Mojtaba Esfahani

Cholesterol regulates the cell surface expression of glycophospholipid-anchored CD14 antigen on human monocytes

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.

Effects of cholesterol and lipoproteins on endocytosis by a monocyte-like cell line

The human monocyte/macrophage-like cell line U937 is a cholesterol auxotroph. Incubation of these cells in the growth medium in which delipidated fetal calf serum has been substituted for fetal calf serum depletes cellular cholesterol and inhibits growth. The cholesterol requirement of these cells for growth can be satisfied by human low-density lipoprotein (LDL), and very-low-density lipoprotein (VLDL), but not by high-density lipoprotein (HDL). U937 cells can bind and degrade LDL via a high-affinity site and this recognition is altered by acetylation of LDL. This indicates that these cells express relatively high LDL receptor activity and low levels of the acetyl-LDL receptor. The cells were used to study the role of cholesterol in lectin-mediated and fluid-phase endocytosis. Growth of the cells in the medium containing delipidated fetal calf serum results in impairment of both concanavalin A-mediated endocytosis of horseradish peroxidase and concanavalin A-independent endocytosis of Lucifer Yellow. Supplementation of the medium with cholesterol prevents cellular cholesterol depletion, supports growth and stimulates Lucifer Yellow endocytosis but fails to restore horseradish peroxidase endocytosis. However, if the cells are incubated in the presence of no less than 40 micrograms LDL protein/ml to maintain normal cell cholesterol levels, concanavalin A-mediated endocytosis of horseradish peroxidase is activated. The effect of LDL is specific since neither VLDL nor HDL3 at the same protein concentration activates horseradish peroxidase uptake by the cells. Furthermore, the activation of endocytosis by LDL is not inhibited by the inclusion of heparin or acetylation of the LDL indicating that binding of LDL to the LDL receptor is not required for these effects. The mediation of activation of horseradish peroxidase endocytosis by the lectin is presumed to involve binding of LDL to concanavalin A associated with the cell surface which in turn stimulates horseradish peroxidase binding and uptake by adsorptive endocytosis. The rate of fluid endocytosis and endosome formation seems to depend on cellular cholesterol content presumably because cholesterol is involved in maintaining the appropriate plasma membrane structure and fluidity.

Effect of low-density lipoprotein on the expression of high affinity Fcγ receptors

Abstract 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 (FcγRI). Measurement of FcγRI expression was performed by immunofluorescence and flow cytometry using the monoclonal antibody (mAb) 32.2, which is specific for an epitope on FcγRI, and monomeric IgG 2a , which binds to the ligand binding site of FcγRI. 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-γ (IFN-γ) increased FcγRI expression, cholesterol depletion after cell growth in media containing delipidated serum and IFN-γ resulted in reduced binding of both mAb 32.2 and IgG 2a . 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 (HDL 3 ), 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 FcγRI, but not for growth. These studies indicate a role for LDL in regulating the expression of FcγRI on the cell surface.

A requirement for cholesterol for phorbol ester-induced adhesion of a human monocyte-like cell line

The human monocyte/macrophage-like cell line U937, which is a cholesterol auxotroph, is nonadherent. However, it becomes adherent after treatment with phorbol 12-myristate 13-acetate (phorbol ester). We investigated the effects of cellular cholesterol depletion and repletion on the effectiveness of phorbol ester to induce adhesion to substratum. Almost 70% of cellular cholesterol is depleted by incubation of the cells for 24 hrs in the growth medium in which delipidated fetal calf serum is substituted for fetal calf serum without affecting viability or the rate of growth. The use of delipidated fetal calf serum inhibited phorbol ester-induced adhesion by 40%. If the cells were preincubated in the medium containing delipidated fetal calf serum 6 hrs prior to addition of phorbol ester, adhesion was inhibited by 90%. Addition of cholesterol to the medium containing delipidated fetal calf serum, which replenishes cellular cholesterol, restored the ability of phorbol ester to induce adhesion to levels seen in cells cultured in the medium containing fetal calf serum. Epicholesterol was not as effective as cholesterol in supporting adhesion. Cholesterol depletion did not inhibit phorbol ester stimulation of superoxide anion production. These observations indicate a function for cholesterol in phorbol ester-induced adhesion that is independent of phorbol ester-induced superoxide anion production. It is proposed that cholesterol is required for synthesis and/or proper orientation and distribution, in the plasma membrane, of macromolecule(s) that mediate phorbol ester-induced adhesion.

Correlation of C3b-receptor activity and diphenylhexatriene polarization in a murine macrophage cell line

Abstract Treatment of a macrophage cell line with lymphokine (LK)-rich lymphocyte culture supernatants activates the cells for C3b receptor-mediated phagocytosis and leads to simultaneous depolarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) incorporated into the cells. Maximal effects were observed after 14 hours of incubation with LK. DPH polarization in liposomes prepared from lipids of untreated and LK-treated cells were the same. Furthermore, LK-treatment had no significant effects on phospholipid fatty acid composition, cholesterol content, or phospholipid composition. These observations indicate LK induces alterations in the organization and dynamics of the plasma membrane that lead to facilitation of phagocytosis.

Effect of lovastatin on cell surface expression of Fc receptors or CD14 antigen in human monocytes.

Lovastatin is a widely used anticholesterolemic drug which exercises its effect by inhibiting hepatic cholesterol synthesis and up-regulating low density lipoprotein (LDL) receptors. In the present study, we determined that the drug has no adverse effects on the expression of three cell surface antigens of human monocytes, i.e. high affinity Fc receptors (Fc gamma RI), low affinity Fc receptors (Fc gamma RII) and CD14 antigen. We have shown previously these antigens are regulated by cholesterol and lipoproteins. At 0.5 micrograms/mL of culture medium, lovastatin did not reduce the percentage of receptor-positive cells or the average number of receptor molecules per cell. These observations add to the attractiveness of the drug as an anticholesterolemic agent and also indicate that endogenous cholesterol biosynthesis by monocytes is not required for expression of Fc gamma RI, Fc gamma RII, or CD14.