Abstract
Human peripheral blood monocytes are the reticuloendotherial cells, which are known to act on the elimination of soluble immune complexes in vivo. Phagocytosis of soluble immune complexes by phagocytes has been detected by radioimmunoassay. We described here new method of the measurement using enzyme-linked immunosorbent assay (ELISA). Peroxidase-anti-peroxidase soluble complex (PAP) was used as a soluble immune complex in this method. Monocytes were isolated from peripheral blood mononuclear cells by their adherent activity to plastic culture dishes. Monocytes (5×104 per well in 200μl of medium) were placed in the wells of a 96-well culture microplate and mixed with 100μl of PAP after culturing for 24 hs. The cells were then incubated in a humidified atmosphere of 5% CO2 at 37°C for 60min. After washing four times with cold phosphate buffered saline, 100μl of 1% Nonidet P 40 was added to each well to destroy the monocytes. Fluid (50μl) from each well was placed in the wells of a flat-bottom microplate for ELISA and then 150μl ABTS at 0.2mg/ml in substrate buffer was added to each well. After 60min at room temperature, the plate was scanned in a microplate ELISA reader at a wavelength of 405nm, with correction being made for medium controls.
The phagocytic activity of monocytes was not observed at 4°C, and was suppressed in the presence of a specific inhibitor of glycolysis (2-deoxyglucose) and blockers of oxidative metabolism (NaN3, KCN). Aggregated IgG also suppressed the phagocytosis. As a result, the phagocytic activity of monocytes detected by this method was thought to be dependent on temperature, cellular metabolism or Fc receptors.
Moreover, we examined the effect of lymphokines on this monocyte activity. The phagocytic activity was enhanced by the addition of 48-h culture supernatant from tonsil lymphocytes stimulated with concanavalin A, and by gamma- and beta-interferon, but not by alpha-interferon or interleukin 2.
