Regulation of Cell-surface Receptors for Human Interferon-gamma on the Human Histiocytic Lymphoma Cell Line U937

Interferon-gamma (IFN gamma) binds to high-affinity receptors on monocytes and is rapidly internalized. This study investigates the ability of the human monocyte-like cell line, U937, to regulate the cell-surface expression of the IFN gamma receptor (IFN gamma R) during endocytosis of ligand. Recombinant IFN gamma was radiolabelled to high specific radioactivity with Bolton-Hunter reagent and used to enumerate IFN gamma R on treated U937 cells. Cells which had internalized IFN gamma for up to 3 h displayed maximal levels of IFN gamma R at all time points tested after all unlabelled IFN gamma had been acid-stripped from the cell at pH 2.78. Therefore there was no evidence of down-modulation of the receptor. After trypsin treatment of the IFN gamma R, the cells were able to synthesize and insert into the cell membrane up to 1000 IFN gamma R molecules/h after a 60 min lag. Since biosynthesis played a minor role during the first 30 min of endocytosis, I examined other possibilities to explain the lack of down-modulation of the receptor. A solubilized-receptor assay revealed the presence of an intracellular pool of receptors equal to about 25% of the number of cell surface receptors. Using trypsin to differentiate between intracellular and surface receptors, I observed that 43% of those receptors that were internalized after a 30 min exposure to IFN gamma (580 molecules) could be recycled back to the plasma membrane. In addition, equal rates of receptor decay (t1/2 = 5 h) were observed in the presence of cycloheximide with or without IFN gamma. All the data taken together suggest that during the first 30 min of endocytosis both the expression of an intracellular source of receptor and recycling of internalized receptors contribute to maintain optimal receptor expression.