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Effects of Oxysterols Upon Macrophage and Lymphocyte Functions In Vitro
Effects of Oxysterols Upon Macrophage and Lymphocyte Functions In Vitro Mikhail Dushkin,1* Yakov Schwartz,* Nikolaj Volsky,† Mikhail Musatov,† Eugeni Vereschagin,* Julia Ragino,* Olga Perminova,† and Vladimir Kozlov† *Laboratory of Atherogenesis, Institute of Internal Medicine, Russian Academy of Medical Science, Siberian Division, Vladimirovski Spusk Street, 2a, 630003 Novosibirsk, Russia †Institute of Clinical Immunology, Russian Academy of Medical Science, Siberian Division, Yadrintsevskaja Street, 14, 630091 Novosibirsk, Russia Oxygenated derivatives of cholesterol (oxysterols), found in high concentrations in atherosclerotic lesions, are potent immunosuppressive agents inhibiting T-cell responses to different stimuli. The action of oxysterols on macrophage functions and macrophage-lymphocyte interaction has been poorly investigated. In this work, the effects of 25hydroxycholesterol (25-OHCh) and 7-ketocholesterol (7-KCh) upon some functions of murine peritoneal macrophage (PM), such as generation of reactive oxygen intermediates (ROI), secretion of neopterin and interleukin-1 (IL-1)-like activity, Fc-receptor (FcR) activity, and murine and human lymphocyte functions, participating in lymphocytemacrophage interactions, such as macrophage-activating factor (MAF) and Ia-inducing factor (IaIF) secretion, were studied in vitro. 7-KCh in concentration of 5 mg/mL culture medium only, but not 25-OHCh, significantly inhibited ROI generation by zymosan-stimulated PM. Pretreatment of PM for 22 h with 25-OHCh and 7-KCh led to the decrease of IL-1-like activity secretion. 25-OHCh and 7-KCh inhibited both FcR-dependent binding and phagocytosis of sheep red blood cells (SRBC). Oxysterols did not change both spontaneous and lipopolysaccharide-stimulated secretion of neopterin by PM. 25-OHCh dose-dependently and more efficiently than 7-KCh inhibited murine spleno1
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Prostaglandins & other Lipid Mediators 55:219 –236, 1998 © 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
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Oxysterols and Macrophage/Lymphocyte Function: Dushkin et al. cyte secretion of MAF, which activity was determined by the ability of splenocyte-conditioned medium to stimulate ROI generation in PM. Both 25-OHCh and 7-KCh inhibited significantly proliferative activity of human mixed lymphocyte culture (MLC), as well as lymphocyte secretion of IaIF, which stimulates the expression of HLA antigens in cultured human monocytes. Purified Ch did not alter these parameters. These data showed, that some inflammatory functions of macrophages and lymphocytes may be modified by such environmental conditions as the presence of oxysterols. Keywords: Oxysterols; macrophages; lymphocytes
Introduction A wide spectrum of biological activity of oxygenated derivatives of cholesterol (Ch), or oxysterols, let to consider them to be an important class of natural sterols (1). Some of these compounds, e.g., 7-keto- or 7bhydroxycholesterol (7b-OHCh), are generated extracellularly by a simple autooxidation of cholesterol (2) and plasma low-density lipoproteins (LDL) (3), whereas the others (e.g., 7a-, 22R-, 24S-, 25- and 27-OHCh) are generated in cytocrome P-450-catalysed reactions in endoplasmatic reticulum and mitochondria (4). Increased formation of oxysterols has been observed in a variety of tissues during such pathological processes as atherosclerosis (5,6), inflammation (7), trauma (8) and oxidative stress (9). It can not be excluded, that oxysterols accumulated in different tissues are involved in situ in the mechanisms of pathological processes including alteration of immunocompetent cell functions. Indeed, some effects of oxysterols upon the early steps of T-cell activation have been studied earlier. For instance, 25-OHCh has been shown to suppress the response to mitogens, alloantigens and tumour antigens, and to inhibit the transformation of native lymphocytes into differentiated cytotoxic lymphocytes (10,11). 7-KCh has been demonstrated to inhibit spleen cell cytotoxicity, natural killer cell activity and lymphocyte blastogenesis (12). Intraperitoneal (i.p.) injection of 7,25-dihydroxycholesterol induces a strong inflammatory response, consisting of massive influx of macrophages and neutrophils, but abolishes stimulated expression of class II major histocompatibility complex (MHC) antigens in macrophages (13). Christ et al. (14) found that 25-OHCh, but not 7b-OHCh, induced DNA fragmentation in cancer RDM4 and normal thymocyte cells. Besides, 25-OHCh and 7b-OHCh were shown to induce apoptotic changes in monocyte THP-1 cells (15). Macrophage system has a pivotal control and instrumental role both in non-specific host defence and primary immune response. However the effects of oxysterols on macrophage functions and macrophage-lymphocytes interaction are studied very poor so far. The aim of this work was to
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Oxysterols and Macrophage/Lymphocyte Function: Dushkin et al. study the effects of 25-OHCh and 7-KCh, the oxysterols known to possess immunomodulatory activity, upon the murine macrophage functions in vitro: the generation of reactive oxygen intermediates (ROI), secretion of neopterin and IL-1 (IL-1), expression of membrane Fc-receptors (FcR). In addition, we studied the effects of these oxysterols upon the secretion of macrophage activating factor (MAF) by murine splenocytes and the secretion of Ia-inducing factor (IaIF) by human blood lymphocytes, that are the factors, participating in the regulation of macrophage functions.
