Student Theses and Dissertations
Date of Award
1981
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Zanvil Cohn
Keywords
phagosome-lysosome (P-L) fusion, macrophages, polyanions, phorbol myristate acetate (PMA), lysosomal membrane, protein synthesis
Abstract
Several approaches were used to study the determinants of phagosome-lysosome (P-L) fusion in intact mouse macrophages. Lysosomes were labeled with the fluorescent vital dye acridine orange and the rate and extent of their fusion with yeast-containing phagosomes was monitored by fluorescence microscopy. Fusion was also assayed by electron microscopy, using horseradish peroxidase or thorium dioxide as markers for secondary lysosomes. Good agreement was found with results obtained from vitally-stained cells, thin section samples using an enzymatic marker, and thorium dioxide-labeled samples evaluated by stereology. The rate of fusion as assayed by fluorescence was not affected by the number of particles ingested, serum concentration, or prior uptake of digestible or non-digestible substances. Fusion was also not affected by enzymatic modification or concanavalin A crosslinking of the plasma membrane or by coating the phagocytic particle with concanavalin A or immune serum. Pretreatment of cells with 10-6 - 10-4 M colchicine, or treatment immediately after ingestion with 1 - 10 μg/ml cytochalasin did not alter P-L fusion; implying that the cytoskeleton does not control fusion in a rate-limiting way. With the fluorescence assay it was possible to observe the rate of fusion separate from and uninfluenced by the phagocytic rate. Both the rate and extent of fusion were dramatically increased after several days in culture and similar changes were found using the EM assays. Fusion was strongly affected by incubation temperature, having a Q10 of 2.5. No detectable fusion occurred below 15°C, and this inhibition was rapidly reversed when cells were returned to 37°C. Fusion was strikingly elevated in 5 hr cultures of activated macrophages from immune-boosted mice. A lower enhancement was seen in cells activated by proteose-peptone, a non-specific inflammatory agent. Treatment with the tumor promoter, phorbol myristate acetate (PMA) caused a striking increase in the rate and extent of P-L fusion. Exposure of cells to phorbol, free myristate, or the monoesters of PMA did not reproduce this effect. Macrophages required from 2 - 3 hr of pretreatment to express maximal P-L fusion and this was maintained for at least 20 hr when cells were returned to PMA-free medium. Catalase, superoxide dismutase, indomethacin and hydrocortisone, agents which are known to block the effect of PMA on H2O2, O2-, prostaglandin or plasminogen activator, did not affect the stimulation of P-L fusion by PMA. The effect of PMA was blocked by 10 μg/ml puromycin or 5 μg/ml cycloheximide when cells were preincubated for 2 hr with the protein synthesis inhibitor and exposure to the inhibitor maintained during incubation with PMA and subsequent assay. Similar treatments with puromycin or cycloheximide had no effect on the high rate of fusion in four day cells. Labeled PMA was rapidly taken up by macrophages, with a plateau of uptake at about 3 hr. When cells were returned to PMA-free medium, cellassociated label was rapidly released, returning to background levels within 1 hr. The released label was found to be a metabolite of PMA by thin layer chromatography. This product migrated between the monoester, phorbol-12-myristate, and free phorbol. Rapid metabolism of PMA was also observed by a macrophage cell line, J774, and to a lesser extent by primary rat embryo fibroblasts. P-L fusion was markedly inhibited by several days of culture in various polyanionic compounds such as dextran sulfate (DS), which were endocytosed and concentrated in secondary lysosomes. Polyanions did not inhibit pinosome-lysosome fusion, as assayed by the intracellular degradation of a pinocytic marker, by electron microscopy, and by monitoring the transfer of a pinocytic marker to a lysosome-enriched fraction obtained from polyanion-treated cells. The inhibition of P-L fusion did not appear to be due to alterations in lysosomal pH, to changes in membrane structure as assayed by freezefracture analysis or to alterations in the cell's phospholipid composition. A tritium-labeled DS was purified and characterized. Soluble 3H-DS was endocytosed by cells at a linear rate for up to 100 hr of culture, and caused P-L fusion inhibition after 3 - 4 d of uptake. More rapid uptake of DS occurred when it was presented to cells in the form of a DS-low density lipoprotein (LDL) complex. Under these conditions, fusion was inhibited after 3 - 4 hr of uptake. Macrophages endocytosed large amounts of LDL after acetylation of the lipoprotein, but LDL by itself did not affect fusion. The inhibition of P-L fusion by polyanions thus may be due to an interaction of the polyanion with the lysosomal membrane.
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Recommended Citation
Kielian, Margaret Carol, "Characterization of Phagosome-Lysosome Fusion in Macrophages" (1981). Student Theses and Dissertations. 478.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/478
Comments
A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy