Methods for Evaluation of the Adhesive and Phagocytic Capacities of Porcine Granulocytes (original) (raw)
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The effects of chemotactic factors on the adhesiveness of rabbit neutrophil granulocytes
Experimental Cell Research, 1979
A range of chemotactic factors has been shown to affect the adhesion of rabbit peritoneal neutrophil granulocytes to cultured endothelial cells and to serum-coated glass. At chemotactically optimal concentrations, cYs-casein, p-casein, alkali denatured human serum albumin (HSA) and several synthetic formyl-peptides reduced the number of adherent neutrophils after 30 min to around 50% of control values. These effects were still observed after neutrophils, but not endothelium or serum-coated glass had been exposed to chemotactic factors and washed before use in assays. Two non-chemotactic analogues, native HSA and a non-formyl-peptide were ineffective. The dose responses for adhesion after 30 min in the presence of Lu,-casein and formyl-methionylleucyl-phenylalanine (FMLP) were found to be inversely related to those for migration towards these substances. After incubation for 60 min in high (10-8-10-7 M) concentrations of FMLP, neutrophil adhesion was found to be enhanced. Neutrophil aggregation was also affected by the presence of chemotactic factors in a similar time-and dose-dependent manner to the adhesion to substratum assays. Using FMLP, it was also shown that the timing of the adhesive changes depended on the concentration of chemotactic factor present.
Flow-cytometric Studies of the Phagocytic Capacities of Equine Neutrophils
Acta Veterinaria Scandinavica, 1995
F low-cytometr ic studies oft he phagocytic capacities of equine neutrophils. Acta vet. scand. 1995, 36, 553-562.-Methodological aspects of flow-cytometric evaluation of the phagocytic properties of equine neutrophils were elucidated. The kinetics of attachment and ingestion were studied, and the phagocytic process was more rapidly completed when serumopsonized yeast cells were used than with use ofigG-opsonized yeast cells. Trypan blue was successfully used to quench fluorescence of non-ingested yeast cells. There were only minor differences in the kinetics of phagocytosis between quenched and unquenched samples, indicating that attachment is rapidly followed by ingestion. Trypan blue quenching caused loss of cells with light scattering properties of granulocytes, although this did not affect the determined frequencies of truly phagocytic neutrophils. Aggregation of yeast cells proved to be a disturbance but not an obstacle to the determination of frequencies of actively phagocytic cells. Flow cytometry is well suited for studies of phagocytosis of yeast cells by equine neutrophils, and the trypan blue quenching provides a means of eliminating false-positive events due to aggregation of yeast cells. The main advantage of the tlow-cytometric method is the possibility ofrapid processing of a large number of samples, making the method useful for studies of herds.
Journal of Clinical Investigation, 1972
A B S T R A C T Polymorphonuclear leukocytes suspended in Krebs-Ringer phosphate medium ingest paraffin oil containing Oil Red 0 emulsified with a variety of substances. Spectrophotometric determination of Oil Red O in the cells after uningested particles have been removed by differential centrifugation provides a quantitative measure of phagocytosis. This system has been used to investigate the effects of several drugs and hormones on the initial rate of phagocytosis and to approach the question of how the surface of a particle influences its acceptability as a substrate for phagocytosis. The rate of uptake of paraffin oil emulsified with bovine albumin was constant for 6 min and was proportional to cell concentration when saturating concentrations of paraffin oil emulsion were used. At lower concentrations of substrate, the initial rate of phagocytosis was directly proportional to paraffin oil concentration. The increment in glucose oxidation associated with phagocytosis varied directly with the initial rate of particle uptake. The rate of ingestion of the albumin emulsion was not altered by serum (2-20%, v/v), glucose (5-20 mM), or omission of potassium from the medium. The rate of phagocytosis was decreased 65% if magnesium was omitted, and was essentially zero in the absence of divalent cations. The initial rate of uptake was inhibited by inhibitors of glycolysis, by Nethylmaleimide (0.05-1 mM), colchicine (0.001-0.1 mM), theophylline (1 and 2 mM), dibutyryl cyclic AMP (1 mM), hydrocortisone (2.1 mM), and ethanol (85 mM). Inhibitors of oxidative phosphorylation and dexamethasone (0.01 mM) were without effect, while insulin (2 mU/ml) slightly stimulated the phagocytic rate. Paraffin oil emulsified with different agents was used to approach the question of how the surface of a particle influences its acceptability as a substrate for phagocytosis. Emulsions prepared with nonionic detergents, methylated proteins, and proteins with a weak net charge at pH 7.4 were poorly ingested. On the other hand emulsions prepared with agents of strong net positive or negative charge were rapidly taken up. The effect of divalent cations on the rate of phagocytosis varied with the nature of the emulsifier, but was not related in any simple, direct fashion to the net surface charge of the particles. However, it has not been conclusively established that charge was the only variable of the emulsion particles employed.
Evaluation In Vitro of Canine Neutrophil Function
The Veterinary Journal, 2001
Polymorphonuclear granulocyte (PMN) phagocytosis may be affected by many pathological changes. A panel of tests requiring relatively small volumes of blood was applied to 16 healthy dogs in order to obtain normal values and to standardize techniques. PMNs were isolated by discontinuous Percoll gradients; chemotaxis was tested in a modified Boyden chamber using the leading front method; fluorescinated yeast uptake was evaluated on a slide and superoxide (SO) production and adherence was carried out on a microtitre plate. The different aspects of phagocytosis showed no correlation with one another. Better results were obtained using a 60 min incubation period using interleukin-8 (25 ng/mL) as an activator for chemotaxis, and incubating plates for 30 min with phorbol myristate acetate (10-6 mol/L) to assess SO production.