Materials and Methods Materials [3H]thymidine (25 Ci/mmol) was purchased from Amersham International (Arlington, IL). 25-OHCh, 7-KCh, Zymosan A, luminol, concanavalin A were purchased from Sigma Chemical Co. (St. Luis, Mo). Culture medium RPMI 1640 and fetal calf serum (FCS) were purchased from Gibco. Lypopolysaccharide (LPS from E. coli 0111:B4) from Difco (Detroit). Animals Male (C57BlxCBA)F1 mice (age 5–10 weeks) were obtained from the Institute of Clinical Immunology (Novosibirsk, Russia) Preparation of Macrophage, Thymocyte and Splenocyte Culture Peritoneal macrophages (PM) were harvested from mice with ice-cold phosphate-buffered saline (PBS), as described previously (16). The cells were transferred into 35 mm Petri dishes or into 24-well plates (2 3 106 cells/dish or 0.8 3 106 cells/well) in RPMI 1640 medium containing 20% FCS, 50 mg/mL gentamicin (medium A). After overnight incubation the nonadherent cells were removed by washing three times with PBS and then cell were used to study oxysterol effects upon PM functions. Thymocyte and splenocyte suspensions were prepared as follows: mice were killed by lethal anaesthesia and their thymus and spleen were aseptically removed. Single-cell suspensions were prepared by teasing the thymus and spleen in RPMI 1640 medium containing 10 mM HEPES and 0.5 mM 2-mercaptoethanol followed by filtration through a nylon membranes. The cells were then placed in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 50 mg/mL gentamicin, 2 mM glutamine, nonessential aminoacids and 1 mM sodium salt of pyruvic acid (medium B). Cell viability (.98%) was assessed with the trypane blue exclusion test. Both 25-OHCh and 7-KCh in concentrations 0.5–5.0 mg/mL (1.2–12.4 mM) did not affect the cell viability. Determination of IL-1-Like Activity of PM-Conditioned Medium PM monolayers in 24-well plate (2 3 106 cell/well) were cultured in medium A with or without 25-OHCh, 7-KCh and Ch in ethanol solution
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Oxysterols and Macrophage/Lymphocyte Function: Dushkin et al. (not more than 5 mL/mL of medium) for 24 h. In control wells the equivalent contents of ethanol were added. Afterwards, the cell monolayers were washed and incubated for 20 h in the same medium in the presence of LPS (20 mg/mL). The levels of IL-1 activity were measured according to the method of Phillips and Rabson, (17). Murine thymocyte suspensions were cultured in medium B in 96-well plates (2 3 105 cell/well) in the presence of suboptimal mitogen concentration of concanavalin A (1 mg/mL) with or without PM supernatants (30%) for 54 h at 37°C in humidified atmosphere of 5% CO2. Incorporation of thymidine into cells was estimated by incubation of cells with [3H]thymidine (1 mCi/well) for 18 h at 37°C. Then the cells were washed and transferred on filters using harvester (Flow Lab.). Radioactivity was measured in vials containing 5 mL of Ultima Gold (Packard) in a liquid scintillation counter (LKB 1211) and the results were expressed as counts per min. (cpm). Determination of Fc-Receptor Expression on PM Membrane and of FcR-Dependent SRBC Phagocytosis Fc-receptor expression and FcR-dependent phagocytotic activity in PM cultures were assessed by method of Rummage and Lev, (18) with slight modifications. PM monolayers in 24-well plate (8 3 105 cell/well) were cultured in medium A with or without 25-OHCh, 7-KCh and Ch in ethanol solution (not more than 5 mL/mL of medium) for 24 h. In control wells the equivalent contents of ethanol were added. Afterwards, the cell monolayers were washed and used in experiments. For phagocytotic activity determination 1 mL of 1% opsonized SRBC suspension was added to PM monolayers and cells were incubated for 30 min. at 30°C, then SRBC suspension was aspirated and 1.5 mL of 0.09% hypotonic saline was added into the wells to lyse non-phagocytized SRBC. The hemolysates were removed, PM were washed out with PBS and solubilized with 0.5 mL of 1% sodium dodecyl sulphate. 100 mL aliquotes of solubilisates obtained were transferred into 96-wells plates and the number of SRBC taken up by PM was assessed photometrically at 405 nm using 8-channel photometer (“Titertek”). The Fc-receptor expression on PM membranes was assessed analogically. Briefly, PM monolayers were incubated with opsonized SRBC for 30 min. at 4°C, then non-binding SRBC were gently washed out with PBS, SRBC bound with macrophage membranes were lysed with hypotonic saline and lysate aliquotes were photometered at 405 nm wave length. Determination of PM Neopterin Secretion PM were incubated in medium A with or without LPS (25 mg/mL) in the presence or absence of oxysterols for 24 h at 37°C, 5% CO2, 100% humidity. Neopterin content was measured in cultural medium by highpressure liquid chromatography method (19) on C-18 column (64 3 2 mm, 5 mm particle size, Nucleosil, Germany) with fluorometric detection
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Oxysterols and Macrophage/Lymphocyte Function: Dushkin et al. (Gilson chromatography system, excitation 350 nm, emission 440 nm) using D (1)-neopterin (Aldrich) as a standard. Determination of ROI Generation ROI generation in PM was assayed in terms of luminol-amplified chemiluminescence (CL) determination as described earlier (16). Briefly, the light emission was recorded continuously in count per min. directly from a dish bottom at 37°C in a two-channel chemiluminometer “Photon” (SOPKTB, USSR). The PM monolayers were washed twice with PBS and supplied with 2 mL of phenol red-free Hank’s balanced solution containing luminol (1 mM). Background CL was measured for 3 min. before addition of 10 mL of oxysterols in ethanol or 10 mL ethanol (control dishes). After 30 min. incubation with sterols the changes in CL were measured during 3 min., after which 100 mL of opsonized zymosan (OZ) in PBS (20 mg/mL) was added, and CL increase was monitored until maximal value was achieved. For the further evaluation, the maximal values obtained from the three measurements were used. Determination of Macrophage Activating Factor (MAF) Secretion by Splenocytes MAF activity in splenocyte-conditioned medium was determined using CL method of Lyubimov et al. (20), based on MAF ability to induce ROI generation in PM in a dose-dependent fashion (21). Murine splenocytes were incubated in the medium B with or without oxysterols for 24 h at 37°C in humidified atmosphere of 5% CO2, then washed with PBS and cultured in fresh medium B containing 5 mg/mL of concanavalin A for 48 h. Then splenocyte culture medium was filtered through 25 mm-pore filters, added to the culture medium of PM (to a final concentration of 10%) and PM were cultured for 20 h. The OZ-stimulated CL was determined in PM monolayers, as described above. MAF activity was defined as values of CL in the presence of concanavalin A-stimulated splenocyte medium minus values of CL in the presence of nonstimulated splenocyte medium. Determination of Proliferative Activity in Mixed Culture of Lymphocytes (MLC) Mononuclear cells (MNC) were isolated from EDTA-stabilized peripheral blood of healthy volunteers using Ficoll-Hypaque gradient centrifugation. Stimulating cells were prepared by treatment of MNC with 25 mg/mL of mitomycin C, then mixed with responding cells (1:1) and MLC was cultured in 96-well plates (“Linbro”). MLC were incubated in RPMI 1640 medium containing 10 mM HEPES, 2 mM L-glutamine, 50 mg/mL gentamicin and 20% IV (AB) human serum (medium C) with or without oxysterols for 6 days at 37°C. Then 1 mCi [3H]thymidine was added into
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Oxysterols and Macrophage/Lymphocyte Function: Dushkin et al. each well, and MLC were incubated for additional 18 h. Cell-associated radioactivity counts were expressed in cpm/min./106 cells. Determination of Ia-Inducing Factor (IaIF) Secretion in MLC Stimulating and responding cells were prepared as described above and incubated separately in medium C with or without oxysterols for 18 h. Then cells were washed twice with PBS, mixed (1:1) and MLC was cultured for 48 h as described above. IaIF activity of culture medium of MLC was determined by method of Seledtsov et al. (22) in human monocyte culture, obtained by standard adhesion method. Monocytes were incubated for 4 days in 100 mL of medium C and 50 mL of MLC culture tested. Then HLA-DR antigen expression was evaluated in monocyte culture using specific fluorescent-labelled monoclonal antibodies. Statistics Results are presented as mean for not less than three replicates per experimental groups. Statistical significance of the differences between the groups was assessed by non-parametrical Mann-Whitney’s test.
Results Effect of Oxysterols on Cell Viability The cell toxicity of oxysterols was assessed by trypane blue exclusion test. Incubation of cells with 5 mg/mL (12.4 mM) of 7-KCh and 25-OHCh influenced non-significantly on viability of cultured PM (98.3 6 0.9%), thymocytes (96.8 6 1.4%), splenocytes (96.1 6 1.5%) and MLC (95.5 6 4.6%). Effect of Oxysterols on ROI Generation by Macrophages PM were incubated for 30 min. at 37°C in Hank’s balanced solution in the presence of 25-OHCh, 7-KCh or Ch (5 mg/mL), then zymosan-induced (2 mg/mL) luminol-dependent CL was measured until maximal value was achieved. 7-KCh at concentration above 2 mg/mL reduced CL response significantly, whereas 25-OHCh and Ch had no significant effect on ROI generation by PM (Fig. 1). Effect of Oxysterols on Production of IL-I-Like Activity by PM PM were preincubated for 24 h in the presence of 25-OHCh, 7-KCh, then monolayers were washed 3 times and cultured for the next 20 h in the presence of 20 mg/mL of LPS. Supernatants were collected and analysed for IL-I-like activity by standard thymocyte comitogenic assay (Fig. 2). Preincubation of PM with 25-OHCh (0.5–5 mg/mL) or 7-KCh (5 mg/mL) led to the decrease of IL-I-like activity production by LPS-stimulated PM. The inhibitory effect of 25-OHCh had concentration-dependent character and at a dose of 5 mg/mL was significantly more pronounced as compared
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FIGURE 1. Influence of the concentration of 25-OHCh, 7-KCh and Ch on PM zymosanstimulated luminol-amplified CL. PM were preincubated for 30 min. at 37°C with 25-OHCh, 7-KCh or Ch, then added zymosan (1 mg/mL) and luminol-amplified CL was assayed until maximal value was achieved. Control PM were incubated in buffer without sterols. Results are expressed as percentage vs. mean control values of CL intensity (100%) obtained in four separate independent experiments performed in triplicates. The bars represent the mean 6 SE. Mean control value was equal to 68.3 6 24.2 3 103 cpm/min./106 cells. *Significant differences vs. control value (p , 0.05).
