Modulation of CALLA and HLA-DR on a non T non B leukemic cell line by lipid treatment

The relationship between membrane lipid fluidity and expression of HLA-DR and cALL (CALLA) antigens was studied in a human non T non B acute lymphoblastic leukemia cell line (Reh). The membrane fluidity was modulated by treatment with cholesteryl hemisuccinate or phospholipids (e.g. egg lecithin) and monitored by fluorescence polarization. HLA-DR and CALLA expression was measured in an indirect immunofluorescence test with a Fluorescence Activated Cell Sorter (FACS 440), on 24, 48, 72 and 96 hour-cultured cells. Significant antigenic modulation was obtained with cholesteryl hemisuccinate treatment on 48 hour-cells where a slight increase in HLA-DR and a marked decrease in CALLA were observed. In contrast no antigenic modification was observed on lecithin-treated cells.     

Cholesteryl hemisuccinate alters membrane fluidity, angiotensin receptors, and responses in adrenal glomerulosa cells

We examined the effects of cholesteryl hemisuccinate on membrane fluidity and angiotensin II (AII) actions in bovine adrenal glomerulosa cells. Incubating cells with cholesteryl hemisuccinate decreased membrane fluidity and markedly inhibited AII binding. The effect on binding was characterized by a decrease in AII receptor number. The effects of AII on phosphatidyl inositol turnover and calcium fluxes, proposed intermediaries of AII actions on aldosterone secretion, were less impaired than AII binding by cholesteryl hemisccinate. AII stimulation of aldosterone secretion was preserved despite the decrease in AII binding after cholesteryl hemisuccinate treatment. These results indicate that AII binding can be dissociated from its effects on aldosteronogenesis by a reagent that alters membrane fluidity.     

Changes in cell-surface expression of MHC and Thy-1.2 determinants following treatment with lipid modulating agents

Treatment of normal mouse spleen cells with lipid fluidity modulators changes the expression of cell-surface H-2 determinants. BALB/c spleen cells treated for 1 to 2 hr with cholesteryl hemisuccinate (CHS) displayed reduced levels of all tested H-2 determinants (H-2L, H-2K, and H-2D) as evaluated by flow microfluorometry and increased membrane lipid packing density as determined by 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence polarization. In contrast, decreasing membrane lipid packing density by phosphatidylcholine treatment decreased DPH fluorescence polarization and increased the expression of MHC determinants. The effects were selective in that expression of Thy-1.2 determinants was decreased by the latter treatment and not increased by CHS. The results are discussed in terms of passive modulation of antigenic expression.     

Relationship between Thylakoid Membrane Fluidity and the Functioning of Pea Chloroplasts : EFFECT OF CHOLESTERYL HEMISUCCINATE

Cholesteryl hemisuccinate has been incorporated into pea chloroplast thylakoids to investigate the relationship between fluidity and functioning of this membrane system. Levels of sterol which increased the apparent viscosity of the membrane, estimated by fluorescence polarization measurements using the lipophilic probe, 1,6-diphenyl-1,3,5 hexatriene, affected several photosynthetic processes. A decrease in fluidity was accompanied by an inhibition of dark limiting steps associated with electron transfer between photosystems two and one (PSII and PSI) as observed by the oxidation of the primary acceptor of PSII and by electron flow to ferricyanide. Also, treatment with cholesteryl hemisuccinate inhibited the saltinduced rise in chlorophyll fluorescence and changed the ionic conductivity of the membrane as judged by measurements of the decay of the lightinduced proton gradient. The results are discussed in terms of the effect of fluidity changes on the lateral diffusion of plastoquinone and chlorophyll protein complexes in the lipid matrix of the membrane.     

Influence of membrane physical state on lysosomal potassium ion permeability

Lysosomal permeability to potassium ions is an important property of the organelle. Influence of the membrane physical state on the potassium ion permeability of isolated lysosomes was assessed by measuring the membrane potential with bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol and monitoring the lysosomal proton leakage with p-nitrophenol. The membrane fluidity of lysosomes was modulated by treatment with membrane fluidizer benzyl alcohol and rigidifier cholesteryl hemisuccinate. Changes in the membrane order were examined by steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene. The measurements of membrane potential and proton leakage demonstrated that the permeability of lysosomes to potassium ions increased with rigidification of their membranes by cholesteryl hemisuccinate treatment at 37 degrees C, and decreased with fluidization of their membranes by benzyl alcohol treatment at 2 degrees C. The changes in ion permeability could be recovered by fluidizing the rigidified membranes and rigidifying the fluidized membranes. The results suggest that the physical states of lysosomal membranes play an important role in the regulation of their K(+) permeability.     

