as a sign of a more homogeneous response to the chemokines. In the absence of evident changes in circulating leucocyte numbers and in serum cortisol concentrations, these findings could be interpreted as a consequence of a different expression of chemoattractant receptors on the membrane of PMNs collected at different times.     

Fucoidan directly regulates the chemotaxis of canine peripheral blood polymorphonuclear cells by activating F-actin polymerization

Fucoidan was recently shown to enhance innate immune functions. The objective of this study was to examine the direct stimulatory effect of fucoidan on the chemotactic activity of canine peripheral blood polymorphonuclear cells (PMNs). The chemotactic activity of PMNs was evaluated in a modified Boyden chamber assay and total cellular filamentous (F)-actin levels were measured using a flow cytometer. The chemotactic response of PMNs was increased by exposure to recombinant canine (rc) interleukin (IL)-8. In vitro treatment with fucoidan increased the chemotactic activity of PMNs in response to rcIL-8 compared with that of untreated PMNs, and also stimulated total cellular F-actin polymerization. The increased chemotactic activity of fucoidan-treated PMNs was suppressed by cytochalasin D, an inhibitor of F-actin polymerization. These results suggest that fucoidan directly regulates PMN chemotaxis, and that this effect is associated with an increase in actin polymerization.     

A preliminary study of uterine derived polymorphonuclear cell function in mares with chronic uterine infections

From 6 mares with chronic uterine infection, polymorphonuclear neutrophils (PMNs) were obtained from the uterus. In order to recover an adequate number of viable PMNs, 0.1% oyster glycogen was infused into the uterus as a mild irritant 12 h prior to the uterine flushing. Phagocytosis and chemotaxis of the uterine derived PMNs were determined. The supernatant from the uterine flushing was compared with autologous serum for its capacity as an opsonin and chemoattractant. There was a significant increase of both phagocytosis and chemotaxis when autologous serum was used compared with the supernatant from the uterine flushing. This study indicates that autologous serum has a greater opsonizing capacity than uterine secretion from mares with chronic uterine infection. Since all mares conceived following this study, the use of oyster glycogen was not considered to have deleterious effect on the uterine mucosa.     

Expression and role of adhesion molecule CD18 on bovine neutrophils.

Expression of CD18 on bovine neutrophils in response to stimulation by zymosan activated serum (ZAS) and phorbol myristate acetate (PMA) and the effects of monoclonal antibodies (MAB) recognizing CD18 or bovine neutrophil surface antigens (S2G8 and S5F8G10) on adherence, chemotactic responses and phagocytosis of bovine neutrophils were evaluated. CD18 expression of neutrophils was increased after ZAS and PMA treatment by 12.2 and 54.2% respectively, and were significantly (p < 0.05, p < 0.01) different from those of untreated neutrophils. CD18 expression by neutrophils from a Holstein-Friesian heifer affected with leukocyte adhesion deficiency was within negative controls when stimulated by ZAS and PMA. Adherence, chemotactic responses, and phagocytosis were significantly decreased (p < 0.01) in neutrophils continuously treated with anti-CD18 MAB (MHM 23). Adherence was also significantly decreased in anti-CD18 pretreated neutrophils. Significant (p < 0.01) differences of chemotactic responses and phagocytosis of neutrophils were found between neutrophils pretreated and continuously treated with anti-CD18 MAB (MHM 23). Monoclonal antibodies to other surface antigens did not significantly alter neutrophil adherence, chemotaxis or phagocytosis. This study demonstrated that CD18 expression on bovine neutrophils is increased significantly by stimulation with ZAS and PMA and that the adhesion molecule CD18 plays an important role in adhesion-related functions.     

Trans-10, cis-12 conjugated linoleic acid directly enhances the chemotactic activity of porcine peripheral blood polymorphonuclear neutrophillic leukocytes by activating F-actin polymerization in vitro

Trans-10, cis-12 conjugated linoleic acid (t10c12-CLA) can reportedly alter the immune responses of phagocytes; however, it is unknown whether t10c12-CLA has a direct effect on the chemotaxis of peripheral blood polymorphonuclear neutrophillic leukocytes (PMNs). Here, we examined the effect of t10c12-CLA on the chemotaxis of porcine PMNs. The chemotactic response of porcine naïve PMNs was increased by porcine recombinant (pr) interleukin (IL)-8. Treatment with t10c12-CLA increased the chemotactic activity of porcine PMNs to IL-8 compared to porcine naïve PMNs, and enhanced their total cellular F-actin level. This increased chemotactic activity of t10c12-CLA-treated porcine PMNs was inhibited by cytochalasin D, an F-actin polymerization inhibitor. These results suggest that t10c12-CLA directly upregulates the chemotaxis of porcine PMNs, and that this effect may be associated with increased actin polymerization.     

