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Summary

Ten healthy first- and second-lactation Holstein cows were observed from 1 week before to 1 week after calving and at postpartum day 30 to determine polymorphonuclear neutrophil (pmn) functional variation and immunoglobulin binding profiles. Blood and mammary pmn were obtained 3 times weekly and within 24 hours of calving. Functional traits measured included phagocytosis of Staphylococcus aureus and in vitro chemotaxis through micropore filters in a Boyden chamber. Additionally, pmn were evaluated for endogenous binding of IgG1, IgG2, IgA, and IgM before and after in vitro chemotaxis. Exogenous binding of the same isotypes was determined after incubation in pooled colostrum, purified immunoglobulin, and pooled sera. Phagocytosis results indicated a significant and transient increase in percentage of milk pmn with associated, rather than phagocytosed, bacteria for 1 week after calving. Blood pmn phagocytosis was not significantly different during this period. Though total chemotaxis was essentially unchanged, the percentage of pmn that were unable to complete migration increased substantially on the day of calving, an effect that disappeared by postpartum day 4. A significant (P < 0.01) positive correlation (r = 0.29) between percentage of pmn migrating completely through the micropore filter and percentage of blood pmn with associated bacteria was observed. Changes were not observed in endogenous immunoglobulin binding, with the exception of a peak in relative fluorescence intensity for IgG1 on the day of calving; this disappeared within 2 days after calving. Correlations between relative intensities of IgG2 and IgM, and percentage of mammary neutrophils phagocytosing were 0.37 and 0.70. Exogenous binding of antibody to blood neutrophils before chemotaxis was generally accomplished most effectively by pooled colostrum, whereas use of pooled sera markedly reduced binding and percentage and intensity of IgM in all cases. Binding of all isotypes was slightly higher before than after calving. Incubation of blood neutrophils in isotypes G1, G2, A, and M after chemotaxis yielded lower immunoglobulin binding among all isotypes, particularly IgM. Fluctuations in neutrophil function were observed immediately around parturition, and these changes correlated strongly with endogenous immunoglobulin-binding profiles.

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