The role of serum factors in the phagocytosis of pneumococci was studied employing a spectrophotometric assay which measures reduced nitro blue tetrazolium (NBT) dye. Dye reduction occurs within the phagocyte shortly after bacterial ingestion as measured by the phagocytic index technique and by the uptake of 125I-pneumococci. Bacteria prepared with γG antibody were not phagocytosed unless a small volume of fresh normal serum was added. Using fresh sera deficient in single complement components, it was demonstrated that the first four components are necessary for optimal bacterial phagocytosis. When highly purified complement components were added to the antibody-coated pneumococci, enhancement of phagocytosis was achieved only with the sequential addition of C1, C4, C2, and C3.
Evidence has been presented that human C3 bound to an immune complex exhibits peptidase activity and that this activity is essential for phagocytosis. A heat-labile, dialyzable serum cofactor which enhances C3 peptidase activity enhanced the phagocytosis of pneumococci prepared with purified complement components. A second phagocytosis-promoting cofactor, which is not a complement component, was found to be a heat-labile, 5–6S, beta pseudoglobulin. This protein may stabilize C3 peptidase activity or inhibit enzymatic inactivation of C3.