to 7-KCh effect. Ch (5 mg/mL) produced no significant effect on IL-I-like activity secretion by PM. Effect of Oxysterols on FcR-Dependent Binding and Phagocytosis of SRBC by PM Monolayers of PM were preincubated for 24 h with or without 5 mg/mL of Ch or its oxygenated derivatives, and then binding of SRBC to macrophage membranes and FcR-dependent phagocytosis of SRBC were studied. 25-OHCh as well as 7-KCh inhibited both FcR-dependent binding and phagocytosis of SRBC, while Ch had no significant effect (Fig. 3). Effect of Oxysterols on Neopterin Production by PM PM were incubated for 24 h with or without 5 mg/mL of oxysterols in the presence or absence of 25 mg/mL LPS, and then neopterin concentration in culture medium was determined. LPS induced 1.5-fold increase of neopterin concentration. Both 25-OHCh and 7-KCh had no significant
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FIGURE 2. Effects of the concentration of oxysterols upon the production of IL-1-like substances by PM. PM were preincubated for 24 h at 37°C with or without (control) Ch or its oxygenated derivatives (25-OHCh or 7-KCh) at different concentrations, washed out and incubated for 20 h in the presence of 20 mg/mL LPS. The supernatants were collected and analysed for the IL-1-like activity by the standard thymocyte comitogenic assay. Results are expressed as percentage vsl mean control values of proliferative activity (100%) obtained in three independent experiments performed in tetraplicates. The bars represent the mean 6 SE. Mean control value corresponded to 12.7 6 4.9 3 103 cpm/min. *Significant differences vs. control value (p , 0.05).
effect on nonstimulated and LPS-stimulated neopterin production by PM (Fig. 4). Effect of Oxysterols on MAF-Production by Murine Splenocytes After preincubation of splenocytes with or without oxysterols for 24 h and further incubation of splenocytes with 5 mg/mL of concanavalin A for 48 h, MAF-activity in splenocyte conditioned culture medium was determined by its ability to induce ROI generation in PM. Control splenocyte (incubation without oxysterols) conditioned medium induced a maximal response at the concentration of 10%, and at this concentration the differences between inhibitory effects of 25-OHCh and 7-KCh were clearly discernible (Fig. 5). So the effect of oxysterols on MAF production was assessed further at 10% supernatant concentration. The inhibitory effect of 25-OHCh had concentration-dependent fashion and at a dose of
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FIGURE 3. PM FcR-dependent binding (f) and phagocytosis of SRBC (M) in the presence of sterols. PM were incubated for 24 h with or without Ch or 25-OHCh or 7-KCh (each at 5 mg/mL concentration) and FcR binding and phagocytosis were determined as described in Materials and Methods section. The results are expressed as percentage compared to mean control values (without sterol treatment). Four independent experiments were performed in duplicates. Mean control values corresponded to 0.584 6 0.110 O.D. (for FcR-dependent binding) and to 0.754 6 0.080 units of O.D. (for phagocytosis). *Significant differences as compared to control (p , 0.05).
5 mg/mL was significantly higher than that of 7-KCh. Ch had, but only slight effect on production of MAF activity (Fig. 6). Effect of Oxysterols on Lymphocyte Proliferation in MLC Effect of oxysterols on proliferative response in primary unidirectional MLC was determined after separate preexposure of stimulating (Fig. 7, III) or responding cells (Fig. 7, II) to oxysterols, or after direct addition of oxysterols to MLC incubation medium (Fig. 7, I). Both 25-OHCh and 7-KCh inhibited MLC proliferation at 5 mg/mL concentration. Direct addition of oxysterols into MLC culture medium proved to be more effective, than separate preincubation of responding or stimulating cells. Effect of Oxysterols on the Production of Ia-Inducing Factor by Human Lymphocytes After preincubation of MLC with oxysterols (1 mg/mL), Ia-inducing activity was estimated by the enhancing effect of MLC conditioned culture medium on the HLA-DR-antigens expression in human monocyte (Fig.
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FIGURE 4. Effect of oxysterols on neopterin production by PM stimulated or non-stimulated with LPS. PM were incubated for 24 h with or without 25 mg/mL LPS in the presence (experiment) or absence (control) of 5 mg/mL of oxysterols. Neopterin content was measured in cultural medium by HPLC method with fluorometric detection (19). Three independent experiments were performed in duplicates.
8). Both 25-OHCh and 7-KCh actually completely inhibited the ability of MLC to produce IaIF. Ch had no significant effect on this parameter.
Discussion The data presented in this study demonstrate oxysterol ability to suppress PM and lymphocyte functions, while Ch at the same concentrations has no comparable effects. It could be assumed that the suppressive effects of oxysterols on PM and lymphocytes is due to their cytotoxicity since 7-KCh and 25-OHCh are cytotoxic toward rapidly proliferating cells, such as tumor cells in culture (23). However, in our experiments oxysterol cytotoxicity is rather unlikely, because such cells as lymphocytes or macrophages have been shown to be resistant to high concentrations of the oxysterols (13,24). Moreover, as it has been recently demonstrated (25), the presence of 5 mg/mL of 7-KCh, 25-OHCh and 27-OHCh in the culture medium for 24 h did not alter the adenine release from the cells, although the higher concentrations of these compounds (10 –100 mg/mL) had well detectable cytotoxic effect. In agreement with this study under our experimental conditions 7-KCh and 25-OHCh also did not affect cell viability at concentration of 5 mg/mL (12.4 mM). Both 25-OHCh and 7-KCh inhibited cell proliferation in MLC and the extent of their inhibitory effect depended on experimental conditions: oxysterols added directly in MLC induced maximal inhibitory effect on proliferative response, oxysterol pretreatment of responding cells caused
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FIGURE 5. Influence of concentration of culture medium conditioned by murine splenocytes incubated in the presence (experiment) or absence (control) of oxysterols on OZ-stimulated CL in PM. Murine splenocytes were incubated for 24 h with or without of 5 mg/mL of sterols, then washed out and cultured for the next 48 h in the presence of 5 mg/mL of concanavalin A. Culture medium was added at different concentrations into PM monolayers, PM were cultured for 20 h and then OZ-stimulated CL in PM was measured (see Material and Methods section). The results are expressed as percentage vs. mean control value obtained at 10% concentration of added splenocyte conditioned medium. Three independent experiments were performed in duplicates. Mean control value corresponded to 114.2 6 29.3 3 103 cpm/min./106 cells. *Significant differences vs. control (p , 0.05).
