Cord blood plasma lipoproteins inhibit mitogen-stimulated lymphocyte proliferation

Lipoproteins were isolated from adult plasma and the umbilical cord blood plasma of newborn infants and were compared for their capacity to inhibit mitogen-stimulated [3H]thymidine uptake of adult peripheral blood mononuclear cells in vitro. Relative to the comparable adult lipoproteins, cord blood low density lipoproteins and high density lipoproteins inhibited mitogen stimulation at twofold to fourfold lower total protein concentrations. Apoproteins AI, B, and E were quantitated by radioimmunoassay of each of the adult and cord blood lipoprotein fractions. A strong correlation was observed between inhibitory activity and the amount of apoprotein E in the cord blood low and high density lipoproteins. Further evidence that lipoproteins containing apoprotein E accounted for the difference in suppressive activity of cord blood low and high density lipoproteins relative to the adult lipoproteins was obtained by selective removal of the apoprotein E-containing lipoproteins by using immunoaffinity chromatography or heparin-agarose adsorption. The results indicated that cord blood lipoproteins containing apoprotein E in association with apoproteins AI or B are capable of suppressing lymphocyte proliferation in vitro.     

Conditions influencing inhibitors of the colony-stimulating factor (CSF)

Lipoprotein inhibitors of myeloid cell-stimulating glucoprotein, the colony-stimulating factor (CSF), occur in normal human serum. Lipoproteins are known to be labile under various physico-chemical influences. The kinetics of changes in the inhibitory activity were studied during common conditions such as storage, freezing-thawing and transient hyperlipemia. Storage for as short a period as one month resulted at both +4 °C and ?20 °C in a decrease in measureable stimulating activity. Both freezing-thawing of sera five times and hyperlipemia approximately halved the stimulating activity. The decrease in stimulating activity is attributed to an increase of inhibitors rather than a decrease in CSF, since activity was fully restored by chloroform treatment, which removes all inhibitory lipoproteins. The decreased activity was shown by preparative ultracentrifugation to be due to changes in the lipoprotein composition of serum. With freshly drawn sera, most of the inhibitory activity was recovered in the light (LDL) and very light density lipoprotein (VLDL) fractions. The pattern was similar in hyperlipemic serum except that inhibitor levels were higher in the fraction containing chylomicrons and VLDL. In stored and frozen-thawed sera, most of the inhibitory activity was still recovered in the LDL fraction but the inhibition was markedly increased in both the heavy (HDL) and the very heavy density lipoprotein (VHDL) fraction. As the changes in inhibitory activity partially reflect unspecific degradation of lipoproteins, some treatment to remove lipoprotein is recommended before assaying for the stimulating activity in serum samples.     

Potent inhibitors of glucagon-stimulated adenylate cyclase associated with serum lipoprotein particles

Lipoprotein fractions from some individuals have inhibitory effects on rat liver adenylate cyclase. Precipitation of the lipoprotein fractions with acetone released an inhibitory factor, which was soluble in acetone-H2O (3:1, v/v). The inhibition was greater against glucagon-stimulated activity than against basal activity. Acetone extraction increased the potency of inhibition. All three lipoprotein fractions, i.e., very low, low, and high density lipoproteins, released some inhibitory component after acetone extraction. The inhibitor was concentrated in the lipoprotein fractions, since acetone extraction of plasma did not release an inhibitor. The acetone extract from the very low density lipoprotein was the most inhibitory. This material was further purified and partially characterized. The inhibitor had a molecular mass of about 500. It was inhibitory at micromolar concentrations. The material was sufficiently hydrophobic to migrate in normal-phase thin-layer chromatography (TLC). Nuclear magnetic resonance results indicated that it was not a polar lipid. There were several different inhibitory factors that were separable by TLC. The sequestration of these inhibitors into lipoproteins reduced their effectiveness in inhibiting the action of counter-regulatory hormones, such as glucagon.     

Suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and of incorporation of acetate into cholesterol in homozygous hypercholesterolemic fibroblasts by ferritin-low density lipoprotein conjugates.

