Chylomicron-retinyl palmitate clearance in type I hyperlipidemic families.

Our primary aim was to determine the extent to which intraplasmic retinyl palmitate (RP) transfers to other lipoprotein particles when chylomicron remnants are not produced and/or the plasma RP residence time is increased. The study was conducted on three familial type I hyperlipoproteinemic patients, four lipoprotein lipase (LpL)-deficient heterozygotes, and three controls on a metabolic research unit. To each subject, a fat load was administered containing 16% of total daily calories in type I patients, 40% in heterozygotes and controls, plus 60,000 U/m2 vitamin A. Triglyceride (TG) and RP levels were evaluated in chylomicron and nonchylomicron fractions. Delay in the clearance of chylomicron fraction RP and the marked deficiency in nonchylomicron-RP (presumed lack of remnant production) in all three type I patients suggests that RP does not demonstrate significant intraplasmic transfer from chylomicrons to existent apolipoprotein B100 particles. In contrast to noncoincident TG and RP peaking in the normal subject, heterozygotes were found to demonstrate coincident plasma TG and RP curves, which is consistent with a common catabolic pathway for both TG and RP and inconsistent with intraplasmic RP transfer. This corroborates reports on compromised chylomicron clearance in heterozygotes. We conclude that RP is an appropriate representative marker for intestinally derived particles in LpL-deficient or partially deficient individuals.

Suppressive effect of antioxidants on intercellular adhesion molecule-1 (ICAM-1) expression in human epidermal keratinocytes.

Intercellular adhesion molecule-1 (ICAM-1) is strongly expressed by human epidermal keratinocytes during the course of inflammatory skin diseases. To test the possibility that reactive oxygen species produced in the skin during an inflammatory response affect ICAM-1 expression, cultured human epidermal keratinocytes were treated with H2O2 at concentrations that did not damage the cells, and cell-surface ICAM-1 expression was analyzed. Expression of ICAM-1 was induced on keratinocytes by treatment with 300 microM H2O2 for 1 h. The antioxidant N-acetyl-L-cysteine strongly inhibited H2O2-induced ICAM-1 expression, whereas the antioxidants pyrrolidine dithiocarbamate and alpha-tocopherol were less inhibitory. N-acetyl-L-cysteine also suppressed keratinocyte surface expression of ICAM-1 induced by the cytokines interferon-gamma (IFN-gamma) or tumor necrosis factor-alpha (TNF-alpha), whereas pyrrolidine dithiocarbamate and alpha-tocopherol suppressed IFN-gamma-induced surface expression but not TNF-alpha-induced expression. We found that N-acetyl-L-cysteine treatment reduced ICAM-1 mRNA levels when keratinocytes were stimulated with either IFN-gamma or TNF-alpha; however, pyrrolidine dithiocarbamate and alpha-tocopherol had no effect on either IFN-gamma- or TNF-alpha-induced ICAM-1 mRNA levels. Our results indicate that reactive oxygen species may be involved in the skin inflammatory process by increasing epidermal ICAM-1 expression and that some antioxidants may be effective in suppressing the epidermal ICAM-1 expression induced by reactive oxygen species and cytokines in inflammatory skin diseases.

Crystals of soluble interleukin-1 receptor complexed with its natural antagonist reveal a 1:1 receptor-ligand complex.

Interleukin-1 is a cytokine involved in the acute phase response against infection and injury. We obtained crystals of a complex of soluble, recombinant human interleukin-1 receptor and recombinant human interleukin-1 receptor antagonist, a naturally occurring antagonist. The crystals are suitable for X-ray analysis and diffract to 2.7 A resolution. Solvent content calculations indicate that the crystals contain one receptor and one antagonist molecule per asymmetric unit. Other receptor to antagonist ratios are highly unlikely. These results suggest that the interleukin-1 antagonist binds a single receptor molecule and does not cause receptor aggregation.

Inhibition of IL-1 beta expression in THP-1 cells by probucol and tocopherol.