Maturation of cellular defence in the respiratory tract of young calves

The maturation of respiratory tract defence was investigated in a longitudinal study of calves during the first 100 days of life. From day 7, the proportions of the cell types identified in bronchoalveolar lavage (BAL) fluid were similar to those found in adults, with a predominance of alveolar macrophages over polymorphonuclear neutrophils (PMNs) and lymphocytes. Functionally, bactericidal activity of BAL cells was defective and for the first 21 days they supported intracellular bacterial growth. At 24 hours of life, the movement of peripheral blood neutrophils to a chemotactic source was poor, but this increased rapidly during the first week of life. Like BAL cells, peripheral blood PMNs supported intracellular bacterial growth for the first two weeks of life. These studies suggest that cellular defence mechanisms may be compromised during the first week of life.     

Streptococcal products and leukocyte activities.

Various streptococcal species are directly responsible for udder infections which should normally be countered by polymorphonuclear neutrophils (PMNs). In order to detect a putative inhibition of streptococcal products on the activities of bovine PMNs, we used a combination of four tests which permits an adequate evaluation of PMNs functions, e.g. PMN adherence on endothelial cells, chemotactic assay, phagocytosis of bacteria labelled with fluorescein isothiocyanate (FITC) and measurement of anion superoxide production. The conclusion is that neither of the two pathogenic streptococcal species isolated from mastitis appeared to produce in vitro factors affecting PMN activities.     

肝素调控猪嗜中性粒细胞对精子的趋化和吞噬作用

为降低由人工授精诱导的猪子宫局部免疫反应,从而提高其人工授精效率,在含有10%血清的培养液中添加不同浓度的肝素(0、100、500、1000μg/mL),检测猪嗜中性粒细胞(PMNs)对猪精子的吞噬性和趋化性。结果表明,100~1000μg/mL的肝素可显著抑制猪PMNs对精子的吞噬性(P<0.01)和趋化性(P<0.05)。提示肝素可作为猪精液稀释液的保护成分,降低由人工授精所导致的子宫炎症反应,从而提高猪的人工授精受胎率。     

Increase in apoptotic polymorphonuclear neutrophils in peripheral blood after intramammary infusion of Escherichia coli lipopolysaccharide

A transient increase in apoptotic polymorphonuclear neutrophils (PMNs) as revealed by the terminal deoxynucleotidyl, transferase-mediated dUTP nick end labeling (TUNEL) technique in bovine jugular and milk vein blood was observed 4 h after intramammary infusion of Escherichia coli lipopolysaccharide (LPS) (jugular vein; before infusion 10.1%, 4h 58.3%: milk vein; before infusion 13.2%, 4 h 76.6%) decrease in PMA-induced oxidative bursts of PMNs was also observed during the same period and continued until 8 h after the infusion. TUNEL-positive cells showed an intention of a Comet tail as detected by a single-cell gel electrophoresis assay (Comet assay) and the morphological apoptotic future, though DNA fragmentation was not clearly detected. A definite decrease in peripheral PMNs and a marked increase in PMNs in the LPS-infused teat cistern were observed during the same period. The migration of milk vein blood-derived PMN and the expression of adhesion receptors (L-selectin and CD18) on PMN were suppressed, accompanied by an increase in apoptotic cells. TUNEL-positive PMN observed in normal animals showed a reduced migration capacity. The increase in apoptotic PMNs observed in the LPS-infused cattle was thought to be due to the remaining intravenous spontaneous apoptotic cells existing under the normal condition (the aging cell), and this increase appeared to lower the expression of adhesion receptors and the migration capacity. Decreased PMA-induced oxidative burst activity in PMN was thought to be derived from these aging cells and immature band cells appearing in the circulation as a subsequent event of leukopenia and/or severe stress associated with mastitis. The results from the present study indicate the possibility that the function of PMN in the circulation at early stages of bovine mastitis is regulated by the kinetics of PMN aging.     