Inflammation, 1983
Studies were undertaken to determine functional properties of neutrophils attracted into involuted mammary glands of Staphylococcus aureus-immunized ewes by soluble antigens prepared from S. aureus (homologous antigen) or from Bacillus cereus (heterologous antigen). The ewes were immunized with an S. aureus vaccine known to stimulate synthesis of cytophilie IgG2 antibody, and the inflammatory responses were elicited 4-8 weeks later by infusing homologous antigen into one gland and heterologous antigen into the other. Inflammatory cells were collected at 2, 4, and 8 h postinfusion. The magnitude of the cellular responses was similar in both glands with high proportions of viable neutrophils. There were no significant differences between neutrophil populations from each gland for proportions of cells bearing cytophilic immunoglobulin, although the proportions of cytophitic immunoglobulin-positive cells from both glands were lower in 2-h exudates than in 4-h or 8-h exudates. In invitro phagocytosis assays using 3H-labeled S. aureus and B. cereus no differences could be detected between the two populations of neutrophils in terms of phagocytic efficacy using a range of bacteria-neutrophil ratios and various opsonizing treatments.
Characterisation of mononuclear phagocytes in sheep
1995
CHAPTER ONE: Introduction 1.1 Mononuclear Phagocytes 12 Origin of Mononuclear Phagocytes 1.3 Classification and Heterogeneity of Macrophages 1.4 Morphology of Mononuclear Phagocytes 1.5 Cytochemistry of Mononuclear Phagocytes 1.6 Mononuclear Phagocyte Functions 1.6.1 Phagocytosis and Microbicidal Activity 1.6.2 Cytotoxic Function 1.6.3 Cytokine Production 1.6.4 Antigen Processing and Presentation Processing for MHC Class I-Restricted Antigen Presentation Processing for MHC Qass II Restricted Antigen Presentation 1.7 Mononuclear Phagocyte Surface Antigens 1.8 CD14 1.8.1 Structural Features of CD14 1.8.2 Distribution of CD 14 1.8.3 Regulation of CD 14 Expression 1.8.4 Functions of CD 14 1.8.5 Signal Transduction via CD 14 1.9 Fc Receptors for IgG 1.9.1 Structural Features of FcyR 1.9.2 Cellular Distribution of FcyR FcyRI FcyRII FctRIII Page No.
Journal of Comparative Pathology, 1991
Antibody-induced damage to neutrophils was studied to elucidate processes associated with destruction of neutrophils in immune-mediated neutropenias. Cytomorphological changes and release of certain cellular constituents were determined for neutrophils treated with an antineutrophil serum in the presence or absence of rabbit complement. Neutrophils exposed to the antineutrophil serum alone showed endocytofic vacuoles and degranulation. In contrast, neutrophils exposed to the antineutrophil serum and complement showed marked morphologic changes. The plasma membrane developed numerous vesicles, villous processes and minute areas ofbilayer discontinuity. Highly damaged cells exhibited cellular and nuclear swellings, disruption of cytoplasmic integrity and disordered distribution of lysosomal granules. Cytoplasmic constituents (K + and lactate dehydrogenase) were released extracellularly from neutrophils exposed to the antineutrophil serum with or without complement. Cytological changes induced by the antineutrophil serum and complement were analogous to those reported for leucocytes exposed to the activated complement components C5b-9 (the membrane attack complex) and bacterial toxins. It was concluded that the cytological abnormalities observed were most probably associated with immunemediated damage to the cell membrane, leading to leakage of cytoplasmic constituents like K +, colloidal osmotic swelling, and disruption of the cytoskeletal system.
Canadian journal of veterinary research = Revue canadienne de recherche vétérinaire
The ability of bovine polymorphonuclear leukocytes (PMN) to phagocytose fluorescent beads in vitro was studied using flow cytometry. The effects of varying laboratory conditions (bead:PMN ratio, length of incubation, and temperature) were first determined, then the effects of lipopolysaccharide (LPS), phorbol myristate acetate (PMA), cytochalasin B, and formyl-met-leu-phe (fMLP) on phagocytosis were evaluated. The recommended bead:PMN ratio, incubation period, and incubation temperature are 20:1, 30 min, and 38.5 degrees C, respectively. Lipopolysaccharide increased phagocytosis at a relatively high minimum dose; PMA increased phagocytosis even at low doses; cytochalasin B increased and decreased phagocytosis at low and high doses, respectively; and fMLP had no significant effect on phagocytosis. Also, the effects of ethylene diamine tetraacetic acid (EDTA) and acid citrate dextrose (ACD) as anticoagulants were compared with heparin-treated blood PMNs. Both EDTA and ACD decreased ph...
The Journal of Cell Biology, 1986
The effect of human serum and some of its components on the process of transepithelial migration of human neutrophils was investigated in an in vitro system. 10% autologous serum caused an increase in neutrophil adherence to and migration across canine kidney epithelial cells. This increase in neutrophil binding also occurred if the epithelium but not the neutrophils had been preincubated with serum. The binding was lost if the serum was either preabsorbed over the kidney epithelium before use or heat inactivated. Indirect immunofluorescence studies indicated that IgG, IgM, and a component of C3 bound to the epithelial surface, whereas IgA, IgE, or C5a were not detectable. The majority of epithelial cells were immunofluorescent, however epithelial cells with varying degrees of reactivity were also apparent and approximately 5% of the epithelial cells did not bind IgG, IgM, and C3. When epithelia were simultaneously tested for the presence of either IgG, IgM, or C3, and bound neutrop...