Conjugates of ferritin with low density lipoproteins (LDL) were prepared and separated by sucrose gradient centrifugation. These conjugates, at cholesterol concentration of 100--132 microgram/ml, caused a greater than 90% suppression of hydroxymethylglutaryl coenzyme A reductase activity and of acetate incorporation into cholesterol in cultured skin fibroblasts from a normal subject as well as from a subject with homozygous familial hypercholesterolemia. The half maximal inhibition concentration was approx. 10 microgram/ml cholesterol for LDL and ferritin . (LDL)2 and 5 microgram/ml for (ferritin)2 . LDL in both cell lines. In contrast, native low density lipoproteins have only a minimal inhibitory effect in homozygous cells. The ability of the conjugates to stimulate the incorporation of oleate into cholesteryl esters was also equal in the two cell lines, although the conjugates were only 10% as active as low density lipoproteins in the normal cells. LDL reduced the ferritin . (LDL)2-mediated suppression of hydroxymethylglutaryl-CoA reductase activity in homozygous cells while ferritin . (LDL)2 reduced the LDL-mediated stimulation of cholesteryl ester formation in normal cells.     

Molecular mechanism of the blockade of plasma cholesteryl ester transfer protein by its physiological inhibitor apolipoprotein CI

Genetically engineered mice demonstrated that apolipoprotein (apo) CI is a potent, physiological inhibitor of plasma cholesteryl ester transfer protein (CETP) activity. The goal of this study was to determine the molecular mechanism of the apoCI-mediated blockade of CETP activity. Kinetic analyses revealed that the inhibitory property of apoCI is independent of the amount of active CETP, but it is tightly dependent on the amount of high density lipoproteins (HDL) in the incubation mixtures. The electrostatic charge of HDL, i.e. the main carrier of apoCI in human plasma, is gradually modified with increasing amounts of apoCI, and the neutralization of apoCI lysine residues by acetylation produces a marked reduction in its inhibitory potential. The inhibitory property of full-length apoCI is shared by its C-terminal alpha-helix with significant electrostratic properties, whereas its N-terminal alpha-helix with no CETP inhibitory property has no effect on HDL electronegativity. Finally, binding experiments demonstrated that apoCI and to a lower extent its C-terminal alpha-helix are able to disrupt CETP-lipoprotein complexes in a concentration-dependent manner. It was concluded that the inhibition of CETP activity by apoCI is in direct link with its specific electrostatic properties, and the apoCI-mediated reduction in the binding properties of lipoproteins results in weaker CETP-HDL interactions and fewer cholesteryl ester transfers.     

Serum lipoproteins as cholesterol donors to mycoplasma membranes.

Mycoplasma hominis andAcholeplasma laidlawii were grown in media in which a fraction of human serum lipoproteins provided the sole source of cholesterol. Increasing levels of very low density lipoproteins had an inhibitory effect on the growth of the organisms. Low and high density lipoproteins in all concentrations proved to be excellent sources of cholesterol. Both organisms were able to limit the amount of cholesterol taken up and to preferentially incorporate free cholesterol despite an excess of esterified cholesterol in the medium. When similar levels of free cholesterol were provided by low density or high density lipoproteins, the organisms incorporated from 20–45% more cholesterol from the former. This preference for cholesterol from low density lipoproteins partially supports the theory that the low density lipoproteins act as a donor while the high density lipoproteins are a scavenger of cholesterol.     

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase by oxysterol by-products of cholesterol biosynthesis. Possible mediators of low density lipoprotein action

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34, reductase) activity was studied in cultured rat intestinal epithelial cells using 3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one ( U18666A ), an inhibitor of 2,3- oxidosqualene cyclase (EC 5.4.99.7, cyclase) that causes cellular accumulation of squalene 2,3:22,23-dioxide ( Sexton , R. C., Panini , S.R., Azran , F., and Rudney , H. (1983) Biochemistry 22, 5687-5692). Treatment of cells with U18666A (5-50 ng/ml) caused a progressive inhibition of reductase activity. Further increases in the level of the drug paradoxically lessened the inhibition such that at a level of 1 microgram/ml, no inhibition of enzyme activity was observed. Cellular metabolism of squalene 2,3:22,23-dioxide to compounds with the chromatographic properties of polar sterols led to an inhibition of reductase activity that could be prevented by U18666A (1 microgram/ml). The drug was unable to prevent the inhibition of enzyme activity by 25-hydroxycholesterol or mevalonolactone, but totally abolished the inhibitory action of low density lipoproteins. Pretreatment with U18666A did not affect the ability of cells to degrade either the apoprotein or the cholesteryl ester component of low density lipoproteins. These results suggest that oxysterols derived from squalene 2,3:22,23-dioxide may act as physiological regulators of reductase and raise the possibility that the suppressive action of low density lipoproteins on reductase may be partially or wholly mediated by such endogenous oxysterols generated through incomplete inhibition of the cyclase.     