Chemotactic response of equine polymorphonuclear leucocytes to Streptococcus equi

Streptococcus equi infection in horses is characterised by intense infiltration of lymph nodes by polymorphonuclear leucocytes (PMNs) suggesting a potent chemotactic response to the organism or its products. Equine PMNs were separated using Ficoll-Hypaque medium and used in an assay of chemotaxis under agarose to study the components of S equi involved in this response. Results showed that complement-derived chemotactic factors generated by activation of the alternative complement pathway were important in chemotactic responses to S equi. Both whole bacteria and peptidoglycan preparations were potent complement activators, whereas purified M protein was less active. In contrast, S equi culture supernatant protein did not activate complement; instead it directly inhibited migration of PMNs. Moreover, PMNs, when incubated with culture supernatant of a non-haemolytic strain, showed signs of cellular degeneration suggesting the presence of a cytotoxin distinct from haemolysin.     

In vitro study of neutrophil apoptosis in dogs

In the present study, to investigate the apoptosis of the polymorphonuclear neutrophil (PMN) from healthy dogs, we carried out TUNEL assay and DNA analysis by electrophoresis on dog PMNs. The TUNEL assay indicated that apoptotic PMNs in dogs were 0.15+/-5% before incubation, 0.3+/-5% at 4h incubation, 1+/-6% at 8h, 9+/-4% at 12h and 28+/-5% at 24h, respectively. The ladder formation was much more clearly observed in DNA from PMNs after 24h incubation at 37 degrees C than that before incubation. The results in this study indicated that healthy dog PMNs undergo apoptosis spontaneously within hours to days, and that the apoptosis of PMNs might be related to the high turnover of these circulating cells in dogs.     

Bovine herpesvirus type 1 infection of bovine bronchial epithelial cells increases neutrophil adhesion and activation

Respiratory infection of cattle with bovine herpesvirus type 1 (BHV-1) predisposes cattle to secondary pneumonia with Mannheimia haemolytica as part of the bovine respiratory disease complex (BRD). One cell type that has received limited investigation for its role in the inflammation that accompanies BRD is the respiratory epithelial cell. In the present study we investigated mechanisms by which BHV-1 infection of respiratory epithelial cells contributes to the recruitment and activation of bovine polymorphonuclear neutrophils (PMNs) in vitro. Primary cultures of bovine bronchial epithelial (BBE) cells were infected with BHV-1 and assessed for cytokine expression by real-time PCR. We found that BHV-1 infection elicits a rapid IL-1, IL-8 and TNF-α mRNA response by BBE cells. Bovine PMNs exhibited greater adherence to BHV-1 infected BBE cells than uninfected cells. The increased adherence was significantly reduced by the addition of an anti-IL-1β antibody or human soluble TNF-α receptor (sTNF-αR). Pre-incubation of bovine PMNs with conditioned media from BHV-1 infected BBE cells increased PMN migration, which was inhibited by addition of an anti-IL-1β antibody, sTNF-αR, or an IL-8 peptide inhibitor. Conditioned media from BHV-1 infected BBE cells activated bovine PMNs in vitro as demonstrated by PMN shape change, production of reactive oxygen species and degranulation. PMNs also exhibited increased LFA-1 expression and susceptibility to M. haemolytica LKT following incubation with BHV-1 infected BBE cell conditioned media. Our results suggest that BHV-1 infection of BBE cells triggers cytokine expression that contributes to the recruitment and activation of neutrophils, and amplifies the detrimental effects of M. haemolytica LKT.     

Evaluation of the polymorphonuclear cell functions of bottlenose dolphins

The functions of polymorphonuclear cells (PMN) are the important non-specific defense mechanisms in the immune system. Especially marine mammals are protected by these mechanisms from the aquatic environment with a large variety of microorganisms. Therefore, we examined the PMN functions of bottlenose dolphins in order to obtain the normal ranges and to standardize the techniques. PMNs were isolated by using lymphocyte isolate solution whose density was 1.077; superoxide production was assessed by nitroblue tetrazolium reduction test (NBT) and phagocytosis was tested by using polystyrene latex beads. We showed that the optimal incubation time was 30 min in NBT assay and 12 hr in phagocytosis assay for dolphin PMNs.     