The cytokine interleukin-1, IL-1, likely plays an important role in the early stages of atherogenesis. The possible action of probucol and tocopherol on the expression and secretion of IL-1 beta was investigated using the human monocytic leukemia cell line, THP-1. Both probucol and D-alpha-tocopherol inhibit the phorbol ester-induced release of IL-1 beta without altering differentiation. Analysis of IL-1 beta mRNA levels revealed that probucol and tocopherol had an inhibitory effect on the activation of expression of the IL-1 beta gene. The data suggest that the beneficial effects of probucol may be related to inhibition of IL-1 at an early phase of atherosclerotic plaque formation.

A fluorometric assay for the quantitation of cell adherence to endothelial cells.

A rapid quantitative fluorometric assay was developed for analysis of leukocyte adherence to endothelial cells. In this method adherent monocyte and T cell lines are labeled with the fluorescent dye Calcein AM without affecting cell function. Following coincubation with endothelial cells and gentle washing to remove nonadhering cells, the relative fluorescence intensity of the adhering cells is determined with a fluorescence microtiter plate reader. Relative fluorescence intensity increases linearly with cell number over a wide range of concentrations. By comparison of fluorescence levels of adhering cells to a dilution series of labeled cells alone, the number of adhering cells can be determined. We compared this adherence assay with the 51Cr-labeling assay and found comparable adherence. However, we found the fluorescence assay to be more rapid as the use and special handling of radioactive material is eliminated. To monitor the reliability and reproducibility of this method, we followed the adherence of Calcein AM-labeled THP-1 cells, a human monocytic cell line, to human endothelial cells treated with interleukin-1.

Identification of a specific receptor for interleukin-1 in vascular smooth muscle cells: regulation by interleukin-1 and interleukin-6.

A fluorescently labeled ligand was utilized to establish the existence of an interleukin-1 (IL-1) receptor in vascular smooth muscle. The binding of the phycoerythrin-labeled IL-1 beta to the murine T cell line, EL-4, was examined as a positive control. The phycoerythrin-labeled IL-1 beta identified a specific IL-1 receptor in the EL-4 cells. Vascular smooth muscle cells were also positively stained by the fluorescent ligand. The binding of phycoerythrin-labeled IL-1 beta to these cells was saturable and reversed by 100-fold excess unlabeled IL-1 beta. Incubation of the vascular smooth muscle cells with IL-1 beta (25 ng/ml) or IL-6 (250 ng/ml) for 18 h increased and decreased, respectively, the percentage of cells positively stained by phycoerythrin-labeled IL-1 beta which suggests these cytokines regulate IL-1 receptor expression in these cells. These data indicate a specific receptor for IL-1 exists in vascular smooth muscle cells.

Human IL-1 receptor antagonist from Escherichia coli: large-scale microbial growth and protein purification.

Interleukin-1 receptor antagonist (IL-1ra) is a recently discovered cytokine which specifically inhibits IL-1 pro-inflammatory activities in various experimental conditions. In this work, the growth conditions of a recombinant E. coli strain which in laboratory studies expressed human IL-1ra mostly in insoluble form, have been optimized at the level of 6-1 bioreactors and then scaled up to a 50-1 process. As a result, a high amount (0.43 g l-1 of microbial culture) of soluble, active IL-1ra has been directly obtained in the large-scale cell lysate with no need for protein solubilization. Also, an efficient purification procedure has been developed for the soluble protein, based on cation exchange expanded bed adsorption directly followed by anion exchange chromatography. This process, which does not include any intermediate dialysis step or gradient elutions, can be easily scaled up to larger production volumes and is therefore well-suited for manufacturing. As a result of the overall optimization study, more than 12 g of pure IL-1ra have been obtained from a single 50-1 fermentation run, without any denaturation/renaturation process. The final product, whose identity and purity have been checked also by MALDI-TOF and ESI-MS, shows full biological activity both in cellular assays and in in vivo experiments with Cynomolgus monkeys.

An ex vivo method for studying inflammation in cynomolgus monkeys: analysis of interleukin-1 receptor antagonist.