an intermediate one, and pretreatment of stimulating cells induced minimal effect. Since even inactivated stimulating cells are able to produce mitogenic substances (26), it is reasonable to suggest, that oxysterols can inhibit production of such substances, thereby suppressing cell proliferation. These results are in agreement with the earlier communications, that oxysterols at non-cytotoxic concentrations are able to depress strongly the lymphocyte response induced by various antigenic and mitogenic stimuli (24). Besides oxysterols were reported to inhibit splenocyte secretion of IL-2, which is required for final T-cell proliferation (27). Moreover, activated lymphocytes are known to produce the macrophage-
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FIGURE 6. Influence of oxysterol concentration on MAF-like activity production by murine splenocyte culture. Murine splenocytes were incubated with (experiment) or without (control) different concentrations of sterols and splenocyte conditioned medium was used at 10% concentration to stimulate OZ-induced CL in PM culture (for details see Fig. 5). Results are expressed in percentage vs. mean control values (without sterol treatments). Negative control (NC) corresponds to the CL of PM incubated without splenocyte conditioned medium addition and sterols. Four independent experiments were performed in triplicates. Mean control value corresponded to 114.2 6 29.3 3 103 cpm/min./106 cells. *Significant differences vs. control (p , 0.05).
priming factors, which increase macrophage ROI production in response to zymosan or LPS (21). The major priming factor of activated lymphocyte origin is IFN-g which is the principal component responsible for total MAF activity (28). Our study demonstrates, that preincubation of lymphocytes with different concentrations (0.5–5 mg/mL) of 25-OHCh reduced the ability of lymphocyte conditioned medium to stimulate zymosan-induced ROI generation in macrophages, whereas 7-KCh had relatively lower inhibitory effect. The data obtained testify, that 25OHCh at low concentration (0.5 mg/mL) is able to depress strongly the splenocyte production of MAF-like factors. Lymphocyte production of IaIF play an important role in macrophagelymphocyte interactions during inflammatory response and is necessary for expression of the class II antigens in macrophages. The abolishment of class II antigen induction in PM of 7,25-OHCh-treated mice has been reported previously by Moog et al. (13). The authors could not explain
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FIGURE 7. Effect of sterols on MLC proliferative activity. MLC was prepared as described in Material and Methods section. I-MLC was incubated for 6 days with or without (control) 5 mg/mL of sterols. II-Responding cells were preincubated for 18 h with or without (control) 5 mg/mL of sterols, washed out and then mixed with stimulating cells (1:1) and MLC was incubated for 6 days. III-Stimulating cells were preincubated with or without (control) 5 mg/mL of sterols, washed out, then mixed with responding cells (1:1) and incubated for 6 days. Proliferative activity of MLC was determined by [3H]-thymidine incorporation during additional incubation for 18 h. The results are expressed as percentage vs. respective controls. Three independent experiments were done in duplicates. Mean control value corresponded to 4.5 6 0.9 3 103 cpm/min. *Significant differences vs. corresponding controls (p , 0.05). M-control, s-25-OHCh, z-7-Kch, u-Ch.
these results by induction of prostaglandin E2 synthesis since injection of inhibitors of PGE2 production did not led to reversion of oxysterol effect. In our in vitro studies, preincubation of responding cells with comparatively low doses (1 mg/mL) of 7-KCh and 25-OHCh led to the loss of the ability of MLC medium to stimulate expression of class II antigens in human macrophages. It could be assumed, that the decrease of PM class II antigens expression in experiments by Moog et al. (13) are accounted, at least in part, for the inhibitory effects of oxysterols on IaIF secretion. Thus, the results obtained testify, that oxysterols can exert their inhibitory effects upon macrophage-linked functions of lymphocytes. Our study also demonstrates, that oxysterols are able to depress directly some PM functions. Indeed, both 7-KCh and 25-OHCh decreased effectively Fc-receptor expression and FcR-dependent phagocytosis in PM. 25-OHCh and, to a lesser extent, 7-KCh dose-dependently inhibited IL-1-like activity production by LPS-stimulated PM. 7-KCh in concentra-
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FIGURE 8. Effect of oxysterols on production of Ia-inducing factor (IaIF) by human lymphocytes. Stimulating and responding cells were preincubated for 18 h with or without (control) 1 mg/mL sterols, washed out, mixed (1:1) and cultured for 48 h. Activity of IaIF was determined in MLC-conditioned medium by its ability to stimulate II class HLA antigen expression in human monocyte culture using fluorescent-labelled monoclonal antibodies to human HLA DR antigens (see Materials and Methods section). Negative control (NC)—incubation of monocyte without MLC-conditioned medium. The results are expressed as percentage of DR-positive cells vs. total cell number. *Significant differences vs. control (p , 0.05).
tion of 5 mg/mL, but not 25-OHCh, decreased zymosan-stimulated ROI generation in PM. However, oxysterols studied had no effect upon unstimulated and LPS-stimulated macrophage secretion of neopterin, GTPcyclohydrolase product, which is non-specific biochemical indicator of inflammatory activation of macrophages (29,30). The differences in the effects of 7-KCh and 25-OHCh on PM function could be accounted for the different mechanisms of action of these sterols. Two mechanisms are known to mediate oxysterol effects: 1) the regulation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase and LDL receptors (31) and 2) direct interaction of these compounds with plasma membrane (32). 25-OHCh has been shown to be more effective than 7-KCh in inhibiting HMG-CoA reductase (33). Existence of cytosolic oxysterol-binding protein for 25-OHCh and a cis-acting sterol regulatory element located in promoter regions of HMG-CoA-reductase, LDL receptor and apoE genes (31,33,34) suggests that this sterol and its structural analogs could act as intracellular regulatory factors. The participation of oxysterols in regulation of macrophage functions has recently been corraborated in our studies demonstrated that oxysterols are able to decrease
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Oxysterols and Macrophage/Lymphocyte Function: Dushkin et al. the expression of IL-1b and TNF-a mRNA in LPS-stimulated PM (Dushkin et al., manuscript in preparation). As to the direct effects of oxysterols upon membranes, 7-KCh is known to induce by far more substantial modification of membrane dynamic properties, which consequently trigger several biological effects (32), associated with alteration of membrane-linked receptors and enzymes. Unlike 7-KCh, 25-OHCh induces much less modifications of the membrane structure, apparently, due to its more polar nature. Indeed, C-7 oxysterols have been shown to decrease packing ability of phospholipid non-polar chains in liposomes (35,36). However this effect has not been observed for 20-, 22S- and 25-OHCh. Theunissen et al. (37) studied the transfer of radiolabelled oxysterols from monolayers to lipoproteins or vesicles and showed, that transfer rate of oxysterols is distributed in the following order: 7-KCh . 7b-OHCh . 7a-OHCh . 25-OHCh. The authors explained the 20-fold differences in membrane incorporation of 7-KCh and 25-OHCh by more polar nature of 25-OHCh. It could be speculated, that our data on the ability of 7-KCh, but not 25-OHCh, to inhibit ROI generation in PM is explained by alteration of membranelinked NADPH-oxidase. Thus, 7-KCh and 25-OHCh are able to inhibit either macrophage and lymphocyte functions. The formation of oxygenated metabolites of cholesterol in situ could be implicated in mechanisms leading to atherosclerosis (1). Atherosclerotic plaques and macrophage-derived foam cells have been recently reported to contain considerable concentrations of oxysterols (5,6). 7-KCh was found to be the major oxysterol in macrophages from rabbit atherosclerotic aorta (6). Intracellular concentrations of this compound reached 1–7 mg/106 cells in rabbit macrophages and 1–5 mg/106 cells in human macrophages. Human foam cells contained high level of 27-OHCh (3–5 mg/106 cells) not found in rabbit arterial macrophages. 25-OHCh was contained only in minor concentrations both in human and rabbit foam cells. Since 7-KCh has been shown to be the major oxysterol formed during LDL oxidation by macrophages and by copper (3), the accumulation of 7-KCh into foam cells may be connected with macrophage uptake of oxydized LDL or lipids of extracellular origin. 27-OHCh has been reported to be produced by mitochondrial 27-hydroxylase (4), and this sterol is secreted by human alveolar macrophages in high concentrations (5– 8 mg/mL) (5). Recently it has been shown, that 27-OHCh, analogically to 25-OHCh, is involved in down-regulation of LDL receptor and HMG-CoA reductase activities (38,39). Although the immunomodulating effects of 27-OHCh have been poorly investigated, this oxysterol could be expected to have the effects similar to those of 25-OHCh. Atherosclerosis is considered to be a kind of chronic inflammatory disease, in which activated macrophages and lymphocytes are involved (40). The lingering pattern and chronization of the inflammatory process
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Oxysterols and Macrophage/Lymphocyte Function: Dushkin et al. during atherosclerosis development may be caused by the alterations of macrophage functions and macrophage-lymphocyte interactions by oxysterols presented in atheroma. This hypothesis is now under our investigation.