Effects of nitric oxide on bovine polymorphonuclear functions.

The effects of nitric oxide (NO) on the functionality of polymorphonuclear neutrophils (PMNs) in bovine milk or blood were investigated. In 2 experiments, mastitis was induced by infusing both hind quarters with saline containing Escherichia coli endotoxins. In addition, the left hind quarter was infused with aminoguanidine, an inhibitor of the inducible form of NO synthase (iNOS). At various times after infusion, somatic cells were isolated from milk samples, and superoxide (O2-) production induced by phorbol myristate acetate was evaluated. In both experiments, the addition of aminoguanidine had no inhibitory effect on the number of milk somatic cells or on their O2- production. The effect of NO and iNOS inhibitors on the functionality of bovine PMNs isolated from blood was investigated in vitro. The neutrophils did not produce NO. A neutrophil:monocyte co-culture system was used to study the effect of NO derived from monocytes on O2- production by bovine neutrophils. Neither NO derived from activated monocytes nor the iNOS inhibitors aminoguanidine and L-N6-(1-iminoethyl)lysine had an effect on the ability of bovine neutrophils to release O2-. Moreover, aminoguanidine did not affect the ability of bovine neutrophils to phagocytose bacteria. These results suggest that inhibition of NO release during inflammation does not interfere with the migration of immune cells to the site of infection or the ability of these cells to destroy pathogens. Thus, NO does not appear to play a major role in the control of the functions of bovine neutrophils.     

Effects of human IL-8 isoforms on bovine neutrophil function in vitro

Interleukin 8 (IL-8) is a potent chemotactic and activating agent for human neutrophils and bovine IL-8 is chemotactic for bovine neutrophils; however, it is unclear whether IL-8 activates bovine neutrophils. Two isoforms of human recombinant (hr) IL-8 protein (77 and 72 amino acid) were used to stimulate bovine neutrophils in vitro. Bovine neutrophils exhibited significant migration in the presence of 0.1, 0.5, 1.0 and 5.0ngml(-1) hr IL-8 when incubated for 30min at 37 degrees C in a modified Boyden chamber assay. Both the 77 and 72 aa forms were equally effective in inducing migration in this assay. At the highest doses of IL-8 examined (1 and 5ngml(-1)), migration was similar to migration in the presence of 20% zymosan-activated serum (ZAS) or 12h lipopolysaccharide (LPS)-stimulated blood monocyte supernatants (CM). Significant (p<0. 05) release of alkaline phosphatase (ALK-P) (from specific granules) occurred but myeloperoxidase (MPO) release and superoxide anion production were not enhanced in bovine neutrophils by either form of hrIL-8 at any of the doses tested. Significant (p<0.05) alkaline phosphatase release was observed in the presence of 10 and 100ngml(-1) for the 72 aa form of IL-8 and only at the higher dose for the 77 aa form of IL-8. The ZAS and CM significantly enhanced neutrophil degranulation of ALK-P and MPO as well as inducing superoxide anion production. These results suggest that IL-8 may play a role in both neutrophil recruitment and activation during bovine inflammatory processes.     

An Acute-phase Protein as a Regulator of Sperm Survival in the Bovine Oviduct: Alpha 1-acid-glycoprotein Impairs Neutrophil Phagocytosis of Sperm In Vitro

We have previously shown that polymorphonuclear neutrophils (PMNs) are present in bovine oviduct fluid under physiological conditions, and that the oviduct provides a microenvironment that protects sperm from phagocytosis by PMNs. Alpha 1-acid glycoprotein (AGP) is a major acute-phase protein produced mainly in the liver that has immunomodulatory functions. AGP mRNA is expressed in extrahepatic organs, such as the lung, kidney, spleen, lymph node, uterus, and ovary. Therefore, in this study, we investigated, 1) the local production of AGP in the bovine oviduct, 2) the effect of AGP on the phagocytic activity of PMNs for sperm and superoxide production and 3) the impact of AGP desialylation on the PMN phagocytosis of sperm. The AGP gene was expressed in cultured bovine oviduct epithelial cells (BOECs) and AGP protein was detected in oviduct fluid. Preexposure of PMNs to AGP at physiological levels impaired PMN phagocytosis for sperm and superoxide generation. The desialylation of AGP eliminated these suppressive effects of AGP on PMN. Scanning electron microscopy revealed that AGP drastically reduced the formation of DNA-based neutrophil extracellular traps (NETs) for sperm entanglement. Additionally, AGP dose-dependently stimulated BOECs to produce prostaglandin E₂ (PGE₂) which has been shown to partially contribute to the regulation of sperm phagocytosis in the bovine oviduct. AGP and PGE₂ at concentrations detected in the oviducts additively suppressed sperm phagocytosis by PMNs. These results provide evidence that locally produced AGP may be involved in protecting sperm from phagocytosis by PMNs in the bovine oviduct.     

