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 bovine blood and mammary neutrophils (PMN) after various treatments and determine their ability to modulate receptor numbers.


METHODS
Cell isolation, activation, and flow cytometric studies.


METHODS
Healthy lactating Holstein cattle.


METHODS
Complement component C3b (18,300 kd) was isolated from bovine serum by column chromatography, and flow cytometric 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 exposed to optimal concentrations of recombinant human C5a, formyl-methyl leucyl phenylalanine, recombinant bovine interferon-gamma, variable concentrations of phorbol myristate acetate (0.01 to 100 ng), calcium ionophore A23187, serum-opsonized zymosan, zymosan-activated serum (ZAS), zymosan-activated plasma (ZAP), and hydrocortisone acetate (25 and 70 ng). Additionally, mammary and blood PMN were preincubated in skim milk and whey.


RESULTS
Variable concentrations of phorbol myristate acetate caused a dose-dependent increase in percentage of PMN binding C3b, and increased the amount of C3b bound per cell. Significant increases were observed after PMN treatment with calcium ionophore, serum opsonized zymosan, ZAS, and ZAP; conversely, incubation of PMN with hydrocortisone acetate resulted in reduced overall binding of C3b. Mammary PMN consistently bound more C3b, which was attributed to their activation during migration into the mammary gland. Binding of C3b was inhibited by skim milk. Activation of blood PMN with PMA, ZAS, and ZAP elicited larger responses than those observed for mammary PMN.


CONCLUSIONS
Modulation of complement receptors on bovine PMN is possible. Additionally, significant difference between the level of binding of C3b to blood and milk PMN, with milk PMN having higher binding, may be attributable to migration of PMN into the mammary gland, causing increased receptor expression.


CONCLUSIONS
Contribution to a greater understanding of the role of complement in bovine immunologic systems, leading to testing for in vivo enhancement of bovine immune responses to invading pathogens.
Di Carlo
A L
AL
Department of Animal Sciences, University of Maryland, College Park 20742, USA.
Paape
M J
MJ
Miller
R H
RH
D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D001773:Blood Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D000001:Calcimycin / Q000494:pharmacology; D002273:Carcinogens / Q000494:pharmacology; D002417:Cattle / Q000502:physiology*; D002469:Cell Separation / Q000662:veterinary; D003179:Complement C3b / Q000378:metabolism* / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D005260:Female; D005434:Flow Cytometry / Q000662:veterinary; D006854:Hydrocortisone / Q000031:analogs & derivatives / Q000494:pharmacology; D007107:Immune System / Q000502:physiology; D008321:Mammary Glands, Animal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008892:Milk / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011951:Receptors, Complement / Q000032:analysis / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D015054:Zymosan / Q000494:pharmacology
D000893:Anti-Inflammatory Agents; D002273:Carcinogens; D011951:Receptors, Complement; D000001:Calcimycin; C021650:hydrocortisone acetate; D003179:Complement C3b; D015054:Zymosan; D013755:Tetradecanoylphorbol Acetate; D006854:Hydrocortisone