Acknowledgments This work was supported by the International Science Foundation (Grant No. NQ 1300), Russian Foundation for Fundamental Investigations and Russian Atherosclerosis Foundation.
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Editor: Dr. B. Vargaftig
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Prostaglandins & other Lipid Mediators 55:219 –236, 1998 © 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
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Oxysterols and Macrophage/Lymphocyte Function: Dushkin et al. cyte secretion of MAF, which activity was determined by the ability of splenocyte-conditioned medium to stimulate ROI generation in PM. Both 25-OHCh and 7-KCh inhibited significantly proliferative activity of human mixed lymphocyte culture (MLC), as well as lymphocyte secretion of IaIF, which stimulates the expression of HLA antigens in cultured human monocytes. Purified Ch did not alter these parameters. These data showed, that some inflammatory functions of macrophages and lymphocytes may be modified by such environmental conditions as the presence of oxysterols. Keywords: Oxysterols; macrophages; lymphocytes
Introduction A wide spectrum of biological activity of oxygenated derivatives of cholesterol (Ch), or oxysterols, let to consider them to be an important class of natural sterols (1). Some of these compounds, e.g., 7-keto- or 7bhydroxycholesterol (7b-OHCh), are generated extracellularly by a simple autooxidation of cholesterol (2) and plasma low-density lipoproteins (LDL) (3), whereas the others (e.g., 7a-, 22R-, 24S-, 25- and 27-OHCh) are generated in cytocrome P-450-catalysed reactions in endoplasmatic reticulum and mitochondria (4). Increased formation of oxysterols has been observed in a variety of tissues during such pathological processes as atherosclerosis (5,6), inflammation (7), trauma (8) and oxidative stress (9). It can not be excluded, that oxysterols accumulated in different tissues are involved in situ in the mechanisms of pathological processes including alteration of immunocompetent cell functions. Indeed, some effects of oxysterols upon the early steps of T-cell activation have been studied earlier. For instance, 25-OHCh has been shown to suppress the response to mitogens, alloantigens and tumour antigens, and to inhibit the transformation of native lymphocytes into differentiated cytotoxic lymphocytes (10,11). 7-KCh has been demonstrated to inhibit spleen cell cytotoxicity, natural killer cell activity and lymphocyte blastogenesis (12). Intraperitoneal (i.p.) injection of 7,25-dihydroxycholesterol induces a strong inflammatory response, consisting of massive influx of macrophages and neutrophils, but abolishes stimulated expression of class II major histocompatibility complex (MHC) antigens in macrophages (13). Christ et al. (14) found that 25-OHCh, but not 7b-OHCh, induced DNA fragmentation in cancer RDM4 and normal thymocyte cells. Besides, 25-OHCh and 7b-OHCh were shown to induce apoptotic changes in monocyte THP-1 cells (15). Macrophage system has a pivotal control and instrumental role both in non-specific host defence and primary immune response. However the effects of oxysterols on macrophage functions and macrophage-lymphocytes interaction are studied very poor so far. The aim of this work was to
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Oxysterols and Macrophage/Lymphocyte Function: Dushkin et al. study the effects of 25-OHCh and 7-KCh, the oxysterols known to possess immunomodulatory activity, upon the murine macrophage functions in vitro: the generation of reactive oxygen intermediates (ROI), secretion of neopterin and IL-1 (IL-1), expression of membrane Fc-receptors (FcR). In addition, we studied the effects of these oxysterols upon the secretion of macrophage activating factor (MAF) by murine splenocytes and the secretion of Ia-inducing factor (IaIF) by human blood lymphocytes, that are the factors, participating in the regulation of macrophage functions.
Materials and Methods Materials [3H]thymidine (25 Ci/mmol) was purchased from Amersham International (Arlington, IL). 25-OHCh, 7-KCh, Zymosan A, luminol, concanavalin A were purchased from Sigma Chemical Co. (St. Luis, Mo). Culture medium RPMI 1640 and fetal calf serum (FCS) were purchased from Gibco. Lypopolysaccharide (LPS from E. coli 0111:B4) from Difco (Detroit). Animals Male (C57BlxCBA)F1 mice (age 5–10 weeks) were obtained from the Institute of Clinical Immunology (Novosibirsk, Russia) Preparation of Macrophage, Thymocyte and Splenocyte Culture Peritoneal macrophages (PM) were harvested from mice with ice-cold phosphate-buffered saline (PBS), as described previously (16). The cells were transferred into 35 mm Petri dishes or into 24-well plates (2 3 106 cells/dish or 0.8 3 106 cells/well) in RPMI 1640 medium containing 20% FCS, 50 mg/mL gentamicin (medium A). After overnight incubation the nonadherent cells were removed by washing three times with PBS and then cell were used to study oxysterol effects upon PM functions. Thymocyte and splenocyte suspensions were prepared as follows: mice were killed by lethal anaesthesia and their thymus and spleen were aseptically removed. Single-cell suspensions were prepared by teasing the thymus and spleen in RPMI 1640 medium containing 10 mM HEPES and 0.5 mM 2-mercaptoethanol followed by filtration through a nylon membranes. The cells were then placed in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 50 mg/mL gentamicin, 2 mM glutamine, nonessential aminoacids and 1 mM sodium salt of pyruvic acid (medium B). Cell viability (.98%) was assessed with the trypane blue exclusion test. Both 25-OHCh and 7-KCh in concentrations 0.5–5.0 mg/mL (1.2–12.4 mM) did not affect the cell viability. Determination of IL-1-Like Activity of PM-Conditioned Medium PM monolayers in 24-well plate (2 3 106 cell/well) were cultured in medium A with or without 25-OHCh, 7-KCh and Ch in ethanol solution
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Oxysterols and Macrophage/Lymphocyte Function: Dushkin et al. (not more than 5 mL/mL of medium) for 24 h. In control wells the equivalent contents of ethanol were added. Afterwards, the cell monolayers were washed and incubated for 20 h in the same medium in the presence of LPS (20 mg/mL). The levels of IL-1 activity were measured according to the method of Phillips and Rabson, (17). Murine thymocyte suspensions were cultured in medium B in 96-well plates (2 3 105 cell/well) in the presence of suboptimal mitogen concentration of concanavalin A (1 mg/mL) with or without PM supernatants (30%) for 54 h at 37°C in humidified atmosphere of 5% CO2. Incorporation of thymidine into cells was estimated by incubation of cells with [3H]thymidine (1 mCi/well) for 18 h at 37°C. Then the cells were washed and transferred on filters using harvester (Flow Lab.). Radioactivity was measured in vials containing 5 mL of Ultima Gold (Packard) in a liquid scintillation counter (LKB 1211) and the results were expressed as counts per min. (cpm). Determination of Fc-Receptor Expression on PM Membrane and of FcR-Dependent SRBC Phagocytosis Fc-receptor expression and FcR-dependent phagocytotic activity in PM cultures were assessed by method of Rummage and Lev, (18) with slight modifications. PM monolayers in 24-well plate (8 3 105 cell/well) were cultured in medium A with or without 25-OHCh, 7-KCh and Ch in ethanol solution (not more than 5 mL/mL of medium) for 24 h. In control wells the equivalent contents of ethanol were added. Afterwards, the cell monolayers were washed and used in experiments. For phagocytotic activity determination 1 mL of 1% opsonized SRBC suspension was added to PM monolayers and cells were incubated for 30 min. at 30°C, then SRBC suspension was aspirated and 1.5 mL of 0.09% hypotonic saline was added into the wells to lyse non-phagocytized SRBC. The hemolysates were removed, PM were washed out with PBS and solubilized with 0.5 mL of 1% sodium dodecyl sulphate. 