Influence of Membrane Physical State on the Lysosomal Proton Permeability

Influence of membrane physical state on the proton permeability of isolated lysosomes was assessed by measuring the membrane potential with 3,3′-dipropylthiadicarbocyanine iodide and monitoring their proton leakage with p-nitrophenol. Changes in the membrane order were examined by the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene. Both the membrane potential and proton leakage increased with fluidizing the lysosomal membranes by benzyl alcohol and decreased with rigidifying the membranes by cholesteryl hemisuccinate. The proton permeability increased to the maximum of 42% by the benzyl alcohol treatment and decreased to the minimum of 38.1% by the cholesteryl hemisuccinate treatment. Treating the lysosomes with protonophore CCCP increased the proton permeability by 58%. The effects of the membrane fluidization and rigidification can be reversed by rigidifying the fluidized membranes and fluidizing the rigidified membranes, respectively. The results indicate that the proton permeability of lysosomes increased and decreased with increasing and decreasing their membrane fluidity, respectively. Moreover, the lysosomal proton permeability did not alter further if the changes, either an increase or a decrease, in the fluidity exceeded some amount. The results suggest that the proton permeability of lysosomes can be modulated finitely by the alterations in their membrane physical state. Received: 27 September 1999 / Revised: 27 December 1999     

Effect of dehydroepiandrosterone and its sulfate and fatty acid ester derivatives on rat brain membranes

The effects of dehydroepiandrosterone (DHEA) as well as its sulfate and fatty acid ester derivatives on rat brain membrane fluidity was investigated by fluorescence depolarization of a lipid probe 1,6-diphenyl-1,3,5-hexatriene and compared to its effect on phospholipid conformation investigated by Fourier transform infrared spectroscopy. In rat brain, membrane fluidity varied rostro-caudally, the frontal cortex showing the highest fluidity compared to the hypothalamus, hippocampus, striatum, thalamus, and hindbrain. As previously reported, it was observed that cholesteryl hemisuccinate and stearic acid rigidify striatal membrane whereas linoleic acid and L-alpha-phosphatidylcholine increase the membrane fluidity. Striatal fluidity was increased in vitro with increasing concentrations of DHEA, this effect was greater with the DHEA fatty acid ester derivatives (DHEA-L), DHEA-undecanoate, and DHEA-stearate, whereas no effect was observed with DHEA-sulfate (DHEA-S). In the frontal cortex only the two DHEA-L derivatives increased membrane fluidity, whereas DHEA and DHEA-S were without effect. The effect of DHEA-L on synthetic dimyristoylphosphatidylcholine-d54 phospholipid membranes indicates a disordering effect of DHEA-undecanoate and DHEA-stearate as reflected by increased trans-gauche isomerization of the acyl chains of the lipid. Hence, DHEA-L increase the disorder and/or fluidity of brain membranes; interestingly, these compounds are abundant in the brain where they are generally considered as storage compounds that slowly release the active unconjugated steroid hormone.     

Influence of membrane fluidity modifiers on lysosomal osmotic sensitivity

Since lysosomes are prone to osmotic lysis, we have examined the correlation between their physical state and sensitivity to osmotic challenge, using agents which modify membrane fluidity. The latency loss of beta-hexosaminidase after an incubation in hypotonic sucrose medium was followed under different conditions of membrane fluidity, recorded by steady-state fluorescence anisotropy of 1,6-diphenyl-1,3, 5-hexatriene. Increasing fluidity of the lysosomal membranes with benzyl alcohol (BA) and greater rigidity caused by cholesteryl hemisuccinate (CHS) increased and decreased the enzyme latency loss, respectively. The effects of BA and CHS treatments on osmotic sensitivity were reversible subsequently by reciprocal treatments of the lysosomes with CHS and BA, respectively. The results indicate that the physical state of the membrane does indeed affect lysosomal osmotic stability.     

Effects of cholesteryl esters on the accessibility of LH/hCG receptors and membrane lipid fluidity in rat testes

Incubation of rat testicular membranes with cholesteryl hemisuccinate resulted in an increase in both membrane lipid microviscosity and 125I-labelled hCG specific binding. The purpose of this investigation was to establish which functional groups of cholesteryl hemisuccinate are important for the stimulatory effects. The data obtained showed that only esters of cholesterol with dicarboxylic acids, not those of monocarboxylic acids, increase the accessibility of LH/hCG receptors and membrane rigidity. Experiments with cholesteryl sulfates showed that there are polar groups on C3 carbon of cholesterol having no stimulatory effect on receptors, although an increase in membrane rigidity occurred. The side-chain of cholesterol is important for the stimulatory action. Androstenolone hemisuccinate was ineffective in this respect. On the other hand, partially modified side-chains (hemisuccinates of beta-sitosterol and stigmasterol) did not result in a marked reduction of the stimulatory action. The carboxyl group of cholesteryl hemisuccinate must be 'free': its esterification abolishes the stimulatory effect of cholesteryl hemisuccinate on both the LH/hCG receptor and membrane microviscosity. These results suggest that an intact carboxyl group of ester and the side-chain of cholesterol are indispensable for the stimulatory effect of cholesteryl hemisuccinate on the accessibility of LH/hCG receptors.     

