Evaluation of assay procedures measuring macrophage stimulation by immunomodulators in vitro

Several assay procedures for measuring the stimulatory effect on macrophages (møs) of bacterial-derived immunomodulators (OM-85, OM-89, OM-163, Laboratoires OM, Meyrin/Geneva, Switzerland) were evaluated with regard to their complexity, speed, and general convenience. To this effect, bone marrow-derived or peritoneal exudate macrophages were exposed to the immunomodulators in vitro, then tested for metabolic stimulation (glucose oxidation through the hexosemonophosphate shunt pathway, synthesis of type E prostaglandins, release of superoxide, and production of L-arginine-derived nitrogen oxidation products), as well as for enhancement of functional activities (production of tumor necrosis factor-alpha, extracellular cytolysis of P815 target cells, and intracellular parasite destruction). All these tests were found to provide adequate measurements of the mø response to the immunomodulators, with significant effects detectable using the compounds in the ng/ml to microgram/ml range. Concomitant incubation with crude macrophage activating factor or with recombinant murine interferon-gamma (IFN-gamma) dramatically increased the sensitivity of møs to the immunomodulators, and was an absolute requirement for induction of mø cytotoxic activities by the bacterial extracts. The measurement of nitrite production by møs exposed to the immunomodulators with or without treatment with 10 U/ml of IFN-gamma was found to be a highly convenient procedure, which correlated well with functional assays.