Modulation of bovine neutrophil functions by monoclonal antibodies

Shashikumar K. Salgar From the Department of Animal Science, University of Maryland, College Park, MD 20742 (Salgar, Peters), Milk Secretion and Mastitis Laboratory, USDA Agricultural Research Service, Beltsville, MD 20705 (Paape), and Department of Animal Science, North Carolina State University, Raleigh, NC 27695 (Alston-Mills).

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Max J. Paape From the Department of Animal Science, University of Maryland, College Park, MD 20742 (Salgar, Peters), Milk Secretion and Mastitis Laboratory, USDA Agricultural Research Service, Beltsville, MD 20705 (Paape), and Department of Animal Science, North Carolina State University, Raleigh, NC 27695 (Alston-Mills).

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Brenda Alston-Mills From the Department of Animal Science, University of Maryland, College Park, MD 20742 (Salgar, Peters), Milk Secretion and Mastitis Laboratory, USDA Agricultural Research Service, Beltsville, MD 20705 (Paape), and Department of Animal Science, North Carolina State University, Raleigh, NC 27695 (Alston-Mills).

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Robert R. Peters From the Department of Animal Science, University of Maryland, College Park, MD 20742 (Salgar, Peters), Milk Secretion and Mastitis Laboratory, USDA Agricultural Research Service, Beltsville, MD 20705 (Paape), and Department of Animal Science, North Carolina State University, Raleigh, NC 27695 (Alston-Mills).

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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.

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.

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