Effect of antimicrobial agents and corticosteroids on bovine polymorphonuclear leukocyte chemotaxis

The effect of certain antimicrobial agents and corticosteroids on bovine polymorphonuclear leukocyte chemotaxis was investigated. Peripheral blood was fractioned by density-gradient centrifugation, using Ficoll-Hypaque. The chemotactic assay was performed in modified Boyden chambers, using Micropore filters, and the chemotactic response was measured by the leading-front technique. Tetracyclines, streptomycin, and penicillin had no effect on chemotaxis at concentrations normally achieved in blood during systemic treatment. However, higher concentrations that were achievable with local therapy, such as intramammary injection or topical application, inhibited the chemotactic response. This inhibition was eliminated by serum. Dexamethasone stimulated polymorphonuclear leukocyte chemotaxis with the effect being manifested after the cells were incubated with the drug for 3 hours. Hydrocortisone caused slight inhibition of chemotaxis, whereas prednisone and prednisolone had no effect.     

动物子宫局部免疫系统及孕体识别

介绍了动物子宫局部免疫系统在不同生理阶段的状况和子宫识别孕体的机理.健康、未孕的动物子宫内最主要的白细胞亚型是T淋巴细胞、巨噬细胞和中性粒细胞,这些细胞的迁移和分布明显受发情周期的影响.配种引起多形态嗜中性白细胞(PMNs)流入子宫,可以消除生殖道内的病菌和多余的精子,从而可提高繁殖性能.目前,人们主要从MHCⅠ抗原在子宫的表达和生殖道内的细胞因子两方面来解释子宫识别孕体的机理.总之,动物子宫局部免疫系统是一个新兴的研究领域,在取得初步进展的同时,尚存在许多疑问和争议.     

Cell-surface lactoferrin as a marker for degranulation of specific granules in bovine neutrophils

OBJECTIVE: To develop a rapid and accurate flow cytometric method for measuring degranulation of specific granules in bovine neutrophils. SAMPLE POPULATION: Blood samples obtained from four 6- to 18-month-old Holstein cattle. PROCEDURE: A monoclonal antibody (BL97) was generated against bovine lactoferrin and tested for applicability in ELISA, immunoprecipitation tests, immunofluorescence microscopy, and flow cytometric analyses. Using this antibody, cell-surface lactoferrin was measured concurrent with amount of secreted lactoferrin from bovine neutrophils activated with phorbol myristate acetate (PMA). Cell-surface lactoferrin also was measured on neutrophils in bovine whole blood stimulated with PMA, platelet-activating factor (PAF), N-formyl-methionyl-leucyl-phenylalanine (fMLF), and interleukin 8 (IL-8). RESULTS: Antibody BL97 recognized bovine lactoferrin in ELISA and western immunoblots and was useful for immunoprecipitation testing, immunofluorescence microscopy, and flow cytometric analyses of bovine leukocytes. Neutrophils activated with PMA had parallel increases in content of secreted lactoferrin (measured by ELISA) and cell-surface lactoferrin (measured by flow cytometry) with increasing PMA concentrations. In addition, fluorescein-conjugated BL97 antibody detected increases in cell-surface lactoferrin on neutrophils in bovine whole blood after activation with PMA, PAF, and IL-8. In contrast, increases in cell-surface lactoferrin were not detected on bovine neutrophils treated with fMLF. CONCLUSION AND CLINICAL RELEVANCE: Measurement of cell-surface lactoferrin on bovine neutrophils by flow cytometry is a valid and rapid method for assessment of release of lactoferrin from specific granules in these cells and represents a means to rapidly measure neutrophil activation. This technique allows for investigation of mechanisms of neutrophil modification in isolated cells as well as in whole blood.