LPS-Induced Macrophage Activation and Plasma Membrane Fluidity Changes are Inhibited Under Oxidative Stress

Macrophage activation is essential for a correct and efficient response of innate immunity. During oxidative stress membrane receptors and/or membrane lipid dynamics can be altered, leading to dysfunctional cell responses. Our aim is to analyze membrane fluidity modifications and cell function under oxidative stress in LPS-activated macrophages. Membrane fluidity of individual living THP-1 macrophages was evaluated by the technique two-photon microscopy. LPS-activated macrophage function was determined by TNFα secretion. It was shown that LPS activation causes fluidification of macrophage plasma membrane and production of TNFα. However, oxidative stress induces rigidification of macrophage plasma membrane and inhibition of cell activation, which is evidenced by a decrease of TNFα secretion. Thus, under oxidative conditions macrophage proinflammatory response might develop in an inefficient manner.     

Stimulation of macrophage by polyanions and its conjugated proteins and effect on cell membrane

Lipophilic anionic copolymer (styrene-maleic acid; SMA) conjugates of albumin and antitumor protein neocarzinostatin (NCS) (smancs) were found to stimulate the release of H2O2 and O-2 from the peritoneal macrophages obtained from mice which had been pretreated with the heat-killed preparation of Streptococcus pyogenes (OK-432) in vivo. Some alkyl esters of SMA exhibited effects similar to protein-polymer conjugates. Among them, butyl-SMA was the most effective followed by ethyl-SMA, whereas hydrolyzed SMA showed no effect. This activity was dose-dependent but exhibited a bell-shape profile. These results suggest that the aliphatic ester residue in SMA as well as the main chain of the copolymer may be important for the activation of macrophages. A strong antitumor effect of smancs reported elsewhere may be attributed partly to the activation of macrophages in addition to the direct damage to the cellular DNA by the NCS component. A preliminary investigation of the subcellular mechanism of macrophage activation was carried out in view of membrane fluidity by the fluorescence polarization method. The results showed that the apparent decrease in the cell membrane fluidity and the degree of macrophage activation paralleled the same dose range and at similar time courses. This indicated the interaction of SMA component and macrophage cell membrane.     

An NMR investigation of the changes in plasma membrane triglyceride and phospholipid precursors during the activation of T-lymphocytes.

Two-dimensional 1H-NMR spectroscopy was used to quantify the level of isotropically tumbling plasma membrane triglyceride and the intracellular concentrations of water-soluble phospholipid precursors during the activation of thymic T-lymphocytes. The concentration of "mobile" triglyceride in the plasma membrane was seen to increase 25-fold during 72 h of activation of murine thymic T-lymphocytes with ionomycin and phorbol myristate acetate. This is the first unequivocal demonstration of such a dramatic increase in mobile plasma membrane triglyceride during T-lymphocyte activation and leads to the suggestion that immune cell activation is associated with increased plasma membrane fluidity. The intracellular concentrations of choline- and ethanolamine-based phospholipid precursors were shown to increase during the early stages of T-lymphocyte activation and then remain at levels above those in resting cells. This may facilitate de novo phospholipid biosynthesis, which is presumably necessary since cell volume, and hence the plasma membrane surface area, was demonstrated to increase significantly during thymocyte activation.     

Membrane structure and surface coat of Entamoeba histolytica. Topochemistry and dynamics of the cell surface: cap formation and microexudate

Treatment of living entamoeba histolytica cells with low concentrations of concanavalin A (con A) and peroxidase results in redistribution of the plasma membrane con A receptors to one pole of the cell where a morphologically distinct region--the uroid--is formed. Capping of con A receptors is not accompanied by parallel accumulation of ruthenium red-stainable components. In capped cells, the pattern of distribution of acidic sites ionized at pH 1.8 (labeled by colloidal iron) at the outer surface and of membrane particles (integral membrane components revealed by freeze-fracture) is not altered over the uroid region. Cytochemistry of substrate-attached microexudate located in regions adjacent to E. histolytica cells demonstrates the presence of con A binding sites and ruthenium red- and alcian blue-stainable components and the absent of colloidal iron binding sites. In a previous report we demonstrated that glycerol-induced aggregation of the plasma membrane particles is accompanied by a discontinuous distribution of colloidal iron binding sites, while con A receptors and acidic sites ionized at pH 4.0 remain uniformly distributed over the cell surface. Taken together, our experiments show that, in E. histolytica cells, peripheral membrane components may move independently of integral components and, also, that certain surface determinants may redistribute independently of others. These results point to the complexity of the membrane structure-cell surface relationship in E. histolytica plasma membranes relative to the membrane of the erythrocyte ghost where integral components (the membrane-intercalated particles) contain all antigens, receptors, and anionic sites labeled so far. We conclude that fluidity of integral membrane components (integral membrane fluidity) cannot be inferred from the demonstration of the mobility of surface components nor, conversely, can the fluidity of peripheral membrane components (peripheral membrane fluidity) be assumed from demonstration of the mobility of integral membrane components.     