100 mL aliquotes of solubilisates obtained were transferred into 96-wells plates and the number of SRBC taken up by PM was assessed photometrically at 405 nm using 8-channel photometer (“Titertek”). The Fc-receptor expression on PM membranes was assessed analogically. Briefly, PM monolayers were incubated with opsonized SRBC for 30 min. at 4°C, then non-binding SRBC were gently washed out with PBS, SRBC bound with macrophage membranes were lysed with hypotonic saline and lysate aliquotes were photometered at 405 nm wave length. Determination of PM Neopterin Secretion PM were incubated in medium A with or without LPS (25 mg/mL) in the presence or absence of oxysterols for 24 h at 37°C, 5% CO2, 100% humidity. Neopterin content was measured in cultural medium by highpressure liquid chromatography method (19) on C-18 column (64 3 2 mm, 5 mm particle size, Nucleosil, Germany) with fluorometric detection
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Oxysterols and Macrophage/Lymphocyte Function: Dushkin et al. (Gilson chromatography system, excitation 350 nm, emission 440 nm) using D (1)-neopterin (Aldrich) as a standard. Determination of ROI Generation ROI generation in PM was assayed in terms of luminol-amplified chemiluminescence (CL) determination as described earlier (16). Briefly, the light emission was recorded continuously in count per min. directly from a dish bottom at 37°C in a two-channel chemiluminometer “Photon” (SOPKTB, USSR). The PM monolayers were washed twice with PBS and supplied with 2 mL of phenol red-free Hank’s balanced solution containing luminol (1 mM). Background CL was measured for 3 min. before addition of 10 mL of oxysterols in ethanol or 10 mL ethanol (control dishes). After 30 min. incubation with sterols the changes in CL were measured during 3 min., after which 100 mL of opsonized zymosan (OZ) in PBS (20 mg/mL) was added, and CL increase was monitored until maximal value was achieved. For the further evaluation, the maximal values obtained from the three measurements were used. Determination of Macrophage Activating Factor (MAF) Secretion by Splenocytes MAF activity in splenocyte-conditioned medium was determined using CL method of Lyubimov et al. (20), based on MAF ability to induce ROI generation in PM in a dose-dependent fashion (21). Murine splenocytes were incubated in the medium B with or without oxysterols for 24 h at 37°C in humidified atmosphere of 5% CO2, then washed with PBS and cultured in fresh medium B containing 5 mg/mL of concanavalin A for 48 h. Then splenocyte culture medium was filtered through 25 mm-pore filters, added to the culture medium of PM (to a final concentration of 10%) and PM were cultured for 20 h. The OZ-stimulated CL was determined in PM monolayers, as described above. MAF activity was defined as values of CL in the presence of concanavalin A-stimulated splenocyte medium minus values of CL in the presence of nonstimulated splenocyte medium. Determination of Proliferative Activity in Mixed Culture of Lymphocytes (MLC) Mononuclear cells (MNC) were isolated from EDTA-stabilized peripheral blood of healthy volunteers using Ficoll-Hypaque gradient centrifugation. Stimulating cells were prepared by treatment of MNC with 25 mg/mL of mitomycin C, then mixed with responding cells (1:1) and MLC was cultured in 96-well plates (“Linbro”). MLC were incubated in RPMI 1640 medium containing 10 mM HEPES, 2 mM L-glutamine, 50 mg/mL gentamicin and 20% IV (AB) human serum (medium C) with or without oxysterols for 6 days at 37°C. Then 1 mCi [3H]thymidine was added into
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Oxysterols and Macrophage/Lymphocyte Function: Dushkin et al. each well, and MLC were incubated for additional 18 h. Cell-associated radioactivity counts were expressed in cpm/min./106 cells. Determination of Ia-Inducing Factor (IaIF) Secretion in MLC Stimulating and responding cells were prepared as described above and incubated separately in medium C with or without oxysterols for 18 h. Then cells were washed twice with PBS, mixed (1:1) and MLC was cultured for 48 h as described above. IaIF activity of culture medium of MLC was determined by method of Seledtsov et al. (22) in human monocyte culture, obtained by standard adhesion method. Monocytes were incubated for 4 days in 100 mL of medium C and 50 mL of MLC culture tested. Then HLA-DR antigen expression was evaluated in monocyte culture using specific fluorescent-labelled monoclonal antibodies. Statistics Results are presented as mean for not less than three replicates per experimental groups. Statistical significance of the differences between the groups was assessed by non-parametrical Mann-Whitney’s test.
Results Effect of Oxysterols on Cell Viability The cell toxicity of oxysterols was assessed by trypane blue exclusion test. Incubation of cells with 5 mg/mL (12.4 mM) of 7-KCh and 25-OHCh influenced non-significantly on viability of cultured PM (98.3 6 0.9%), thymocytes (96.8 6 1.4%), splenocytes (96.1 6 1.5%) and MLC (95.5 6 4.6%). Effect of Oxysterols on ROI Generation by Macrophages PM were incubated for 30 min. at 37°C in Hank’s balanced solution in the presence of 25-OHCh, 7-KCh or Ch (5 mg/mL), then zymosan-induced (2 mg/mL) luminol-dependent CL was measured until maximal value was achieved. 7-KCh at concentration above 2 mg/mL reduced CL response significantly, whereas 25-OHCh and Ch had no significant effect on ROI generation by PM (Fig. 1). Effect of Oxysterols on Production of IL-I-Like Activity by PM PM were preincubated for 24 h in the presence of 25-OHCh, 7-KCh, then monolayers were washed 3 times and cultured for the next 20 h in the presence of 20 mg/mL of LPS. Supernatants were collected and analysed for IL-I-like activity by standard thymocyte comitogenic assay (Fig. 2). Preincubation of PM with 25-OHCh (0.5–5 mg/mL) or 7-KCh (5 mg/mL) led to the decrease of IL-I-like activity production by LPS-stimulated PM. The inhibitory effect of 25-OHCh had concentration-dependent character and at a dose of 5 mg/mL was significantly more pronounced as compared
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FIGURE 1. Influence of the concentration of 25-OHCh, 7-KCh and Ch on PM zymosanstimulated luminol-amplified CL. PM were preincubated for 30 min. at 37°C with 25-OHCh, 7-KCh or Ch, then added zymosan (1 mg/mL) and luminol-amplified CL was assayed until maximal value was achieved. Control PM were incubated in buffer without sterols. Results are expressed as percentage vs. mean control values of CL intensity (100%) obtained in four separate independent experiments performed in triplicates. The bars represent the mean 6 SE. Mean control value was equal to 68.3 6 24.2 3 103 cpm/min./106 cells. *Significant differences vs. control value (p , 0.05).
