Evidence for hapten recognition in receptor-mediated intracellular uptake of a hapten-protein conjugate by murine macrophage.

Fluorescein-derivatized bovine serum albumin (FITC-BSA) was used as an exogenous antigen and fluorescent probe to measure the kinetics of antigen uptake into the endocytic pathway of murine macrophage, J774, using flow cytometry. Results revealed dependency of the rate of antigen uptake on epitope density (moles FITC/mole BSA) implicating a role for FITC in the endocytosis of the derivatized antigen. In addition, inhibition of clathrin-coated pit formation in macrophage resulted in significantly reduced uptake of differentially labeled FITC BSA probes indicating receptor-mediated endocytosis via clathrin-coated pits. Fluoresceinamine (I) was found to inhibit the endocytic uptake of FITC-BSA at 10(-6) M. Determination of fractional receptor occupancies in macrophage upon binding different FITC BSA probes and calculation of the corresponding association rates (k(on)) for these binding events yielded values of 4.2+/-0.2 x 10(6)/M/min for FITC5BSA and 1.9+/-0.1 x 10(7)/M/min for FITC22BSA, respectively, at 37 degrees C. The five-fold difference in the rates of binding and endocytosis between the two probes was discussed on the basis of receptor cross-linking by a multivalent ligand (FITC22BSA), in contrast to monovalent ligand binding, on the cell surface that would lead to more rapid and efficient internalization of the FITC22BSA antigen.