Studies on regenerating liver and hepatoma plasma membranes--II. Membrane fluidity and enzyme activity

1. Rat hepatocyte plasma membranes isolated from Morris hepatoma 7288C, normal and regenerating liver were labelled with the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene. 2. Steady-state fluorescence polarisation measurements indicated an increased fluidity of the membranes in the early stages of regeneration, returning to normal levels after 48 hr. 3. There was a decrease in hepatoma plasma membrane fluidity compared to normal hepatocytes. Changes in fluorescence polarisation with temperature (Arrhenius studies) indicate an increase in the lower critical temperature for the membrane lipid thermotropic transition of hepatoma compared to normal liver plasma membranes. 4. These changes in membrane lipid fluidity alter the activation of some intrinsic and extrinsic membrane bound enzymes.     

Membrane dynamics of differentiating cultured embryonic chick skeletal muscle cells by fluorescence microscopy techniques

Changes in membrane fluidity during myogenesis have been studied by fluorescence microscopy of individual cells growing in monolayer cultures of embryonic chick skeletal muscle cells. Membrane fluidity was determined by the techniques of fluorescence photobleaching recovery (FDR), with the use of a lipidsoluble carbocyanine dye, and by fluorescence depolarization (FD), with perylene used as the lipid probe. The fluidity of myoblast plasma membranes, as determined from FPR measurements in membrane areas above nuclei, increased during the period of myoblast fusion and then returned to its initial level. The membrane fluidity of fibroblasts, also found in these primary cultures, remained constant. The fluidity in specific regions along the length of the myoblast membrane was studied by FD, and it was observed that the extended arms of the myoblast have the highest fluidity on the cell and that the tips at the ends of the arms had the lowest fluidity. However, since the perylene probe used in the FD experiments appeared to label cytoplasmic components, changes in fluidity measured with this probe reflect changes in membrane fluidity as well as in cytoplasmic fluidity. The relative change in each of these compartments cannot yet be ascertained. Tips have specialized surface structures, filopodia and lamellipodia, which may be accompanied by a more immobile membrane as well as a more rigid cytoplasm. Rounded cells, which may also have a more convoluted surface structure, show a lower apparent membrane fluidity than extended cells.     

Calcium alters the acyl chain composition and lipid fluidity of rat hepatocyte plasma membranes in vitro

Calcium ion decreases the lipid fluidity of isolated rat hepatocyte plasma membranes by modulating the activity of membrane enzymes which alter the lipid composition. To explore the mechanism of the effect of the cation, eight fluorophores were used to assess lipid fluidity via estimations of either steady-state fluorescence polarization or excimer fluorescence intensity. The results demonstrate that the reduction in fluidity occurs in the hydrophobic interior of the bilayer and that both the dynamic and static (lipid order) components of fluidity are affected by treatment with calcium. Analysis of the membrane lipids demonstrates that calcium treatment decreases the arachidonic acid content of the polar lipid fraction and, thereby, reduces the double-bond index of the fatty acids. This change in composition, which is expected to reduce the lipid fluidity, may result from activation by calcium of the endogenous hepatocyte plasma membrane phospholipase A2.     

Platelet plasma membrane in subjects with primary nocturnal enuresis: Effect of desmopressin

Plasma membrane fluidity of platelets (PLT) obtained from subjects with primary nocturnal enuresis (PNE) and healthy controls was investigated before and after addition of desmopressin (DDAVP). Membrane fluidity was studied by measuring steadystate fluorescence anisotropy of 1-(4-trimethylammoniumphenyl)-6-phenyl-1, 3, 5-hexatriene incorporated into PLT plasma membrane. Our results show an increase in membrane fluidity at the surface level of PLT from subjects with PNE. Moreover, the addition of DDAVP induces a stable and significant decrease of membrane fluidity in both groups. These results suggest alterations of the lipid order in the exterior part of the PLT plasma membrane from patients with PNE.     

