Regulation of inflammation-primed activation of macrophages by two serum factors, vitamin D3-binding protein and albumin

Abstract

A very small amount (0.0005 to 0.001%) of an ammonium sulfate [50%o saturated (NH4)2SO41-precipitable protein fraction of c2-globulin efficiently supported inflammation-primed activation of macrophages. This fraction contains vitamin D3-binding protein essential for macrophage activation. Comparative macrophage activation studies with fetal calf serum, a2-globulin fraction, 50%o (NH4)2S04 precipitate, and purified bovine vitamin D3-binding protein revealed that fetal calf serum and %-globulin fraction appear to contain an inhibitor for macrophage activation while ammonium sulfate precipitate contains no inhibitor. This inhibitor was found to be serum albumin. When bovine serum albumin (25 ,ug/ml) was added to a medium supplemented with 0.0005 to 0.05% (NH4)2SO4 precipitate or 1 to 10 ng of vitamin D3-binding protein per ml, activation of macrophages was inhibited.

Structurally well‐defined macrophage activating factor derived from vitamin D3‐binding protein has a potent adjuvant activity for immunization

“…After 10 s abdominal massage, peritoneal lavage was aspirated, washed and assayed for cell counts and the superoxide generating capacity of macrophages. 12,13 Treatment of mice with GcMAF…”

“…Two and 4 days after the ®rst administration, peritoneal macrophages were harvested and assayed for cell counts and superoxide generating capacity. 12,13 Assay for antibody secreting cells A modi®ed procedure of Jerne et al was used to quantify the number of IgM antibody secreting cells (plaque-forming cells (PFC)) in the spleens of GcMAF-treated mice. 26 Mice were treated with various amounts of GcMAF and immunized with intraperitoneal injection of sheep erythrocytes (8´10 8 cells/mouse) 6 h after GcMAF administration.…”

“…12,13 In vitro treatment of mouse peritoneal adherent cells (macrophages) with lysophosphatidylcholine (lyso-Pc) results in no enhancement of macrophage phagocytic activity 5±10 and superoxide generating capacity. 12,13 However, incubation of peritoneal cells (mixture of adherent and non-adherent cells) with lyso-Pc in a serum-supplemented medium for 30 min, followed by 3 h cultivation of adherent cells alone, markedly enhances Fc receptor-mediated phagocytic and superoxide generating capacities of macrophages, 9±13 implying a participation of non-adherent (B and T) cells and a serum component in the activation of macrophages. 6,9±15 Analysis of macrophage activating signal generation from the non-adherent cells showed that vitamin D 3 -binding protein (DBP; human DBP is known as group-speci®c component, Gc) is a serum factor required for the in¯ammation-primed activation of macrophages.…”

A defect in inducible β‐galactosidase of B lymphocytes in the osteopetrotic (mi/mi) mouse

“…The detailed procedures for isolation and cultivation of mouse peritoneal cells (mixture of adherent and nonadherent cells), enumeration of adherent cells (macrophages) and superoxide generation assay have been described previously. [25][26][27][28] For in vitro treatment of mouse peritoneal cells with lyso-Pc, resident peritoneal cells were isolated from untreated wild type or mi mutant mice. The desired cell number (6 to 9 2 l0 5 /ml) of the washed peritoneal cells were laid on 16 mm plastic culture wells and incubated at 378 in a humidified 5% CO 2 incubator for 30 min to allow macrophage adherence to the substrata.…”

“…After 3 hr cultivation, superoxide generating capacity of macrophages was determined. 27,28 Assay of membranous b -galactosidase on B lymphocytes The isolation procedure for splenic B cells has been described previously. 25,26 B cells were incubated with 1 m g lyso-Pc/ml in EA medium at 378.…”