Nonhuman primates have been used as models for testing the role of interleukin-1 (IL-1) in inflammatory diseases, including endotoxemia. The objective of this investigation was to develop a reproducible and rapid method for in vivo evaluation of IL-1 antagonists using cynomolgus monkeys. IL-1 alone can induce many of the symptoms of endotoxemia in monkeys including fever, loss of appetite, and lethargy, however, test animals are slow to recover and may become desensitized to IL-1. We have developed an ex vivo method using whole blood for analysis of IL-1 antagonists administered in vivo to the monkeys and report here results for the naturally occurring IL-1 receptor antagonist, IL-1ra. In this procedure, animals are given an i.v. infusion of IL-1ra, and blood samples are taken preinfusion and during the infusion. The samples are incubated with or without IL-1 beta and the subsequent ex vivo induction of IL-6 determined. This allows analysis of the effects of in vivo pharmacodynamics on the efficacy of antagonists without exposing the test animals to IL-1. In this ex vivo protocol, each animal serves as its own control, eliminating from the assessment the large animal to animal variation observed with in vivo responses. By testing various doses, we estimate that 50% inhibition of IL-1 induced IL-6 can be achieved with an infusion of IL-1ra at 5 micrograms/kg/15 min. This method allows simple and efficient analysis of inhibitors and antagonists of IL-1 and, potentially, other effectors.

The selective activation of T8+ cells by neuraminidase and galactose oxidase is mediated by activated T4+ cells.

The response of highly enriched populations of human T8+ lymphocytes to the oxidative mitogenic enzymes neuraminidase (NA) and galactose oxidase (GO) was enhanced by NAGO-primed T4+ lymphocytes. No similar enhancement occurred when the cells were primed with phytohemagglutinin (PHA). In the absence of subclass contamination (1%), the T8+ and T4+ cells responded equally to NAGO by the criterion of DNA replication. The addition of a small number, 2-10%, of NAGO-T4+ cells to the NAGO-T8+ cells enhanced DNA synthesis by as much as 8.5-fold. Augmentation of the cellular response did not occur unless the T4+ cells were activated by NAGO. The converse situation, 2-10% of NAGO-T8+ cells in a primarily NAGO-T4+ cell population, did not increase the DNA synthetic response of the NAGO-T4+ cells. The NAGO-T4+ cells did not augment the early event of increased phosphatidylinositol metabolism or the midcycle event of induction of receptors for interleukin 2 (IL2) and transferrin. The NAGO-T4+ cells therefore increased the probability that fully activated T8+ lymphocytes crossed the G1/S boundary. The basis for this effect was not an enhanced responsiveness of the NAGO-T8+ cells to IL2 or to other soluble growth mediators in medium conditioned by NAGO-activated lymphocytes. The results of this investigation thus implicate a control point in the NAGO-T8+ lymphocyte cell cycle that is positively modulated by the NAGO-T4+ cells themselves or by a product of their activation.

The phosphatidylinositol response and proliferation of oxidative enzyme-activated human T lymphocytes: suppression by plasma lipoproteins.

The phosphatidylinositol (PI) response and DNA synthesis of neuraminidase and galactose oxidase (NAGO)-stimulated human T lymphocytes are suppressed by low density lipoproteins (LDL). To understand the mechanism of lymphocyte activation more fully, the PI response and DNA synthesis and suppression of these events by LDL in NAGO-stimulated T lymphocytes were characterized. Between 30 min and 6 hr after NAGO stimulation, there was an increase of 32Pi incorporation into PI without increased incorporation into the phosphorylated forms of PI or into other phospholipids. DNA synthesis as determined by [3H]thymidine incorporation depended on the lymphocyte-accessory monocyte ratio and total cell density. Optimal stimulation of the PI response and DNA synthesis occurred at the same concentration of neuraminidase and galactose oxidase. While the PI response was only partially suppressed by LDL with optimal suppression at 10 to 20 micrograms of protein/ml, DNA synthesis was completely suppressed although at much higher LDL concentrations, greater than 100 micrograms protein/ml. As monocyte numbers are increased, LDL suppression of DNA synthesis is decreased. The ability of NAGO to stimulate the PI response and DNA synthesis in a similar way, and the suppression of both events by LDL, suggests the PI response is important for lymphocyte activation and proliferation. Stimulation of human T lymphocytes by oxidative mitogens, neuraminidase, and galactose oxidase caused increased phosphatidylinositol metabolism and increased DNA synthesis. Both responses were suppressed by low density lipoproteins.

