Regulation of activation of bovine neutrophils by aggregated immunoglobulin G.

OBJECTIVES

To investigate the role of extracellular Ca2+ and Mg2+ in aggregated IgG (aIgG)-mediated cellular activation, and to determine how aIgG-induced activation is coupled to Ca2+ homeostasis in bovine neutrophils.

SAMPLE POPULATION

4 clinically normal, lactating Holstein cows, in their second lactation, which ranged between 60 and 150 days.

PROCEDURE

aIgG was prepared by heating bovine IgG, and C5a was obtained by activating fetal bovine serum with zymosan. Luminol-amplified chemiluminescence (CL) of isolated neutrophils was measured in the presence of aIgG or phorbol 12-myristate 13-acetate (PMA). The reaction mixture contained either Hanks' balanced salt solution or Ca(2+)- and Mg(2+)-free Hanks' balanced salt solution. Binding of aIgG to neutrophils was measured by flow cytometry after incubation with fluorescein isothiocyanate-conjugated second antibody. Intracellular-free concentration [Ca2+]i was measured in a fluorescence spectrofluorometer after incubation of neutrophils, loaded with the fluorescent dye fura-2 acetoxymethyl ester, with either aIgG or C5a.

RESULTS

In a Ca(2+)- and Mg(2+)-containing reaction mixture, aIgG induced strong CL responses, whereas removal of extracellular divalent cations almost abolished the respiratory burst activity. The CL emission on stimulation with PMA was independent of extracellular Ca2+ and Mg2+. Examination of cells by flow cytometry after incubation with aIgG indicated that the binding of aIgG was identical in the presence and absence of extracellular Ca2+ and Mg2+. No increase in [Ca2+]i was seen in fura-2 acetoxymethylester-loaded neutrophils after stimulation with aIgG. C5a induced a typical transient increase in [Ca2+]i.

CONCLUSIONS

aIgG-induced activation of bovine neutrophils is highly dependent on presence of extracellular divalent cations. This dependency is not caused by the need of divalent cations for binding of aIgG by neutrophils or because the influx of Ca2+ from the extracellular space is an integral component of aIgG-mediated activation pathway. Because need for extracellular Ca2+ and Mg2+ could be partially circumvented by pretreating neutrophils with PMA, it is possible that this activation pathway may involve a protein kinase C, which is not directly coupled to receptors for aIgG.