Chlamydia pneumoniae infected macrophages exhibit enhanced plasma membrane fluidity and show increased adherence to endothelial cells

Chlamydia pneumoniae, an intracellular prokaryote, is known to have requirement for some lipids which it is incapable of synthesizing, and these lipids have important fluidizing roles in plasma membrane. We decided to examine if the trafficking of these lipids to C. pneumoniae alters the physicochemical properties of macrophage plasma membrane, affects the expression of genes and proteins of enzymes associated with metabolism of some of these lipids and assess if Ca2+ signaling usually induced in macrophages infected with C. pneumoniae modulates the genes of these selected enzymes. Chlamydia pneumoniae induced the depletion of macrophage membrane cholesterol, phosphatidylinositol and cardiolipin but caused an increase in phosphotidylcholine resulting in a relative increase in total phospholipids. There was increased membrane fluidity, enhanced macrophage fragility and heightened adherence of macrophages to endothelial cells despite the application of inhibitor of adhesion molecules. Also, there was impairment of macrophage 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase gene and protein expression independent of Ca2+ signaling, while phospholipase C gene and protein were up-regulated in a manner minimally dependent on Ca2+ signaling. The implications of these findings are that macrophages infected with C. pneumoniae have altered membrane physicochemical characteristics which may render them atherogenic.     

HDL derived from the different phases of conjugated diene formation reduces membrane fluidity and contributes to a decrease in free cholesterol efflux from human THP-1 macrophages

Oxidized HDL (ox-HDL) has been reported to reduce free cholesterol efflux from cells. In this study we investigate the effect of different stages of ox-HDL on macrophage membrane fluidity and its effect on free cholesterol efflux from macrophages as a cell function influenced by ox-HDL. HDL was oxidized by means of conjugated diene production using copper as a prooxidant. Fluidity of HDL and human THP-1 macrophage membranes was evaluated by changes in fluorescence anisotropy (r) by DPH probe where lower (r) values give higher fluidity. We found that ox-HDL derived from the propagation phase (PP-HDL) and the decomposition phase (DP-HDL) became less fluid ((r): 0.263+/-0.001, 0.279+/-0.002, respectively) than HDL from the lag phase (LP-HDL) and native HDL (nat-HDL) ((r): 0.206+/-0.001) (P<0.05). Macrophages incubated with PP-HDL and DP-HDL had less fluid membranes ((r): 0.231+/-0.001, 0.243+/-0.002, respectively) than those incubated with LP-HDL and nat-HDL ((r): 0.223+/-0.001) (P<0.05). Consequently, fluidity was reduced not only in ox-HDL but also in the cell membranes exposed to ox-HDL. A significant negative correlation was observed between macrophage membrane fluorescence anisotropy (r) and free cholesterol efflux from these cells (-0.876; P<0.05). Thus, lower membrane fluidity was associated with lower free cholesterol efflux from cells. In conclusion, the increase in the HDL oxidation process leads to a lost of macrophage membrane fluidity that could contribute to an explanation of the reduction of free cholesterol efflux from cells by ox-HDL.     

Plasma membrane fluidity measurements in intact endothelial cells: effect of hyperoxia on fluorescence anisotropies of 1-[4-(trimethylamino)phenyl]-6-phenyl hexa-1,3,5-triene

Fluorescence anisotropy measurements are widely used as sensitive indicators of cell membrane fluidity. 1-[4-(trimethylamino)phenyl]-6-phenyl hexa-1,3,5-triene (TMA-DPH) is a cationic fluorescent aromatic hydrocarbon that anchors at the lipid-water interface of membrane lipid bilayers. Its uptake into porcine pulmonary artery and aortic endothelial cells was monitored and the probe remained specifically localized on the cell surface for at least 4 h. It can therefore be recommended for use for specific plasma membrane lipid fluidity measurements in these cells. The effect of hyperoxia on plasma membrane fluidity was measured by using TMA-DPH. In both cell types, hyperoxic damage resulted in decreases in plasma membrane fluidity. Recovery was achieved 48 h after a 42-h hyperoxic exposure. These results indicate that TMA-DPH is a sensitive probe of plasma membrane lipid domains of pulmonary artery and aortic endothelial cells and that hyperoxia causes reversible changes in the physical state of superficial lipid domains of the plasma membrane of these cells.     

