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  • Author or Editor: Brenda Alston-Mills x
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Summary

The neutrophil plasma membrane has a major role in migration, phagocytosis, and destruction of microorganisms. Neutrophils isolated from blood and mammary secretions were homogenized, and the plasma membrane fraction was isolated on discontinuous sucrose gradient (20, 32, and 50%). Purity of plasma membrane preparation was determined by use of marker enzyme analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) of the membrane proteins was performed under reducing conditions for polypeptide characterization. The membrane proteins were also labeled with 125I externally, using 1,3,4,6-tetrachloro-3α-6α-diphenylgly-couril, and proteins were separated by sds-page and autoradiographed.

Compared with whole cell homogenate, the plasma membrane fraction obtained at the 20/32% interface was enriched for the marker enzymes, 5′-nucleotidase (16-fold), alkaline phosphatase (5.5-fold), and total phosphatase (26-fold). The membrane fraction had minimal specific activity for β-glucuronidase (0.4-fold), compared with whole cell homogenate. Plasma membrane protein yield was about 500 μg/ 109 bovine blood neutrophils. The sds-page of plasma membrane proteins revealed 25 protein bands, of which 13 were major bands. There were 3 distinct bands (18, 36, and 65 kd) in the plasma membrane-enriched fraction (20/32 interface) that were not seen in other fractions (30/50% and pellet). Further, 125I-labeling identified 5 distinct protein bands (205, 140, 65, 35, and 30 kd). Blood and mammary neutrophils had similar polypeptide patterns, except that 36- and 65-kd bands were more prominent for blood neutrophils than for mammary neutrophils.

Free access
in American Journal of Veterinary Research

Summary

Enzyme-linked immunosorbent assay and flow cytometric methods to screen hybridoma culture supernatants for antibodies to bovine neutrophils (surrace antigen-specific) were optimized. Sensitivity of the 2 methods was compared. A panel of 14 murine monoclonal antibodies (mab) to surface antigens of bovine polymorphonuclear neutrophilic leukocytes (neutrophils) was produced by hybridoma technology, and their isotypes were determined by wholecell elisa. Monoclonal antibody reactivity with neutrophils, eosinophils, and lymphocytes isolated on phosphate-buffered saline solution and on Ficoll-sodium diatrizoate were compared. Biochemical characterization of antigens recognized by mab was performed by immunoblot analysis. Neutrophil plasma membranes were isolated on sucrose gradients (20, 32, and 50%) and purified for polypeptide characterization. Neutrophil surface proteins were characterized by external labeling with 125I.

The flow cytometric method was proven to be more sensitive and rapid than elisa to screen hybridoma supernatants. This method allowed light-scatter gating of live neutrophil populations for analysis, which eliminated nonspecific binding of antibodies to contaminating cells and dead neutrophils. The optimal conditions for flow cytometric analyses were 5 × 105 neutrophils and 1 µg of fluorescein-labeled F(ab′)2/assay as the second antibody. The optimal conditions for hybridoma screening by elisa were neutrophil concentration of 2.5 × 105/well, using a 96-well polystyrene microtitration plate as solid support, and 2,2′-azino-di[3-ethyl-benzthiazoline sulfonate (6)] with H2O2 as the chromogenic substrate. Tissue culture plates as solid support and 3,3′, 5,5′- tetramethyl benzidine, with H2O2 as the chromogenic substrate, were equally as sensitive.

Panel mab reacted differently with neutrophils, eosinophils, and lymphocytes. Isolation of these cells from blood on Ficoll-sodium diatrizoate generally did not alter mab reactivity.

Coomassie blue-stained gels of neutrophil plasma membrane proteins contained about 25 polypeptide bands, 13 of which were major bands. Autoradiography revealed about 11 surface proteins, 5 of which were heavily labeled with Monoclonal antibody S7G8 identified a 65-kd protein and mab S8G10 identified 65- and 70-kd proteins. On the basis of molecular weight, mab S7G8 and S8G10 are comparable to human CD15, CDl6, and CD64 molecules. The mab generated in this study are potential candidates to discern bovine neutrophil function and heterogeneity.