Expression of HLA-DR antigens on tumour cells does not contribute to skin reactivity to autologous cholesteryl hemisuccinate (CHS)-treated tumour cells in patients with metastatic melanoma

Summary Skin tests with autologous cholesteryl hemisuccinate (CHS)-treated and untreated cells were performed in ten metastatic melanoma patients. In the majority of cases evident reaction was noted with CHS-treated cells (9/10) while the reaction with untreated cells was mostly negative (7/10). Tumour cell suspensions used for skin tests were characterized for reactivity with monoclonal antibody TAL 1B5 detecting the HLA-DR alpha chain. There were no differences between CHS-treated and untreated cells with respect to HLA-DR expression and no correlation was found between grade of skin reaction to CHS-treated cells and the proportion of HLA-DR positive cells in the injected cell sample.     

Cholesteryl hemisuccinate's inductive effect on membrane rigidization regarding both, its remodelling of the cells' surface receptor pattern and its decreasing the natural killer susceptibility of K-562 cells.

The relationship of changes in membrane fluidity to natural killer susceptibility of K-562 target cells was investigated. Membrane rigidization was performed by the chemical modulator cholesteryl hemisuccinate. Steady-state fluorescence polarization measurements of the diphenyl hexatriene labelled, modified K-562 cells revealed that cholesteryl hemisuccinate increased the structural order of the hydrophobic region of membranes in a dose dependent way. Investigation of natural killer susceptibility followed by 51Cr release assay indicated that modified cells are less sensitive to natural killer attack. To elucidate whether surface structures such as transferrin and lectin receptors are associated with the altered susceptibility, the surface density of these receptors was followed by (I-125)-transferrin binding assay and quantitative immunofluorescence. We found that the number of transferrin and concanavalin A receptors increased by a factor of 2.44 and 2.00, respectively, whereas that of the wheat germ agglutinin receptor failed to exhibit any changes upon rigidization. From the results we concluded that i the membrane structural order does influence the natural killer susceptibility, ii changes in membrane structural order result in alteration of the number of cell surface transferrin and lectin receptors, iii however, no direct relationship seems to exist between these two events.     

The effect of age and cholesterol on the rat lung beta-adrenergic system

To assess the influence of membrane lipid composition on beta-adrenergic receptor number and adenylate cyclase activity in aging, we investigated the effect of cholesteryl hemisuccinate on these parameters in lung membranes of 3-, 12-, and 24-month-old CDF (F-344) rats. When cholesteryl hemisuccinate (0.5 mg/ml) was incubated with lung membranes, beta-adrenergic receptor density was increased by 70%. This effect was the same for each age group studied and indicated that the density of both basal and CHS-sensitive receptors is unaltered in rat lung with age. Forskolin, NaF, p[NH]ppG, and isoproteronol-stimulated adenylate cyclase activity is 30% lower in lung membranes from aged rats. Since enzyme activity is affected by the lipid environment and membrane composition often changes with age, we assessed adenylate cyclase activity following cholesteryl hemisuccinate incorporation. There was up to a 75% decrease in adenylate cyclase activity following cholesteryl hemisuccinate incorporation in lung membranes in each of the three age groups. In untreated membranes, there was no significant difference in cholesterol or lipid phosphate content with age. These data suggest that cholesterol content does not account for alterations in senescent rat lung adenylate cyclase activity.     

