Macrophage mediated processing of an exogenous antigenic fluorescent probe: time-dependent elucidation of the processing pathway.

To elucidate time-dependent pathways and mechanisms involved in antigen processing, a fluorescent probe suitable to monitor several steps within this pathway was developed. Previous studies utilizing two-photon fluorescence microscopy with time resolved and intensity imaging demonstrated that the probe, fluorescein derivatized BSA, was localized to the endocytic system and degraded over an extended period of time. However, an additional method, flow cytometry, was required to monitor the kinetics of these intracellular events and to better assess the total cell population. Flow cytometric studies indicated that the antigen entered an acidic intracellular environment consistent with the endocytic system of the macrophage. Additional experiments suggested that minimal proteolytic degradation began 10 min after addition of the antigenic probe while extensive enzymatic degradation did not occur until 180-200 min. Inhibitor studies indicated that degradation of the probe was dependent upon both acidic pH and ATP synthesis as well as all four classes of proteases. Experiments involving specific protease inhibitors also revealed that various classes of proteases were active at different time points throughout the processing of the probe. By combining these results with additional kinetic data, a model for the sequence of events involved in the processing of FITC10BSA was proposed. More importantly, these studies represented some of the first time-dependent kinetic measurements of antigen processing in living cells.