Inhibition of the lytic activity of perforin by lipoproteins

Cytoplasmic granules isolated from cytolytic T lymphocytes (CTL) lyse red blood cells or tumor cell lines in a nonspecific manner. The activity of highly purified granules was inhibited by human or rabbit serum at dilutions as high as 1/10,000. The main inhibitory activity of human serum was isolated by chromatography and was determined to be high density lipoprotein (HDL). HDL not only inhibited at a concentration of 70 ng/ml the lytic activity of isolated granules, but also of the purified, pore-forming protein perforin present in the granules. Purified low density lipoprotein was equally active. Because the CTL granule activity was inhibited by pure egg lecithin vesicles at a concentration equivalent to the phospholipid content of lipoproteins, the lipid portion of lipoproteins is the likely candidate for granule inactivation. Lipoproteins also decreased in a dose-dependent manner the cytotoxic activity of intact cytolytic T cells. However, cytotoxicity was not completely suppressed, and only in the case of CTL exhibiting low efficiency in killing their targets. It is proposed that lipoproteins inactivate perforin and may thereby inhibit a possible lysis of innocent bystander cells.     

Mechanism of regulation of steroidogenesis in the adrenals by blood serum lipoproteins

It has been shown that high density lipoproteins obtained by ultracentrifugation and loaded with cholesterol and pregnenolone exert a stimulant effect on in vitro production of glucocorticoids by rat adrenals. Low density native lipoproteins possess an inhibitory effect connected with the protein component. The action of various blood serum lipoproteins on steroidogenesis in rat adrenals has significantly different mechanisms.     

A synthetic peptide mimic of plasma apolipoprotein E that binds the LDL receptor.

Human plasma apolipoprotein E (apoE) is a low density lipoprotein (LDL) receptor ligand. It targets cholesterol-rich lipoproteins to LDL receptors on both hepatic and peripheral cells. The region of apoE responsible for its binding to the LDL receptor has been localized to amino acids 140-160. An apoE 141-155 monomeric peptide and a dimeric 141-155 tandem peptide were synthesized and tested for their inhibition of 125I-LDL degradation by human fibroblasts and human monocytic-like cells, THP-1. The monomer had no activity at 250 microM, but the dimer inhibited 125I-LDL degradation by 50% at 5 microM. The inhibition was specific for the LDL receptor because the dimer did not inhibit the degradation of 125I-acetylated LDL by scavenger receptors expressed by phorbol ester-stimulated THP-1 cells. As reported for native apoE, amino acid substitutions of Lys-143----Ala, Leu-144----Pro, and Arg-150----Ala decreased the inhibitory effectiveness of the dimer. Furthermore, a trimer of the 141-155 sequence had a 20-fold greater inhibitory activity than the dimer. Studies with a radioiodinated dimer indicated that some of the inhibitory activity could be a result of the interaction of the dimer with LDL. However, direct binding of the 125I-dimeric peptide to THP-1 cells was observed as well. This binding was time-dependent, linear with increasing cell number, Ca(2+)- but not Mg(2+)-dependent, saturable, inhibited by lipoproteins, and increased by preculture of the cells in lipoprotein-depleted medium. Therefore, a synthetically prepared dimeric repeat of amino acid residues 141-155 of apoE binds the LDL receptor.     

Erythroid and granulocytic colony growth in cultures supplemented with human serum lipoproteins.

The ability of human serum to support erythroid and granulocytic colony formation has been investigated. It was found that normal human serum could replace fetal calf serum in the cultures and was able to support the growth of these hemopoietic colonies. Serum fractions enriched for low density lipoproteins, either by precipitation with Heparin-Mn++ or by ultracentrifugation, was found to contain this growth supporting activity of human serum.     

Study of the relationship between the glucocorticoid function of the adrenal cortex and blood lipoproteins]

Single administration of hydrocortisone to rats led to elevation in the blood serum of lipoproteins of very low density; and repeated administration - of lipoproteins of very low density, low density and of high density. Lipoproteins with d less than 1.063 g/ml produced an inhibitory action on the production of steroid hormones by the adrenal glands of rats in vitro.     

Evaluation of gel chromatography for plasma lipoprotein fractionation

The fractionation of lipoproteins of normal and hyperlipidemic subjects on a column of 2% agarose was compared with ultracentrifugation and paper electrophoresis procedures. The following results were obtained. (a) Plasma lipoproteins were eluted successively from the column in the four overlapping peaks of chylomicrons, very low density lipoproteins, low density lipoproteins, and high density lipoproteins. (b) Very low density lipoproteins and high density lipoproteins (d > 1.063, containing nonlipoprotein proteins) showed continuous progressive changes in lipid composition as these fractions emerged, while low density lipoproteins showed a relatively constant lipid composition. (c) A discontinuous transition of lipid composition was observed when consecutive ultracentrifugal fractions were placed on the column. (d) The "trail" of pre-beta lipoprotein seen on paper electrophoresis was shown to consist of particles whose molecular sizes range between chylomicrons and pre-beta lipoproteins. A reverse relationship was observed between electrophoretic mobilities of "trail" components and their particle size. (e) Gel with an agarose content of 2% seemed to fractionate chylomicrons and very low density lipoproteins more effectively than other lipoprotein classes.     

