Analysis of rates of receptor-mediated endocytosis and exocytosis of a fluorescent hapten-protein conjugate in murine macrophage: Implications for antigen processing

A novel fluorescent hapten-protein conjugate was constructed to monitor the events required for CD 4+ T lymphocyte recognition of antigenic proteins. Previous studies utilizing the probe demonstrated that the hapten-protein was localized to an acidic endocytic compartment within the macrophage and that the hapten-protein was sensitive to multiple intracellular events including enzymatic degradation, acidification, and disulfide bond reduction. More importantly, recent experiments indicated that efficient internalization of the probe was dependent upon specific recognition of the hapten. Therefore, the present report addressed the effect of receptor-mediated endocytosis upon the processing of the hapten-protein within murine peritoneal macrophage. These studies determined that the rate of endocytosis was significantly faster than the rate of exocytosis. Specifically, the rate of exocytosis was estimated to be 3.4 × 104 s−1 based on a unimolecular rate constant. Although at higher concentrations, a slightly slower rate was observed (1.9 × 104 s−1). This study also represented one of the first efforts to measure the intracellular concentration effect typically associated with receptor-mediated endocytosis. Experiments involving a radioactively labeled hapten-protein conjugate revealed that the probe was at 100-fold higher concentration within the endocytic vesicles when compared to the extracellular media. The intracellular mechanism involved in this phenomenon was discussed as well as the implications of these findings upon MHC II-peptide binding.