REACTIVITY OF PURIFIED COMPLEMENT COMPONENT 3B WITH BOVINE NEUTROPHILS AND MODULATION OF COMPLEMENT RECEPTOR-1

Objective-To study binding of purified complement component C3b to bov ine blood and mammary neutrophils (PMN) after various treatments and d etermine their ability to modulate receptor numbers. Design-Cell isola tion, activation, and flow cytometric studies. Animals-Healthy lactati ng Holstein cattle. Procedure-Complement component C3b (18,300 kd) was isolated from bovine serum by column chromatography, and flow cytomet ric assays using fluorescein isothiocyanate-labeled C3b were developed to evaluate binding to PMN complement receptor 1. Multiple substances were tested to determine their overall effect on C3b binding to PMN. Blood and milk PMN were isolated by differential centrifugation and ex posed to optimal concentrations of recombinant human C5a, formyl-methy l leucyl phenylalanine, recombinant bovine interferon-gamma, variable concentrations of phorbol myristate acetate 10.01 to 100 ng), calcium ionophore A23187, serum-opsonized zymosan, zymosan-activated serum (ZA S), zymosan-activated plasma (ZAP), and hydrocortisone acetate (25 and 70 ngi. Additionaiiy, mammary and blood PMN were preincubated in skim milk and whey. Results-Variable concentrations of phorbol myristate a cetate caused a dose-dependent increase in percentage of PMN binding C 3b, and increased the amount of C3b bound per ceil. Significant increa ses were observed after PMN treatment with calcium ionophore, serum op sonized zymosan, ZAS, and ZAP; conversely, incubation of PMN with hydr ocortisone acetate resulted in reduced overall binding of C3b. Mammary PMN consistently bound more C3b, which was attributed to their activa tion during migration into the mammary gland. Binding of C3b was inhib ited by skim milk. Activation of blood PMN with PMA, ZAS, and ZAP elic ited larger responses than those observed for mammary PMN. Conclusions -Moduiation of complement receptors on bovine PMN is possible, Additio nally, significant difference between the level of binding of C3b to b lood and milk PMN, with milk PMN having higher binding, may be attribu table to migration of PMN into the mammary gland, causing increased re ceptor expression. Clinical Relevance-Contribution to a greater unders tanding of the role of complement in bovine immunologic systems, leadi ng to testing for in vive enhancement of bovine immune responses to in vading pathogens.