Free access
in American Journal of Veterinary Research

Summary

The main objective of the study reported here was to generate a panel of monoclonal antibodies (mab) to bovine neutrophil surface antigens, and to identify mab that modulate neutrophil chemotaxis, respiratory burst, and phagocytosis. A further objective was to study mab reactivity with resting and activated neutrophils, to identify activation antigens and adhesion molecules.

A panel of 14 mab was generated by producing murine hybridomas. Neutrophils incubated with mab at 4 C for 2 hours were used in chemotaxis, respiratory burst, and phagocytosis assays. Chemotaxis was evaluated in Boyden chambers, using Escherichia coli endotoxin-activated fetal bovine serum as the chemoattractant. Respiratory burst was determined by measuring chemoluminescence of neutrophils incubated with 5-amino-2,3-dihydro-l,4-phthalazinedione, and serum opsonized zymosan. Phagocytosis was determined by flow cytometry, using fluorescein-labeled Staphylococcus aureus. The mab S7G8, S5F8G10, S7E10, and S5F8B8 enhanced chemotaxis (to > 125% of control). The mab S7E10 and S8D9 enhanced respiratory burst activity (to > 125% of control), whereas mab S2G8, S4G10, S8G10, and S5F8B8 caused inhibition (to < 75% of control). The mab S2G8, S4G10, S8G10, and S5F8G10 enhanced phagocytosis (to > 125% of control). Chemotaxis, respiratory burst, and phagocytosis values of neutrophils not bound with mab served as controls for comparison.

The mab binding for nonactivated neutrophils (at 4 C) ranged from 9 to 100%, and for activated neutrophils (at 37 C; at 37 C with phorbol myristate acetate) from 90 to 100%. Binding of mab S4F5, S5F8B8, S6C6, S7E10, S8D9, and S5F8G10 increased when neutrophils had been incubated at 37 C. Binding of these mab was further increased after incubation with phorbol myristate acetate (100 ng/ml) at 37 C, indicating recognition of activation antigens by mab. The mab generated in this study appeared to be potential candidates for studying mechanisms of neutrophil function and for enhancing neutrophil function in vitro and in vivo.

Free access
in American Journal of Veterinary Research

Summary

A flow cytometric procedure was evaluated to measure the oxidative burst activity (hydrogen peroxide formation) of bovine neutrophils. The method involves measuring the oxidation of intracellular dichlorofluorescin to fluorescent dichlorofluorescein (dcf). Phorbol myristate acetate (pma) was used to perturb the neutrophil plasma membrane.

The sources of variation introduced into the dcf assay were also examined. The sources of variation were attributable to the isolation of neutrophils from blood, variation between duplicate assays and duplicate flow cytometric determinations of oxidative product formation, variation in neutrophil oxidative product formation among cows, and the variation (over repeated daily and weekly neutrophil isolations) in neutrophil oxidative product formation.

A final objective was to determine effects of dexamethasone on oxidative product formation, and whether differences existed between blood and mammary neutrophils in oxidative product formation.

There was an increasing trend in the formation of dcf with increasing time of incubation and with increasing pma concentration. Increasing the concentration of pma decreased lag time and increased the rate of oxidative product formation. The increase in dcf formation was statistically significant up to a pma concentration of 10 ng/ml. This concentration was considered optimal for bovine neutrophils.

Examination of the sources of variation indicated that (i) the neutrophil isolation technique was a major source of variation (17.2 to 28.4% of the total variation), and that more than one neutrophil isolation within a cow would be required to obtain an accurate estimation of dcf formation in neutrophils; (ii) duplicate assays and duplicate readings on the flow cytometer accounted for < 0.05% of the total variation and would not be necessary when performing the dcf assay; (iii) large variation (62.4 to 70.8%) existed among cows in neutrophil oxidative product formation, indicating that any treatment being compared should be done either within or preferably repeated across a large number of cows; and (iv) the variation over repeated daily (0.3%), but not weekly (19.6%) determinations of neutrophil oxidative product formation, were small enough to allow for the evaluation of major physiologic and environmental effects.

Intramuscular administration of dexamethasone (50 μg/kg of body weight) resulted in an approximate 80% decrease in neutrophil oxidative product formation. Oxidative product formation was 75% less for neutrophils isolated from mammary secretions when compared with neutrophils from blood. These results indicated that the dcf procedure was responsive to factors known to interfere with oxidative metabolism of bovine neutrophils.

Free access
in American Journal of Veterinary Research