REGULATION OF ACTIVATION OF BOVINE NEUTROPHILS BY AGGREGATED IMMUNOGLOBULIN-G

Objectives-To investigate the role of extracellular Ca2+ and Mg2+ in a ggregated IgG (algG)-mediated cellular activation, and to determine ho w algG-induced activation is coupled to Ca2+ homeostasis in bovine neu trophils. Sample Population-4 clinically normal, lactating Holstein co ws, in their second lactation, which ranged between 60 and 150 days. P rocedure-algG was prepared by heating bovine IgG, and C5a was obtained by activating fetal bovine serum with zymosan. Luminol-amplified chem iluminescence (CL) of isolated neutrophils was measured in the presenc e of algG or phorbol 12-myristate 13-acetate (PMA). The reaction mixtu re contained either Hanks' balanced salt solution or Ca2+- and Mg2+-fr ee Hanks' balanced salt solution. Binding of algG to neutrophils was m easured by flow cytometry after incubation with fluorescein isothiocya nate-conjugated second antibody. Intracellular-free concentration [Ca2 +](i) was measured in a fluorescence spectrofluorometer after incubati on of neutrophils, loaded with the fluorescent dye fura-2 acetoxymethy l ester, with either algG or C5a. Results-In a Ca2+- and Mg2+-containi ng reaction mixture, algG induced strong CL responses, whereas removal of extracellular divalent cations almost abolished the respiratory bu rst activity. The CL emission on stimulation with PMA was independent of extracellular Ca2+ and Mg2+. Examination of cells by flow cytometry after incubation with algG indicated that the binding of algG was ide ntical in the presence and absence of extracellular Ca2+ and Mg2+. No increase in [Ca2+](i) was seen in fura-2 acetoxymethylester-loaded neu trophils after stimulation with algG. C5a induced a typical transient increase in [Ca2+](i). Conclusions-algG-induced activation of bovine n eutrophils is highly dependent on presence of extracellular divalent c ations. This dependency is not caused by the need of divalent cations for binding of algG by neutrophils or because the influx of Ca2+ from the extracellular space is an integral component of algG-mediated acti vation pathway. Because need for extracellular Ca2+ and Mg2+ could be partially circumvented by pretreating neutrophils with PMA, it is poss ible that this activation pathway may involve a protein kinase C, whic h is not directly coupled to receptors for algG.