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The CD14 antigen which is expressed on human monocytes and macrophages is a phosphatidylinositol-linked surface protein. We investigated the effects of cellular cholesterol depletion and repletion on cell surface expression of this glycoprotein. Adherent normal human monocytes were cultured for four days in media containing delipidated fetal calf serum which depleted cellular cholesterol. Immunofluorescence analysis demonstrated a markedly diminished surface expression of CD14 on cells cultured in delipidated serum compared to normal serum. Expression of CD64 (high-affinity Fc receptors, Fc gamma RI) also was reduced under these conditions. This inhibition of CD14 expression was overcome by addition to the culture medium of cholesterol, low density lipoprotein, or very low density lipoprotein. All of these supplements replenished cellular cholesterol. Expression of CD64(Fc gamma RI) was not restored by addition of cholesterol. These observations indicate that cholesterol can regulate the surface expression of some phosphatidylinositol-anchored glycoproteins.

A substrain of the human monocyte-like cell line U937, which is a cholesterol auxotroph, was used to study the effect of cellular cholesterol depletion on the expression of the type I Fc receptor for IgG (Fc gamma RI). Measurement of Fc gamma RI expression was performed by immunofluorescence and flow cytometry using the monoclonal antibody (mAb) 32.2, which is specific for an epitope on Fc gamma RI, and monomeric IgG2a, which binds to the ligand binding site of Fc gamma RI. Incubation of these cells for 24 h in growth medium containing delipidated fetal calf serum depletes cellular cholesterol without affecting growth or viability. While incubation of U937 cells with human interferon-gamma (IFN-gamma) increased Fc gamma RI expression, cholesterol depletion after cell growth in media containing delipidated serum and IFN-gamma resulted in reduced binding of both mAb 32.2 and IgG2a. A significant decrease in the number of cell surface binding sites, as measured by mean fluorescence intensity, was observed after cholesterol depletion. Supplementation of the delipidated serum medium with pure cholesterol in an ethanol/bovine serum albumin mixture, which replenished cellular cholesterol and supported growth, failed to restore antibody binding significantly. In contrast, low-density lipoprotein (LDL) which also delivered cholesterol to the cells restored binding both in terms of the number of the reactive cells and cell surface receptor density. High-density lipoprotein (HDL3), which does not deliver cholesterol to the cells, showed results similar to those obtained with pure cholesterol. This indicates that either LDL cholesterol is better utilized for membrane synthesis than pure cholesterol or that LDL provides another component, in addition to cholesterol, which is required for expression of Fc gamma RI, but not for growth. These studies indicate a role for LDL in regulating the expression of Fc gamma RI on the cell surface.

The lipid requirements of a human macrophagelike cell line were studied. The cells grew only about one generation in a medium supplemented with delipidated serum; during the growth the cholesterol content of the cells was depleted. Growth was restored by including in the medium serum lipids subjected to alkaline hydrolysis or cholesterol. The extent of growth was dependent on cholesterol concentration. No growth was obtained with 5-cholestene, 5-cholesten-3-one, cholesteryl chloride, coprostanol, beta-sitosterol, or stigmasterol. Very limited growth occurred with cholesterol methylether, epicholesterol, or beta-cholestanol. Therefore, for optimal growth of these cells there is a stringent requirement for the structural features of cholesterol, which include a 3-beta OH group, a delta 5-double bond, a trans ring A/B configuration, and freedom of the side chain from bulky groups. This stringency far exceeds what was previously reported for other cells. Of the six sterols that failed to support growth at all, five were incorporated into cells moderately to extensively. This suggests that assembly of a functional membrane is impaired when these sterols are used as substrates for growth.