Effect of altered membrane fluidity on NK cell-mediated cytotoxicity. I. Selective inhibition of the recognition or post recognition events in the cytolytic pathway of NK cells

NK cell-mediated cytotoxicity results from membrane interactions between NK effector and target cells. The role of membrane fluidity in these events is not known. The present study was undertaken to investigate the effect of changes in membrane lipid fluidity of NK effector and NK-sensitive target cells on the lytic pathway of NK cell-mediated cytotoxicity. Fluidity was modulated by various lipids and measured by fluorescence polarization. NK effector cells treated with phosphatidylcholine complexed with polyvinylpyrrolidone (PVP) and bovine serum albumin (BSA) showed increased membrane fluidity. This fluidization of the effector cell membrane resulted in a significant inhibition of cytotoxic activity in the 51Cr-release assay. Single cell analysis revealed that the inhibition was due to a decrease in the frequency of NK target conjugates and reduced killing of conjugated targets. Rigidification of the NK effector cell membranes by treatment with cholesteryl hemisuccinate complexed with PVP and BSA also resulted in inhibition of cytotoxicity. This inhibition was post binding, because binding was increased and lysis was abrogated. Fluidization of K562 target cell membranes caused a slight but insignificant increase in their lysis by NK cells without affecting the binding step. On the other hand, rigidification of K562 membranes decreased the sensitivity of these target cells to lysis. Single cell analysis revealed that this inhibition of NK lysis is post binding, because the frequency of killers was significantly decreased. It was also shown that membrane rigidification of target cells that were programmed for lysis during the lethal hit stage and subsequently separated from effector cells, rendered the programmed cells resistant to killing during the killer cell-independent lysis step. These results demonstrate that fluidization or rigidification of the plasma membrane of either effector or target cells affect different stages of the NK cell-mediated cytolytic events.     

Influence of the physical states of membrane surface area and center area on lysosomal proton permeability

The physical state of the lysosomal membrane was modulated with the membrane fluidizers n-propanol and n-octanol and with the membrane rigidifiers cholesteryl hemisuccinate and cholesterol. Membrane fluidity was examined by the steady-state fluorescence anisotropy of 2-(9-anthroyloxy) palmitic acid and 16-(9-anthroyloxy) palmitic acid. Fluidizing the membranes at the surface and center areas increased the proton permeability coefficient by 92.8 and 18.0%, respectively. Rigidifying the membranes at the surface and center areas decreased the coefficient by 68.2 and 40.2%, respectively. Proton leakage of the lysosomes increased and decreased similar to the coefficient changes with the treatments. The results indicate that lysosomal proton permeability is affected by its membrane's physical state, and the physical state of the membrane surface area affects the proton permeability more markedly. The proton permeability coefficient of liposomes was similar to that of lysosomes, suggesting that efflux of lysosomal protons might occur through the lipid part of the bilayer but not transmembrane proteins.     

Modulation of rat striatal membrane fluidity: effects on age related differences in dopamine receptor concentrations

Rat striatal membrane fluidity and dopamine receptor concentrations were modulated in vitro by ethanol or cholesterol hemisuccinate treatment. In general, available receptor levels are directly proportional to membrane fluidity. However, receptor concentrations remained higher in mature membranes at all levels of fluidity attained. These results suggest that apparent loss of striatal dopamine receptors during aging is not due to membrane sequestration.     

Structure/function studies of the common acute lymphoblastic leukemia antigen (CALLA/CD10) expressed on human neutrophils

The common acute lymphoblastic leukemia antigen (CALLA/CD10) is a nonintegral membrane glycoprotein expressed on normal and neoplastic cells of hematopoietic and nonhematopoietic origin. We have undertaken a series of experiments to examine 1) the structural homology between leukemia cell and neutrophil CALLA/CD10 and 2) the putative function CALLA/CD10 subserves to human neutrophils. Biosynthetic labeling, peptide mapping, and two-dimensional gel electrophoresis indicate that neutrophils synthesize and express a CALLA/CD10 molecule that is similar, but not identical, to leukemic cell CALLA/CD10. The level of CALLA/CD10 expression is similar on the two cell populations, and neutrophil CALLA/CD10 (like its leukemic cell counterpart) undergoes antigenic modulation. Finally, we report that neutrophil cell surface-bound anti-CALLA/CD10 monoclonal antibodies inhibit the chemotactic response to both N-Formyl-methionyl-leucyl-phenylalanine (F-mlp) and zymosan-activated sera (ZAS), but had no inhibitory effect on random migration, degranulation, or aggregation. The anti-class I monoclonal antibody W6/32 exerted a similar effect on chemotaxis. We conclude that CALLA/CD10 has no clearly defined role in neutrophil function but may play a role in some distal event in chemotaxis.     