Normal and mutant human adenosine deaminase genes.

Adenosine deaminase (ADA) deficiency in humans is one cause of severe combined immunodeficiency. When ADA fails to catalyze the deamination of adenosine and deoxyadenosine, the levels of deoxyadenosine that accumulate are toxic to lymphoid cells. Patients with complete ADA deficiency (e.g., with less than 5% normal ADA catalytic activity) lack both B- and T-lymphocyte function. B-lymphoblast cell lines derived from patients with ADA deficiency have been analyzed at multiple levels. Blot hybridization and S1 nuclease analysis of ADA messenger RNA (mRNA) indicates that the majority of ADA-deficient cell lines have ADA mRNA in the same abundance and size as in normal cell lines. Sequence analysis of ADA cDNAs derived from these mRNAs shows that the majority of mutations are single base changes that alter the amino acid sequence. Expression analysis proves that these point mutations lead to deficiency of ADA catalytic activity. Several cell lines have mutations that alter mRNA transcription or processing. These include a point mutation in one allele of an ADA-deficient cell line that leads to deletion of exon 4 during mRNA splicing. In addition, two cell lines are homozygous for large deletions of the gene that are the result of homologous recombination. Subjects with partial ADA deficiency have undetectable ADA activity in their erythrocytes, variable activity in their lymphoid cells, and normal immunological function. Analysis of the ADA catalytic activity of partially deficient cell lines indicates that the mutations involved affect protein stability. However, the mutations causing partial ADA deficiency are as yet undefined.

Synthetic inhibitors of alcohol dehydrogenase. Pyrazoles containing an unsaturated hydrocarbon residue in the 4-position.

A series of pyrazoles containing an unsaturated hydrocarbon residue in the 4-position has been synthesized and tested for ability to inhibit the activity of the enzyme liver alcohol dehydrogenase. These compounds were found to be less active than the corresponding saturated analogues.

Steroid oxidoreductase activity of alcohol dehydrogenases from horse, rat, and human liver.

Alcohol dehydrogenase from horse (isoenzyme SS and ES, but not EE), rat and human liver were found to catalyze the NAD-dependent oxidation of 3beta-hydroxy groups in 5alpha- and 5beta-steroids of the C19, C21, and C24 series. The enzymes from horse and rat liver were more active on 5beta-than on 5alpha-steroids. This difference was most marked with the enzyme from rat liver, especially with 3beta-hydroxyandrostan-17-ones and 3beta-hydroxypregnan-20-ones as substrates. The Km of isoenzyme ES from horse liver was lower for 3beta-hydroxy-5alpha-cholanoic acid (0.4 muM) than for 3beta-hydroxy-5beta-cholanoic acid (0.9 muM). 3alpha-Hydroxysteroids were not substrates for the enzymes from horse and rat liver. Human liver alcohol dehydrogenase had low affinity for 3beta-hydroxy-5alpha (and 5beta)-cholanoic acids, but oxidation could be clearly demonstrated by gas chromatographic analysis of the products.

Suppression of interleukin-1 beta and LDL scavenger receptor expression in macrophages by a selective protein kinase C inhibitor.

