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 monito r the events required for CD 4(+) T lymphocyte recognition of antigeni c proteins. Previous studies utilizing the probe demonstrated that the hapten-protein was localized to an acidic endocytic compartment withi n 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 ind icated that efficient internalization of the probe was dependent upon specific recognition of the hapten. Therefore, the present report addr essed the effect of receptor-mediated endocytosis upon the processing of the hapten-protein within murine peritoneal macrophage. These studi es determined that the rate of endocytosis was significantly faster th an the rate of exocytosis. Specifically, the rate of exocytosis was es timated to be 3.4 x 10(4) s(-1) based on a unimolecular rate constant. Although at higher concentrations, a slightly slower rate was observe d (1.9 x 10(4) s(-1)). This study also represented one of the first ef forts to measure the intracellular concentration effect typically asso ciated with receptor-mediated endocytosis. Experiments involving a rad ioactively labeled hapten-protein conjugate revealed that the probe wa s at 100-fold higher concentration within the endocytic vesicles when compared to the extracellular media. The intracellular mechanism invol ved in this phenomenon was discussed as well as the implications of th ese findings upon MHC II-peptide binding. ((C) Elsevier, Paris).