RESUMO
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.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Membrana Celular/efeitos dos fármacos , Colesterol/farmacologia , Glicosilfosfatidilinositóis/metabolismo , Monócitos/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica , Humanos , Receptores de Lipopolissacarídeos , Lipoproteínas LDL/farmacologia , Lipoproteínas VLDL/farmacologia , Monócitos/metabolismo , Receptores de IgG/efeitos dos fármacos , Receptores de IgG/metabolismoRESUMO
Plasma lipoproteins have been implicated in immunoregulation. Here we report that LDL and VLDL up-regulate high affinity Fc receptors (Fc gamma RI) in normal human monocytes. Adherent monocytes were cultured for 4 days in media containing fetal calf serum or delipidated serum. Immunofluorescence analysis showed a significant decrease in percentage of Fc gamma RI-positive cells from 85 +/- 3 in medium containing normal serum to 54 +/- 9 in medium containing delipidated serum. The decrease in the fraction of cells expressing Fc gamma RI was parallel to a decrease in the average number of receptor molecules per cell as indicated by a decrease in the mean value fluorescence intensity from 234 +/- 20 to 112 +/- 14. The inhibition of Fc gamma RI expression was overcome by addition to the culture medium of LDL or VLDL. Since pure cholesterol is ineffective, it is proposed that these lipoproteins deliver a component(s) such as apolipoprotein B-100 which triggers a signal leading to up-regulation of Fc gamma RI in monocytes and macrophages.
Assuntos
Lipoproteínas LDL/fisiologia , Lipoproteínas VLDL/fisiologia , Monócitos/metabolismo , Receptores de IgG/metabolismo , Regulação para Cima/fisiologia , Células Cultivadas , Meios de Cultura , Citometria de Fluxo , Imunofluorescência , Humanos , MasculinoRESUMO
Optimal expression of the high-affinity Fc receptor for IgG (FcRI) by the human monocyte cell line U-937 requires the presence of low density lipoprotein (LDL), and neither cholesterol nor high density lipoprotein can provide the component necessary for optimal FcRI expression. Here we show that FcR-mediated phagocytosis also requires LDL. U-937 cells were cultured in medium containing interferon gamma and either fetal calf serum (FCS) or delipidated FCS (DLFCS). The phagocytosis of IgG-coated erythrocytes was measured by a colorimetric assay. U-937 cells cultured in DLFCS medium had less than 16% of the phagocytic activity of cells cultured in normal FCS medium. Phagocytosis of IgG-coated erythrocytes could be inhibited 85% by the addition of murine IgG2a myeloma protein (5 micrograms/ml). U-937 cells cultured in DLFCS medium supplemented with pure cholesterol in ethanol (10 micrograms/ml) had only 30% of the phagocytic activity of cells grown in FCS medium. Addition of very low density lipoprotein (0.2 mg of protein per ml) to DLFCS medium also failed to increase phagocytosis. However, the addition of LDL (0.2 mg of protein per ml) to DLFCS medium restored 90% of the phagocytic activity. Since neither pure cholesterol nor very low density lipoprotein restored normal phagocytic function to U-937 cells despite a normalization of cellular cholesterol content, the restoration of phagocytosis observed with LDL replacement cannot be explained by mere delivery of cholesterol by LDL. Thus, LDL is required for the expression of FcRI and FcR-mediated phagocytosis by U-937 cells and may be an important regulator of phagocytic activity of monocytes and macrophages in vivo.
Assuntos
Lipoproteínas LDL/fisiologia , Fagocitose/imunologia , Receptores Fc/fisiologia , Animais , Bovinos , Linhagem Celular , Colesterol/análise , Colesterol/farmacologia , Meios de Cultura , Eritrócitos/imunologia , Humanos , Imunoglobulina G , Cinética , Lipoproteínas/farmacologia , Lipoproteínas LDL/sangue , Fagocitose/efeitos dos fármacos , Receptores Fc/efeitos dos fármacosRESUMO
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.
Assuntos
Antígenos de Diferenciação/biossíntese , LDL-Colesterol/fisiologia , Receptores Fc/biossíntese , Anticorpos Monoclonais , Antígenos de Diferenciação/metabolismo , LDL-Colesterol/sangue , Citometria de Fluxo , Humanos , Imunoglobulina G/metabolismo , Técnicas In Vitro , Interferon gama/farmacologia , Masculino , Receptores Fc/metabolismo , Receptores de IgGRESUMO
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.
Assuntos
Adesão Celular/efeitos dos fármacos , Colesterol/fisiologia , Acetato de Tetradecanoilforbol/farmacologia , Linhagem Celular , Colesterol/farmacologia , Meios de Cultura , Humanos , Macrófagos , Monócitos , Superóxidos/metabolismoRESUMO
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.
Assuntos
Colesterol/farmacologia , Endocitose/efeitos dos fármacos , Lipoproteínas LDL/farmacologia , Monócitos/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Cloroquina/farmacologia , Colesterol/metabolismo , Concanavalina A/farmacologia , Heparina/farmacologia , Peroxidase do Rábano Silvestre/metabolismo , Humanos , Lipoproteínas/metabolismo , Lipoproteínas/farmacologia , Lipoproteínas LDL/metabolismo , Linfoma Difuso de Grandes Células B , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Monócitos/metabolismoRESUMO
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.