Studies of inhibitors of bone marrow colony formation in normal human sera and during a viral infection

Various methods are compared for removing inhibitors of bone marrow colony stimulating activity from the serum of normal individuals. The best method proved to be chloroform treatment of sera, which is easily performed on a large scale. Chloroform treatment had no effect in itself on the colony-stimulating factor (CSF) and no additional inhibitors were detected after such treatment. Inhibitors were characterized by preparative ultracentrifugation. After fractionation, most of the inhibitory activity was recovered in the light (LDL) and very light density lipoprotein (VLDL) fractions. Treatment with specific anti human β-lipoprotein serum removed most of the inhibitory activity. Increased CSF levels have been reported to accompany the acute phase of infectious diseases. Application of the chloroform treatment to sera from patients with mumps showed that the mean CSF in these sera did not differ significantly from that found in sera from normal individuals. High CSF levels in acute infections therefore seem to reflect variations in inhibitor levels rater than a true increase in CSF.     

Release of endothelial cell lipoprotein lipase by plasma lipoproteins and free fatty acids

Lipoprotein lipase (LPL) bound to the lumenal surface of vascular endothelial cells is responsible for the hydrolysis of triglycerides in plasma lipoproteins. Studies were performed to investigate whether human plasma lipoproteins and/or free fatty acids would release LPL which was bound to endothelial cells. Purified bovine milk LPL was incubated with cultured porcine aortic endothelial cells resulting in the association of enzyme activity with the cells. When the cells were then incubated with media containing chylomicrons or very low density lipoproteins (VLDL), a concentration-dependent decrease in the cell-associated LPL enzymatic activity was observed. In contrast, incubation with media containing low density lipoproteins or high density lipoproteins produced a much smaller decrease in the cell-associated enzymatic activity. The addition of increasing molar ratios of oleic acid:bovine serum albumin to the media also reduced enzyme activity associated with the endothelial cells. To determine whether the decrease in LPL activity was due to release of the enzyme from the cells or inactivation of the enzyme, studies were performed utilizing radioiodinated bovine LPL. Radiolabeled LPL protein was released from endothelial cells by chylomicrons, VLDL, and by free fatty acids (i.e. oleic acid bound to bovine serum albumin). The release of radiolabeled LPL by VLDL correlated with the generation of free fatty acids from the hydrolysis of VLDL triglyceride by LPL bound to the cells. Inhibition of LPL enzymatic activity by use of a specific monoclonal antibody, reduced the extent of release of 125I-LPL from the endothelial cells by the added VLDL. These results demonstrated that LPL enzymatic activity and protein were removed from endothelial cells by triglyceride-rich lipoproteins (chylomicrons and VLDL) and oleic acid. We postulate that similar mechanisms may be important in the regulation of LPL activity at the vascular endothelium.     

Inhibitory activity of interleukin 2-activated lymphocytes on human granulocyte precursors (CFU-g)

Interleukin 2-activated lymphocytes (lymphokine-activated killer [LAK] cells) cultured from 2 to 14 days were added to the cultures of granulocyte precursors (CFU-g). The LAK cells inhibited colony formation of granulocyte precursors; LAK cells cultured for five days showed the strongest inhibitory activity on colony formation. The presence of cell-to-cell interaction between LAK cells and bone marrow mononuclear cells (BMNC) in CFU-g assays emphasized the LAK cell-derived colony inhibitory activity (LAK-CIA), but cell-to-cell interaction was not always a requirement for LAK-CIA, since LAK cells were also found to inhibit colony formation without such interaction. This report shows that LAK cells can inhibit in vitro colony formation of granulocyte precursors. We therefore concluded that the observed CIA is caused by soluble factor(s) derived from LAK cells, and that E-rosette-forming cells are manifesting LAK-CIA.     

Interaction of plasma lipoproteins with erythrocytes. II. Modulation of membrane-associated enzymes.