to 7-KCh effect. Ch (5 mg/mL) produced no significant effect on IL-I-like activity secretion by PM. Effect of Oxysterols on FcR-Dependent Binding and Phagocytosis of SRBC by PM Monolayers of PM were preincubated for 24 h with or without 5 mg/mL of Ch or its oxygenated derivatives, and then binding of SRBC to macrophage membranes and FcR-dependent phagocytosis of SRBC were studied. 25-OHCh as well as 7-KCh inhibited both FcR-dependent binding and phagocytosis of SRBC, while Ch had no significant effect (Fig. 3). Effect of Oxysterols on Neopterin Production by PM PM were incubated for 24 h with or without 5 mg/mL of oxysterols in the presence or absence of 25 mg/mL LPS, and then neopterin concentration in culture medium was determined. LPS induced 1.5-fold increase of neopterin concentration. Both 25-OHCh and 7-KCh had no significant
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FIGURE 2. Effects of the concentration of oxysterols upon the production of IL-1-like substances by PM. PM were preincubated for 24 h at 37°C with or without (control) Ch or its oxygenated derivatives (25-OHCh or 7-KCh) at different concentrations, washed out and incubated for 20 h in the presence of 20 mg/mL LPS. The supernatants were collected and analysed for the IL-1-like activity by the standard thymocyte comitogenic assay. Results are expressed as percentage vsl mean control values of proliferative activity (100%) obtained in three independent experiments performed in tetraplicates. The bars represent the mean 6 SE. Mean control value corresponded to 12.7 6 4.9 3 103 cpm/min. *Significant differences vs. control value (p , 0.05).
effect on nonstimulated and LPS-stimulated neopterin production by PM (Fig. 4). Effect of Oxysterols on MAF-Production by Murine Splenocytes After preincubation of splenocytes with or without oxysterols for 24 h and further incubation of splenocytes with 5 mg/mL of concanavalin A for 48 h, MAF-activity in splenocyte conditioned culture medium was determined by its ability to induce ROI generation in PM. Control splenocyte (incubation without oxysterols) conditioned medium induced a maximal response at the concentration of 10%, and at this concentration the differences between inhibitory effects of 25-OHCh and 7-KCh were clearly discernible (Fig. 5). So the effect of oxysterols on MAF production was assessed further at 10% supernatant concentration. The inhibitory effect of 25-OHCh had concentration-dependent fashion and at a dose of
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FIGURE 3. PM FcR-dependent binding (f) and phagocytosis of SRBC (M) in the presence of sterols. PM were incubated for 24 h with or without Ch or 25-OHCh or 7-KCh (each at 5 mg/mL concentration) and FcR binding and phagocytosis were determined as described in Materials and Methods section. The results are expressed as percentage compared to mean control values (without sterol treatment). Four independent experiments were performed in duplicates. Mean control values corresponded to 0.584 6 0.110 O.D. (for FcR-dependent binding) and to 0.754 6 0.080 units of O.D. (for phagocytosis). *Significant differences as compared to control (p , 0.05).
5 mg/mL was significantly higher than that of 7-KCh. Ch had, but only slight effect on production of MAF activity (Fig. 6). Effect of Oxysterols on Lymphocyte Proliferation in MLC Effect of oxysterols on proliferative response in primary unidirectional MLC was determined after separate preexposure of stimulating (Fig. 7, III) or responding cells (Fig. 7, II) to oxysterols, or after direct addition of oxysterols to MLC incubation medium (Fig. 7, I). Both 25-OHCh and 7-KCh inhibited MLC proliferation at 5 mg/mL concentration. Direct addition of oxysterols into MLC culture medium proved to be more effective, than separate preincubation of responding or stimulating cells. Effect of Oxysterols on the Production of Ia-Inducing Factor by Human Lymphocytes After preincubation of MLC with oxysterols (1 mg/mL), Ia-inducing activity was estimated by the enhancing effect of MLC conditioned culture medium on the HLA-DR-antigens expression in human monocyte (Fig.
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FIGURE 4. Effect of oxysterols on neopterin production by PM stimulated or non-stimulated with LPS. PM were incubated for 24 h with or without 25 mg/mL LPS in the presence (experiment) or absence (control) of 5 mg/mL of oxysterols. Neopterin content was measured in cultural medium by HPLC method with fluorometric detection (19). Three independent experiments were performed in duplicates.
8). Both 25-OHCh and 7-KCh actually completely inhibited the ability of MLC to produce IaIF. Ch had no significant effect on this parameter.
Discussion The data presented in this study demonstrate oxysterol ability to suppress PM and lymphocyte functions, while Ch at the same concentrations has no comparable effects. It could be assumed that the suppressive effects of oxysterols on PM and lymphocytes is due to their cytotoxicity since 7-KCh and 25-OHCh are cytotoxic toward rapidly proliferating cells, such as tumor cells in culture (23). However, in our experiments oxysterol cytotoxicity is rather unlikely, because such cells as lymphocytes or macrophages have been shown to be resistant to high concentrations of the oxysterols (13,24). Moreover, as it has been recently demonstrated (25), the presence of 5 mg/mL of 7-KCh, 25-OHCh and 27-OHCh in the culture medium for 24 h did not alter the adenine release from the cells, although the higher concentrations of these compounds (10 –100 mg/mL) had well detectable cytotoxic effect. In agreement with this study under our experimental conditions 7-KCh and 25-OHCh also did not affect cell viability at concentration of 5 mg/mL (12.4 mM). Both 25-OHCh and 7-KCh inhibited cell proliferation in MLC and the extent of their inhibitory effect depended on experimental conditions: oxysterols added directly in MLC induced maximal inhibitory effect on proliferative response, oxysterol pretreatment of responding cells caused
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FIGURE 5. Influence of concentration of culture medium conditioned by murine splenocytes incubated in the presence (experiment) or absence (control) of oxysterols on OZ-stimulated CL in PM. Murine splenocytes were incubated for 24 h with or without of 5 mg/mL of sterols, then washed out and cultured for the next 48 h in the presence of 5 mg/mL of concanavalin A. Culture medium was added at different concentrations into PM monolayers, PM were cultured for 20 h and then OZ-stimulated CL in PM was measured (see Material and Methods section). The results are expressed as percentage vs. mean control value obtained at 10% concentration of added splenocyte conditioned medium. Three independent experiments were performed in duplicates. Mean control value corresponded to 114.2 6 29.3 3 103 cpm/min./106 cells. *Significant differences vs. control (p , 0.05).
an intermediate one, and pretreatment of stimulating cells induced minimal effect. Since even inactivated stimulating cells are able to produce mitogenic substances (26), it is reasonable to suggest, that oxysterols can inhibit production of such substances, thereby suppressing cell proliferation. These results are in agreement with the earlier communications, that oxysterols at non-cytotoxic concentrations are able to depress strongly the lymphocyte response induced by various antigenic and mitogenic stimuli (24). Besides oxysterols were reported to inhibit splenocyte secretion of IL-2, which is required for final T-cell proliferation (27). Moreover, activated lymphocytes are known to produce the macrophage-
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FIGURE 6. Influence of oxysterol concentration on MAF-like activity production by murine splenocyte culture. Murine splenocytes were incubated with (experiment) or without (control) different concentrations of sterols and splenocyte conditioned medium was used at 10% concentration to stimulate OZ-induced CL in PM culture (for details see Fig. 5). Results are expressed in percentage vs. mean control values (without sterol treatments). Negative control (NC) corresponds to the CL of PM incubated without splenocyte conditioned medium addition and sterols. Four independent experiments were performed in triplicates. Mean control value corresponded to 114.2 6 29.3 3 103 cpm/min./106 cells. *Significant differences vs. control (p , 0.05).
