Phagocytosis by macrophages. II. The dissociation of the attachment and ingestion steps.

The phagocytic process of mouse peritoneal macrophages was dissociated, using bovine serum albumin (BSA)-coated particles containing spin-labelled cholestanone, into 2 steps: attachment of particles to the cell surface and ingestion of the particles into the cytoplasm. The number of particles was estimated from electron spin resonance (e.s.r.) measurements. The particles ingested into the cytoplasm were distinguished from those attached to the cell surface by treatment with a membrane-impermeable reducing agent, ascorbate. The validity of the assay method was tested under various conditions. The measurements provided accurate and reproducible data. The phagocytic reaction was followed as a function of time and the rate constants for the attachment and ingestion steps were obtained from the initial phase. Both steps were highly dependent on temperature. Divalent cations in the incubation medium were essential for the attachment step but apparently had no effect on the ingestion step. The metabolic inhibitors, KCN and 2-deoxyglucose, inhibited both steps. Cytochalasin B inhibited both steps, while colchicine inhibited only the attachment step but apparently had no effect on the ingestion step.