Progesterone induces transient changes in plasma membrane fluidity of amphibian oocytes during the first meiotic division

Progesterone acts at the surface of the amphibian oocyte to induce resumption of the meiotic divisions. Progesterone binding leads to a transient dose-dependent decrease in the fluidity (increase in order parameter) of the Rana oocyte plasma membrane, which was detected by electron spin resonance in isolated plasma membranes using either 5- or 16-DOXYL stearic acid probes. The 5-DOXYL probe, which inserts into the membrane with the spin label nearest the surface, showed an increase in the order parameter within minutes, a maximum change by 2 h, and a return to control levels by 6 h. The order parameter for the 16-DOXYL probe, which reflects the fluidity deeper within the plasma membrane, increased slowly and remained elevated during the first meiotic division. RU 38486, a synthetic steroid that blocks progesterone receptors, prevents progesterone-induced fluidity changes. These findings indicate that the binding of progesterone to its receptor changes the oocyte plasma membrane structure resulting in a differential decrease in mobility near the membrane surface compared to that deeper in the membrane.     

Microsomal desaturation of stearic acid in relation to lymphocyte activation

The conversion of stearic acid to oleic acid (delta 9-desaturase) was followed in mouse thymocytes stimulated by either concanavalin A or concanavalin A + interleukin-2 resulting in different rates of cell proliferation. To estimate the plasma membrane turnover of oleic acid as compared to that of a saturated fatty acid, double-label experiments ([14C]oleic acid, [3H]palmitic acid) were performed. Following an inhibition delta 9-desaturase was found to be activated from the fourth hour of stimulation. In the early period of cell activation this process proved to be independent of protein synthesis, whereas in the stage of proliferation it was dependent on it. Increased membrane fluidity in the first 30 min of activation is not likely due to enrichment of oleic acid. Cell proliferation and microsomal desaturation seem to be coupled and an increasing amount of oleic acid is at least one of the factors resulting in increased fluidity of the surface membrane of proliferating cells.     

Binding of amyloidogenic transthyretin to the plasma membrane alters membrane fluidity and induces neurotoxicity

Transthyretin (TTR) can deposit as amyloid in the peripheral nervous system and induce a peripheral neuropathy. We examined the mechanism of TTR amyloid neurotoxicity on SH-SY5Y neuroblastoma cells. Wild-type (WT) TTR and two amyloidogenic mutants (V30M and L55P) were expressed in Escherichia coli. Incubation (aging) of WT TTR at 37 degrees C for 1 week caused no significant aggregation. However, there was a significant increase in the extent of amyloid fibril formation after the amyloidogenic mutants had been aged. L55P TTR aggregated more readily than V30M TTR. Both amyloidogenic mutants were neurotoxic after aging. The order of neurotoxicity was as follows: L55P > V30M > WT. As binding of amyloid proteins to the plasma membrane may cause cytotoxicity, we studied the binding of TTR to a plasma membrane-enriched preparation from SH-SY5Y cells by surface plasmon resonance. All three forms bound to the plasma membrane through electrostatic interactions. The binding of the amyloidogenic mutants was increased by aging. The amount of binding correlated closely with the amount of aggregation and with the cytotoxicity of each form. As membrane fluidity can influence cell viability, we also examined the effect of TTR on membrane fluidity using a fluorescence anisotropy method. Binding of the amyloidogenic TTR mutants increased membrane fluidity, and once again, the order of potency was as follows: L55P > V30M > WT. These results demonstrate that TTR can bind to the plasma membrane and cause a change in membrane fluidity. Altered membrane fluidity may be the cause of the neurotoxicity.     

Fluidity and lipid composition of oat and rye shoot plasma membrane: effect of sterol perturbation by xenobiotics

Oat and rye plants were treated with either tetcyclacis (an experimental plant growth regulator), nuarimol (a fungicide) or gamma-ketotriazole (an experimental herbicide). These treatments reduced shoot growth and changed the lipid composition of the shoot plasma membranes. In oat, both tetcyclacis and nuarimol treatments increased plasma membrane cholesterol and increased the phosphatidylethanolamine/phosphatidylcholine (PE/PC) ratio, whereas gamma-ketotriazole treatment reduced cholesterol and the PE/PC ratio. In rye, all treatments reduced the PE/PC ratio. Generally, the sterol/phospholipid ratio was less in oat than in rye but the cholesterol/phospholipid ratio was greater. With all treatments in oat and rye, increases were observed in unsaturation of the phospholipid acyl chains. The fluidity of membranes was measured by steady-state fluorescence polarisation of the probe diphenylhexatriene; oat membranes were more fluid than rye. Membrane fluidity was greater in plasma membranes from plants treated with the xenobiotics than the controls. The results are discussed in the context of the effect of plasma membrane lipid composition on membrane fluidity, and it is concluded that there appears to be no overall simple relationship between membrane lipid composition and fluidity that holds for all treatments in both species.     

