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 antige n processing, a fluorescent probe suitable to monitor several steps wi thin this pathway was developed. Previous studies utilizing two-photon fluorescence microscopy with time resolved and intensity imaging demo nstrated that the probe, fluorescein derivatized BSA, was localized to the endocytic system and degraded over an extended period of time. Ho wever, an additional method, flow cytometry, was required to monitor t he kinetics of these intracellular events and to better assess the tot al cell population. Flow cytometric studies indicated that the antigen entered an acidic intracellular environment consistent with the endoc ytic system of the macrophage. Additional experiments suggested that m ini mal proteolytic degradation began 10 min after addition of the ant igenic probe while extensive enzymatic degradation did not occur until 180-200 min. Inhibitor studies indicated that degradation of the prob e was dependent upon both acidic pH and ATP synthesis as well as all f our classes of proteases. Experiments involving specific protease inhi bitors also revealed that various classes of proteases were active at different time points throughout the processing of the probe. By combi ning these results with additional kinetic data, a model for the seque nce of events involved in the processing of FITC(10)BSA was proposed. More importantly, these studies represented some of the first time-dep endent kinetic measurements of antigen processing in living cells.