When incubated with intact erythrocytes, low density lipoproteins (LDL) decrease the phosphate content of erythrocyte spectrin allowing the cells to undergo morphological transformation. The phosphate content of spectrin depends on the balance between the activity of membrane-associated cyclic AMP-independent protein kinases and phosphoprotein phosphates. LDL do not influence the activity of membrane-associated cyclic AMP-independent protein kinases; these lipoproteins activate by 2-fold and greater membrane-associated phosphatases as determined by hydrolysis of p-nitrophenyl phosphate and by phosphate hydrolysis of phosphorylated erythrocyte membrane proteins. We conclude that LDL interact at the exterior surface of the erythrocyte to stimulate dephosphorylation of spectrin. The significance of this conclusion is augmented by the fact that spectrin, the target for LDL-induced dephosphorylation, specifies cell morphology and modulates the distribution of cell-surface receptors. LDL also render erythrocyte acetylcholinesterase less susceptible to inhition by F-. Lipoproteins in the high density class (HDL) do not stimulate dephosphorylation of spectrin, and they are consequently unable to alter erythrocyte morphology. HDL do prevent the LDL-induced activation of membrane phosphatase. The inhibitory capacity of HDL is observed over the range of LDL:HDL (w/w) which exists in the plasma of normolipemic humans.     

Studies on inhibitors of bone marrow colony formation in normal human sera and during a viral infection

Various methods are compared for removing inhibitors of bone marrow colony stimulating activity from the serum of normal individuals. The best method proved to be chloroform treatment of sera, which is easily performed on a large scale. Chloroform treatment had no effect in itself on the colony-stimulating factor (CSF) and no additional inhibitors were detected after such treatment. Inhibitors were characterized by preparative ultracentrifugation. After fractionation, most of the inhibitory activity was recovered in the light (LDL) and very light density lipoprotein (VLDL) fractions. Treatment with specific anti human β-lipoprotein serum removed most of the inhibitory activity. Increased CSF levels have been reported to accompany the acute phase of infectious diseases. Application of the chloroform treatment to sera from patients with mumps showed that the mean CSF in these sera did not differ significantly from that found in sera from normal individuals. High CSF levels in acute infections therefore seem to reflect variations in inhibitor levels rater than a true increase in CSF.     

Apoprotein E-rich high density lipoproteins inhibit ovarian androgen synthesis

The influence of high density lipoproteins (HDL) on luteinizing hormone-stimulated rat ovarian theca/interstitial cell steroidogenesis was studied. Without HDL the cells produced primarily androgens from progestin precursors. In the presence of rat or human HDL steroid output increased 3-5-fold, but the type of steroid produced was dependent on the source of the HDL. Human HDL nonselectively amplified luteinizing hormone-stimulated steroid production, whereas rat HDL promoted progestin production without a concomitant increase in androgen output. Comparisons of the activities of apoprotein E-rich HDL (e.g. HDL from intact mature rats) with apoprotein E-poor HDL (e.g. human HDL or rat HDL from hypophysectomized immature rats) suggested that apoprotein E was responsible for the inhibition of androgen production. Furthermore, the inhibitory activity of rat HDL was abolished by depleting apoprotein E-containing lipoproteins with heparin affinity chromatography. Direct evidence that apoprotein E was the inhibitory constituent of rat HDL was obtained by showing that isolated lipid-free rat apoprotein E inhibited androgen production, whereas isolated rat apoproteins A-I and A-IV did not. The possible paracrine function of apoprotein E in ovarian follicular maturation of the ovary is discussed.     

Role of plasma lecithin:cholesterol acyltransferase in the metabolism of high density lipoproteins

The role of the plasma lecithin:cholesterol acyltransferase reaction in the esterification of the cholesterol of human and baboon plasma high density lipoproteins has been studied. Human plasma was incubated in vitro, and the initial rate of cholesterol esterification in lipoprotein fractions obtained by chromatography on hydroxylapatite was determined. The rate of esterification was greater in the high density lipoprotein fraction than in the low density lipoprotein fraction. High density lipoproteins from human and baboon plasma were filtered through columns of Sephadex G 200, and the relative concentrations in the effluent of key lipids involved in the acyltransferase reaction were determined. The ratio of esterified to unesterified cholesterol varied across the lipoprotein peak obtained from either type of plasma. The relative concentration of lecithin compared to sphingomyelin also varied across the peaks obtained with human high density lipoproteins. When human or baboon plasma was incubated with cholesterol-(14)C and the high density lipoproteins were filtered through Sephadex, the specific activity of the esterified cholesterol varied across the lipoprotein peak. Similar results were obtained when plasma esterified cholesterol was labeled in vivo by the injection of labeled mevalonate into baboons. The data suggest that the acyltransferase reaction is the major source of the esterified cholesterol of the high density lipoproteins.