Functional properties of the acetylcholine receptor incorporated in model lipid membranes. Differential effects of chain length and head group of phospholipids on receptor affinity states and receptor-mediated ion translocation

Torpedo acetylcholine receptor was reconstituted into liposomes of pure synthetic lipids in order to study the influence of the lipid environment on affinity state transitions and the ion translocation function of the receptor. A critical concentration of 30 to 40% of cholesteryl hemisuccinate was necessary in liposomes made of cholesteryl hemisuccinate and dimyristoyl phosphatidylcholine to mimic the kinetics of agonist-induced state transitions observed in native membranes. With increasing chain length of the saturated lecithins, a marked increase in carbamylcholine dissociation constants was observed. Substitution by other dimyristoyl phospholipids for dimyristoyl phosphatidylcholine had the same, though quantitatively less pronounced effects. Introduction of unsaturation in the acyl chains reverted the effect of increasing chain length. Unsaturated phosphatidylethanolamines in combination with 28-35 mol% of cholesteryl hemisuccinate was the best lipid mixture for reconstitution of the receptor-gating function. When phosphatidylethanolamine was replaced totally or partially by other phospholipids with the same or different acyl chain composition, a marked decrease of ion transport was apparent, even when similar vesicle size, receptor incorporation, and agonist-induced affinity transitions were obtained. Therefore, the maintenance of the affinity state transitions of the reconstituted receptor is a necessary but not sufficient condition for the manifestation of the ion-gating receptor activity. On the other hand, the more unsaturated the acyl chains of phosphatidylethanolamine are, the higher the response that was observed, suggesting that a critical lipid packing is essential for the ion translocation function of the receptor.     

Incorporation of sterol into chloroplast thylakoid membranes and its effect on fluidity and function

The sterol, cholesteryl hemisuccinate, has been incorporated into isolated thylakoid membranes of pea and lettuce chloroplasts in order to modify the fluidity of the lipid matrix. Changes in fluidity have been monitored using fluorescence polarization of the hydrophobic probe, 1,6-diphenyl-1,3,5-hexatriene and the electron-spin-resonance, spin-label probe, 5-doxyl stearate. Both methods indicate that incorporation of increasing levels of sterol reduces the fluidity of the thylakoid lipid matrix. At room temperature the thylakoid lipid matrix is relatively fluid and the effect of increasing the viscosity is to inhibit partially the maximum rate of steady-state electron flow and reduce the dark rate of reduction of flash-oxidised cytochrome f. The results are discussed in terms of lipid fluidity influencing the rate of lateral diffusion of reduced plastoquinone from photosystem II to photosystem I.     

Distribution and modulation of a human leukemia-associated antigen (CALLA)

CALLA is a 100,000-dalton surface glycoprotein expressed by malignant cells of patients with clinically important subtypes of acute leukemia. Incubation of human leukemic cells expressing CALLA with specific monoclonal antibody (J5) at 37 degrees C causes rapid and selective internalization and degradation of this antigen (antigenic modulation). In these studies we show that CALLA-specific monoclonal antibodies also identify a cell surface glycoprotein having a m. w. of approximately 100,000 on 2 to 6% of nonmyeloid nucleated cells of normal adult bone marrow, on normal fibroblasts in tissue culture, and on cells of several nonhematopoietic human tumor cell lines. J5 antibody similarly modulates the surface expression of CALLA on nonleukemic cell populations, although the extent of modulation at a given concentration of antibody varied considerably. Modulation was almost complete for CALLA on cells of normal bone marrow, but was highly variable for cells of nonhematopoietic cell lines, possibly reflecting variability in antibody access to surface antigen. Using fluoresceinated or iodinated J5 antibody to modulate expression of CALLA on cells of leukemic cell lines, we show that antibody-antigen complexes undergo a temperature-dependent redistribution on the cell surface during modulation to form microaggregates. Antibody as well as antigen is then internalized. Studies of [35S]methionine-labeled cells indicate that synthesis of CALLA continues despite modulation of its surface expression by specific antibody, implying that the presence of CALLA on the cell surface reflects a dynamic equilibrium between the processes of surface expression of newly synthesized glycoprotein and its spontaneous and antibody-mediated clearance. The implications of these observations for immunotherapy are discussed.     

Partial characterization of six chlorophyll a-protein complexes isolated from a blue-green alga by a nondetergent method

Incorporation of cholesterol hemisuccinate into thylakoid membranes decreased the membrane fluidity as measured by polarized fluorescence from 1,6-diphenyl-1,3,5-hexatriene. Increasing membrane viscosity in this manner did not inhibit the thylakoid membrane protein kinase. In contrast the effects of the protein phosphorylation on State I-State II transitions, which were observed in untreated membranes, were abolished. This observation is interpreted as indicating that protein phosphorylation-induced energy transfer changes are sensitive to membrane viscosity because they might require a lateral migration of the light-harvesting complex serving Photosystem II from grana to stromal lamellae. Cation effects on room- and low-temperature fluorescence emission properties and membrane adhesion were not abolished in these cholesterol hemisuccinate-treated membranes.