priming factors, which increase macrophage ROI production in response to zymosan or LPS (21). The major priming factor of activated lymphocyte origin is IFN-g which is the principal component responsible for total MAF activity (28). Our study demonstrates, that preincubation of lymphocytes with different concentrations (0.5–5 mg/mL) of 25-OHCh reduced the ability of lymphocyte conditioned medium to stimulate zymosan-induced ROI generation in macrophages, whereas 7-KCh had relatively lower inhibitory effect. The data obtained testify, that 25OHCh at low concentration (0.5 mg/mL) is able to depress strongly the splenocyte production of MAF-like factors. Lymphocyte production of IaIF play an important role in macrophagelymphocyte interactions during inflammatory response and is necessary for expression of the class II antigens in macrophages. The abolishment of class II antigen induction in PM of 7,25-OHCh-treated mice has been reported previously by Moog et al. (13). The authors could not explain
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FIGURE 7. Effect of sterols on MLC proliferative activity. MLC was prepared as described in Material and Methods section. I-MLC was incubated for 6 days with or without (control) 5 mg/mL of sterols. II-Responding cells were preincubated for 18 h with or without (control) 5 mg/mL of sterols, washed out and then mixed with stimulating cells (1:1) and MLC was incubated for 6 days. III-Stimulating cells were preincubated with or without (control) 5 mg/mL of sterols, washed out, then mixed with responding cells (1:1) and incubated for 6 days. Proliferative activity of MLC was determined by [3H]-thymidine incorporation during additional incubation for 18 h. The results are expressed as percentage vs. respective controls. Three independent experiments were done in duplicates. Mean control value corresponded to 4.5 6 0.9 3 103 cpm/min. *Significant differences vs. corresponding controls (p , 0.05). M-control, s-25-OHCh, z-7-Kch, u-Ch.
these results by induction of prostaglandin E2 synthesis since injection of inhibitors of PGE2 production did not led to reversion of oxysterol effect. In our in vitro studies, preincubation of responding cells with comparatively low doses (1 mg/mL) of 7-KCh and 25-OHCh led to the loss of the ability of MLC medium to stimulate expression of class II antigens in human macrophages. It could be assumed, that the decrease of PM class II antigens expression in experiments by Moog et al. (13) are accounted, at least in part, for the inhibitory effects of oxysterols on IaIF secretion. Thus, the results obtained testify, that oxysterols can exert their inhibitory effects upon macrophage-linked functions of lymphocytes. Our study also demonstrates, that oxysterols are able to depress directly some PM functions. Indeed, both 7-KCh and 25-OHCh decreased effectively Fc-receptor expression and FcR-dependent phagocytosis in PM. 25-OHCh and, to a lesser extent, 7-KCh dose-dependently inhibited IL-1-like activity production by LPS-stimulated PM. 7-KCh in concentra-
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FIGURE 8. Effect of oxysterols on production of Ia-inducing factor (IaIF) by human lymphocytes. Stimulating and responding cells were preincubated for 18 h with or without (control) 1 mg/mL sterols, washed out, mixed (1:1) and cultured for 48 h. Activity of IaIF was determined in MLC-conditioned medium by its ability to stimulate II class HLA antigen expression in human monocyte culture using fluorescent-labelled monoclonal antibodies to human HLA DR antigens (see Materials and Methods section). Negative control (NC)—incubation of monocyte without MLC-conditioned medium. The results are expressed as percentage of DR-positive cells vs. total cell number. *Significant differences vs. control (p , 0.05).
tion of 5 mg/mL, but not 25-OHCh, decreased zymosan-stimulated ROI generation in PM. However, oxysterols studied had no effect upon unstimulated and LPS-stimulated macrophage secretion of neopterin, GTPcyclohydrolase product, which is non-specific biochemical indicator of inflammatory activation of macrophages (29,30). The differences in the effects of 7-KCh and 25-OHCh on PM function could be accounted for the different mechanisms of action of these sterols. Two mechanisms are known to mediate oxysterol effects: 1) the regulation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase and LDL receptors (31) and 2) direct interaction of these compounds with plasma membrane (32). 25-OHCh has been shown to be more effective than 7-KCh in inhibiting HMG-CoA reductase (33). Existence of cytosolic oxysterol-binding protein for 25-OHCh and a cis-acting sterol regulatory element located in promoter regions of HMG-CoA-reductase, LDL receptor and apoE genes (31,33,34) suggests that this sterol and its structural analogs could act as intracellular regulatory factors. The participation of oxysterols in regulation of macrophage functions has recently been corraborated in our studies demonstrated that oxysterols are able to decrease
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Oxysterols and Macrophage/Lymphocyte Function: Dushkin et al. the expression of IL-1b and TNF-a mRNA in LPS-stimulated PM (Dushkin et al., manuscript in preparation). As to the direct effects of oxysterols upon membranes, 7-KCh is known to induce by far more substantial modification of membrane dynamic properties, which consequently trigger several biological effects (32), associated with alteration of membrane-linked receptors and enzymes. Unlike 7-KCh, 25-OHCh induces much less modifications of the membrane structure, apparently, due to its more polar nature. Indeed, C-7 oxysterols have been shown to decrease packing ability of phospholipid non-polar chains in liposomes (35,36). However this effect has not been observed for 20-, 22S- and 25-OHCh. Theunissen et al. (37) studied the transfer of radiolabelled oxysterols from monolayers to lipoproteins or vesicles and showed, that transfer rate of oxysterols is distributed in the following order: 7-KCh . 7b-OHCh . 7a-OHCh . 25-OHCh. The authors explained the 20-fold differences in membrane incorporation of 7-KCh and 25-OHCh by more polar nature of 25-OHCh. It could be speculated, that our data on the ability of 7-KCh, but not 25-OHCh, to inhibit ROI generation in PM is explained by alteration of membranelinked NADPH-oxidase. Thus, 7-KCh and 25-OHCh are able to inhibit either macrophage and lymphocyte functions. The formation of oxygenated metabolites of cholesterol in situ could be implicated in mechanisms leading to atherosclerosis (1). Atherosclerotic plaques and macrophage-derived foam cells have been recently reported to contain considerable concentrations of oxysterols (5,6). 7-KCh was found to be the major oxysterol in macrophages from rabbit atherosclerotic aorta (6). Intracellular concentrations of this compound reached 1–7 mg/106 cells in rabbit macrophages and 1–5 mg/106 cells in human macrophages. Human foam cells contained high level of 27-OHCh (3–5 mg/106 cells) not found in rabbit arterial macrophages. 25-OHCh was contained only in minor concentrations both in human and rabbit foam cells. Since 7-KCh has been shown to be the major oxysterol formed during LDL oxidation by macrophages and by copper (3), the accumulation of 7-KCh into foam cells may be connected with macrophage uptake of oxydized LDL or lipids of extracellular origin. 27-OHCh has been reported to be produced by mitochondrial 27-hydroxylase (4), and this sterol is secreted by human alveolar macrophages in high concentrations (5– 8 mg/mL) (5). Recently it has been shown, that 27-OHCh, analogically to 25-OHCh, is involved in down-regulation of LDL receptor and HMG-CoA reductase activities (38,39). Although the immunomodulating effects of 27-OHCh have been poorly investigated, this oxysterol could be expected to have the effects similar to those of 25-OHCh. Atherosclerosis is considered to be a kind of chronic inflammatory disease, in which activated macrophages and lymphocytes are involved (40). The lingering pattern and chronization of the inflammatory process
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Oxysterols and Macrophage/Lymphocyte Function: Dushkin et al. during atherosclerosis development may be caused by the alterations of macrophage functions and macrophage-lymphocyte interactions by oxysterols presented in atheroma. This hypothesis is now under our investigation.
Acknowledgments This work was supported by the International Science Foundation (Grant No. NQ 1300), Russian Foundation for Fundamental Investigations and Russian Atherosclerosis Foundation.
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Received: 11-21-97
Accepted: 12-11-97
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