Membrane fluidity and the probability of complement fixation

We develop a mathematical theory of the role of membrane fluidity in the initiation of the IgG mediated complement cascade. The basic assumption is that C1q must be at least doubly bound to activate C1r, but that once C1q is doubly bound, C1r still requires some mean finite time tau to become enzymatically active. If C1q dissociates during this time interval, C1r cannot be activated. We consider the consequences of the simplest model of fluidity--one in which the difference between "fluid phase" lipids and "non-fluid phase" lipids is to allow protein mobility, but not a change in protein conformation. We show that under these conditions fluidity will effect C1r activation only if the rate of formation of multiply bound C1q is limited by diffusion in the membrane. If diffusion in the membrane is not rate-limiting, then, within the framework of this model, fluidity has no effect whatsoever on C1r activation. Thus, an experimental determination that C1q binding is not rate-limited by diffusion in the surface, but that fluidity does effect activation, would suggest a protein conformational change resulting perhaps from altered lipid composition. If diffusion in the surface does rate limit multiple C1q binding, we predict the possibility of an optimum diffusion coefficient for activation. For suitably chosen and reasonable parameter values this optimum will occur in the range (10(-11) less than or equal to D less than or equal to 10(-8) cm2/sec. We predict further, under these circumstances, a precipitous drop in the probability of activation above the optimum. The abrupt switch from a high probability of activation to essentially no probability of activation suggests the possibility of a very sensitive control mechanism exploitable by relatively small changes in membrane lipid composition.     

伴刀豆球蛋白A和层粘连蛋白与膜受体结合下膜分子运动及微丝组装变化的比较

研究伴刀豆球蛋白A和层粘连蛋白分别与小鼠腹腔巨噬细胞膜受体结合下引起细胞膜分子运动的变化和对微丝组装的影响.结果表明,伴刀豆球蛋白A和层粘连蛋白作用下均导致膜表面蛋白分子的侧向扩散速率减慢,膜脂流动性降低,加快膜内微丝组装并使微丝含量增加.两配体作用下引起细胞上述反应有相似性.     

Cepharanthine inhibited HIV-1 cell–cell transmission and cell-free infection via modification of cell membrane fluidity

The anti-HIV-1 activity of cepharanthine (CEP), a natural product derived from Stephania cepharantha Hayata, was evaluated. CEP stabilized plasma membrane fluidity and inhibited HIV-1 envelope-dependent cell-to-cell fusion of HIV-1-infected cells as well as cell-free infection. It is suggested that CEP inhibited the HIV-1 entry process by reducing plasma membrane fluidity, and the plasma membrane is therefore an identical target to prevent viral infection.     

Dimethylnitrosamine inhibits the glucagon-stimulated adenylate cyclase activity of rat liver plasma membranes and decreases plasma membrane fluidity

The effect of the hepatocarcinogen dimethylnitrosamine on rat liver plasma membrane adenylate cyclase activity and lipid fluidity was assessed. Glucagon-stimulated adenylate cyclase activity exhibited a complex response to increasing concentrations of dimethylnitrosamine, whereas fluoride-stimulated adenylate cyclase activity was progressively inhibited. Maximal inhibitory effects were observed at a concentration of 15 mM in both cases. The activity of detergent-solubilized adenylate cyclase was unaffected by dimethylnitrosamine. ESR analysis using a fatty acid spin probe showed that dimethylnitrosamine produced a marked, dose-dependent reduction in the fluidity of the plasma membrane with a maximal effect occurring at 20 mM. Dimethylnitrosamine also elevated the temperature at which the lipid phase separation occurred in rat liver plasma membranes, from 28 degrees C to 31 degrees C. The non-carcinogenic but structurally similar compound, dimethylamine hydrochloride neither inhibited adenylate cyclase nor decreased plasma membrane fluidity. It is suggested that the decrease in membrane fluidity, induced by dimethylnitrosamine, via its effects on membrane fluidity, could influence plasma membrane function and cellular regulation.     

伴刀豆球蛋白A和层粘连蛋白与膜受体结合下膜分子运动及微丝组装变化的比较

研究伴刀豆球蛋白A和层粘连蛋白分别与小鼠腹腔巨噬细胞膜受体结合下引起细胞膜分子运动的变化和对微丝组装的影响.结果表明,伴刀豆球蛋白A和层粘连蛋白作用下均导致膜表面蛋白分子的侧向扩散速率减慢,膜脂流动性降低,加快膜内微丝组装并使微丝含量增加.两配体作用下引起细胞上述反应有相似性.