Date of Award

8-1-1988

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biomedical Sciences

First Advisor

John A. Duerre

Abstract

The human promyelocytic leukemia cell line HL-60 has been studied extensively since the time of it's discovery and has been proven to be a valuable tool for the study of the differentiation process. The capacity of the HL-60 cell line to differentiate to macrophage-like cells, which may be immunologically competent, has proven to be invaluable in the study of these cells as a model for macrophage function. A number of hydrolytic enzymes are characteristically found in macrophages. In an effort to further characterize diferentiated HL-60 cells as macrophages, the location and concentration of a number of these enzymes was determined. HL-60 cells were cultured in RPMI-1640 medium with 5% fetal calf serum and antibiotics. The cells were treated with the differentiation agent, 12-O-tetradecanoylphorbol-13-acetate (TPA), for 48 hours and cell free lysates were prepared. Elastase, chymotrypsin, ribonuclease, β-glucuronidase and lysozme concentrations were determined in the lysosomal fraction and whole cell lysates. The amount of chymotrypsin and elastase increased significantly in cells which had differentiated to macrophage-like cells. The intrracellular concentrations of lysozyme, β-glucuronidase, and ribonuclease decreased or remained the same in differentiated and undifferentiated cells. Macrophages characteristically secrete a number of hydrolytic enzymes including lysozyme and β-glucuronidase. Assay of the media revealed that the TPA treated cells secreted significantly greater amounts of lysozyme and β-glucuronidase than the undifferentiated cells. Differentiated cells which had ingested opsonized yeast particles were harvested and the lysates were tested for the lysosomal enzymes. The concentration of intracellular elastase in these cells was increased over control HL-60 cells and TPA treated cells. The amount of β-glucuronidase and lysozyme secreted into the medium remained the same as that of differentiated cells which had not phagocytized yeast.

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