A comparative study of fluid-phase and adsorptive endocytosis of horseradish peroxidase in lymphoid cells

Adsorptive and fluid-phase endocytosis of horseradish peroxidase (PO) was studied in lymph node cells depleted of macrophages, taken from popliteal lymph nodes of rats immunized against PO (anti-PO cells) and against rabbit IgG (anti-rIgG cells) respectively. The enzymatic activity of PO enabled us to measure the amount of PO endocytosed in the cells and to determine its subcellular localization by means of light and electron microscopy. Uptake of PO by anti-PO cells was a saturable process which reached a plateau at approx. 50 μg/ml of PO. After exposure for 3 h to 50–100 μg/ml of PO, anti-PO cells had endocytosed 5–6 ng of PO per 10⁷ cells. Internalized PO was distributed in cells carrying surface receptors for PO, representing about 6% of the total cell population and consisting mainly of large immunocytes (lymphoblasts, plasma cells). Anti-rIgG cells cultured for 3 h with 100 μ/ml of PO endocytosed a very minute, barely detectable amount of PO. The fluid-phase endocytosis of PO was observed by increasing the PO concentration in the culture medium of anti-rIgG cells. Anti-rIgG cells cultured for 3 h with 500 μg/ml of PO endocytosed about 6 ng of PO/10⁷ cells, but no (or very few) stained cells were found. A large number of PO-internalizing anti-rlgG cells were observed only after culture with high PO concentrations (2 or 5 mg/ml), being both large immunocytes and small-to-medium lymphocytes. Endocytic sites of PO in anti-PO large immunocytes and in anti-rIgG small lymphocytes or large immunocytes were the same and consisted of vesicles, tubules or cisternae located near the Golgi apparatus and round or oval bodies scattered throughout the cytoplasm or localized near the Golgi apparatus (lysosomes?). After exposure to PO, anti-PO and anti-rIgG cells were transferred into PO-free medium. The level of intracellular peroxidase activity did not change during the first 6 h of culture. Then a decrease in enzymatic activity occurred, most probably due to a degradation of PO, at the same rate in anti-PO as in anti-rIgG cells. In conclusion, our results show that intracellular pathways of endocytosis and rates of inactivation of PO entering lymphoid cells are the same, whether specific receptors are present or not. This suggests that endocytosis of antigen in antigen-binding cells could reflect a native membrane recycling event.