A human monocytic cell line, THP-1, stimulated with 40 nM phorbol myristate acetate (PMA), differentiated to macrophage-like cells, and exhibited increased expression and release of interleukin-1 beta and expression of acetylated low density lipoprotein (ac-LDL) receptors. A selective inhibitor, MDL 29,152 (4-propyl-5-(4-quinolinyl)-2(3H)-oxazolone) was used to show that this induction required activation of protein kinase C. MDL 29,152 acts in the catalytic domain of protein kinase C and is at least 200-fold selective for protein kinase C over cAMP-dependent protein kinase in THP-1 cells. MDL 29,152 (50 microM) reduced levels of interleukin-1 beta mRNA in PMA-stimulated cells by 76% and eliminated detectable interleukin-1 beta in the media. Flow cytometric analysis showed that 48 h after THP-1 activation, approximately 50% of the cells expressed ac-LDL receptors, while in the presence of 100 microM MDL 29,152, less than 5% of the cells expressed receptors. The relationship between THP-1 differentiation and protein kinase C activation was determined by following the expression of the cell surface antigen MO-1. Expression of MO-1 antigen increases as monocytes differentiate to macrophages. After 48 h of phorbol activation, 90% of the THP-1 population was MO-1-positive; less than 16% of the population was MO-1-positive when 100 microM MDL 29,152 was present. By dual analysis, it was found that within the differentiated, MO-1-positive population, only approximately 50% of the cells also expressed ac-LDL receptors. Based on these findings, we conclude that protein kinase C promotes processes important in THP-1 activation and differentiation to macrophage-like cells including interleukin-1 beta expression and secretion, ac-LDL receptor and MO-1 expression.

In vitro model for developmental progression from vasculogenesis to angiogenesis with a murine endothelial precursor cell line, MFLM-4.

In the embryo, vascular networks are developed through both vasculogenesis, the assembly of vessels from endothelial progenitor cells or hemangioblasts, and angiogenesis, the sprouting of vessels from preexisting capillaries. Cell culture models using endothelial cells (EC) and various extracellular matrix components have been useful in understanding the cellular and molecular factors involved in angiogenesis. However, there are few models of vasculogenesis. Using a murine endothelial precursor cell line, MFLM-4, derived from e14.5 lung mesenchyme, we have developed a culture system that not only recapitulates many of the characteristics of vasculogenesis but also progresses into angiogenesis. By 8 h, MFLM-4 cultured on the basement membrane preparation Matrigel invade the matrix, coalesce, and assemble into large clusters of cells resembling blood islands. During vascular development, blood islands are the focal areas for coalescence of endothelial precursors. For MFLM-4, this phase of in vitro vasculogenic clustering does not require proliferation. If proliferation is not blocked, MFLM-4 progresses into an angiogenic phase with the clusters forming multicell angiogenic sprouts. Through 3 days of culture, lumens form within the clusters, adjacent clusters are connected with tube-like structures, and eventually an extensive network or plexus of clusters connected by capillary-like tubes is formed. MFLM-4 cultured on Matrigel provides an in vitro system for analysis of the multistage, concurrent processes of vasculogenesis and angiogenesis.

Accessory cells induce a polyphosphatidylinositol response when cultured with mitogen-activated T lymphocytes.

Monocytes (MO) influenced phosphoinositide metabolism when human T lymphocytes, isolated from peripheral blood, were activated by polyclonal mitogens. In the 3 hr immediately following mitogenic challenge, the synthesis of phosphatidylinositol (PI) was augmented and the synthesis of PI-4-phosphate (PIP) and PI-4,5-bisphosphate (PIP2) was induced in cultures of T lymphocytes and MO. In addition, MO induced a rapid and transient degradation of PIP and PIP2 in T cells prelabeled with [32P]PL and subsequently activated by mitogen. Induction of a PIP/PIP2 response correlated well with induction of DNA replication by MO when T cells were activated by phytohemagglutinin or by neuraminidase plus galactose oxidase. MO did not influence polyphosphoinositide metabolism when T cells were stimulated by the nonmitogenic lectin wheat germ agglutinin. Interleukin 1 could not substitute for monocytes in inducing a polyphosphoinositide response. By causing a rapid and transient release of the second messengers diacylglycerol and inositol phosphates and by subsequently increasing their cellular precursors, MO may induce the interleukin 2 responsive state in T lymphocytes.