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R Y Hampton

Publications and source records attributed to R Y Hampton.

31 records · Page 2Linked to original sources
PubMed1991

Lipid A-like molecules that antagonize the effects of endotoxins on human monocytes.

Lipopolysaccharide (LPS) endotoxin is implicated as the bacterial product responsible for the clinical syndrome of Gram-negative septicemia. Although the lipid A domain of LPS appears to be responsible for the toxicity of endotoxin, lipid A from the photosynthetic bacterium Rhodobacter sphaeroides (RSLA) and a disaccharide precursor of lipid A from enteric bacteria, termed lipid IVA, have little activity on human cells. Using the human promonomyelocytic cell line THP-1 and human monocytic cells, we now show that both lipid IVA and RSLA are antagonists of LPS. Complete, apparently competitive, inhibition of LPS activity is possible at a 10-100-fold excess of antagonist, as judged by measuring the release of cytokines and prostaglandin E2. Both antagonists prevent monocyte stimulation by endotoxin extracted from a variety of Gram-negative bacteria. Cells pretreated with either inhibitor and subsequently washed still show attenuated responses to LPS. Stimulation of monocytes by whole Gram-negative bacteria is also antagonized in a dose-dependent manner. Lipid X has no inhibitory effect in the same dose range as lipid IVA and RSLA. These findings rule out LPS sequestration as the explanation for the observed antagonism. Neither inhibitor alters monocyte stimulation by phorbol 12-myristate 13-acetate, Staphylococcus aureus, or purified protein derivative, demonstrating specificity for LPS. Although RSLA appears to inhibit LPS when tested with macrophages from both humans and mice, lipid IVA had the unique ability to act as an LPS antagonist with human-derived cells but to exhibit LPS-like effects with murine-derived cells. Like LPS, lipid IVA stimulated the release of both tumor necrosis factor alpha and arachidonic acid from murine-derived RAW 264.7 macrophage tumor cells. The range of concentrations necessary for lipid IVA to induce LPS-like effects in murine cells was similar to that necessary to antagonize the actions of LPS in human monocytes. The agonist activities of lipid IVA were completely inhibitable by RSLA. This unique species-dependent pharmacology observed with lipid IVA may reflect differences between human and murine LPS receptors. RSLA and lipid IVA may be useful in defining the role of LPS in Gram-negative bacterial infections and may prove to be prototypical therapeutic agents for the treatment of Gram-negative septicemia.

Animals↗
PubMed1991

Macrophage catabolism of lipid A is regulated by endotoxin stimulation.

Lipopolysaccharide (LPS) is a Gram-negative bacterial glycolipid that is believed to cause, by virtue of its stimulatory actions on macrophages and other eukaryotic cells, the life-threatening symptoms associated with Gram-negative infections. Macrophages both respond to and catabolically deactivate LPS. The lipid A moiety of LPS is responsible for the stimulatory actions of LPS on macrophages. We have previously developed methods employing a radiolabeled bioactive lipid A precursor, 4'-32P-lipid IVA, to study the interaction of this class of lipids with animal cells (Hampton, R. Y., Golenbock, D. T., and Raetz, C. R. H. (1988). J. Biol. Chem. 263, 14802-14807). In the current work, we have examined the uptake and catabolism of 4'-32P-lipid IVA by the RAW 264.7 cell line in serum-containing medium at physiological temperatures and have studied the effect of LPS stimulation on the ability of these cells to catabolize lipid IVA. RAW 264.7 macrophage-like cells avidly take up 4'-32P-lipid IVA under cell culture conditions at nanomolar concentrations. Uptake of lipid IVA was accompanied by lysosomal dephosphorylation of a fraction of the lipid to yield 4'-monophosphoryl lipid IVA. Chemically generated 4'-monophosphoryl lipid IVA was found to be substantially less bioactive than lipid IVA in the RAW cell, indicating that this catabolic dephosphorylation results in detoxification. In uptake experiments of 3-4 h duration, all metabolism of lipid IVA is blocked by ligands of the macrophage scavenger receptor. In longer experiments (24 h), both scavenger receptor-dependent and -independent uptake are responsible for the lysosomal catabolism of lipid IVA. Preincubation of RAW 264.7 cells with LPS caused dose-dependent inhibition of lipid IVA dephosphorylation. Sufficient LPS stimulation resulted in essentially complete inhibition of lipid IVA catabolism in both short- and long-term uptake experiments. This effect occurred at physiologically relevant concentrations of LPS (IC50 less than 1 ng/ml), and our data indicate that LPS-induced blockade of lipid IVA catabolism was due to the resultant physiological stimulation of the cells, and not inhibition of dephosphorylation by competition for uptake or enzymatic sites or by simple sequestration of labeled lipid IVA by LPS aggregates. We suggest that in the macrophage, LPS can modulate its own catabolism by virtue of its pharmacological properties. This effect of LPS could play a role in LPS pathophysiology as well as in macrophage biology.

Animals↗
PubMed1991

Recognition and plasma clearance of endotoxin by scavenger receptors.

Lipid A is the active moiety of lipopolysaccharide (LPS, also referred to as endotoxin), a surface component of Gram-negative bacteria that stimulates macrophage activation and causes endotoxic shock. Macrophages can bind, internalize and partially degrade LPS, lipid A and its bioactive precursor, lipid IVA. We report here that lipid IVA binding and subsequent metabolism to a less active form by macrophage-like RAW 264.7 cells is mediated by the macrophage scavenger receptor. Scavenger-receptor ligands inhibit lipid IVA binding to, and metabolism by, RAW cells, and lipid IVA binds to type I and type II bovine scavenger receptors on transfected Chinese hamster ovary cells. Although in vitro competition studies with RAW cells indicate that scavenger receptor binding is not involved in LPS or lipid IVA-induced stimulation of macrophages, in vivo studies show that scavenger-receptor ligands greatly inhibit hepatic uptake of lipid IVA in mice. Thus, scavenger receptors expressed on macrophages may have an important role in the clearance and detoxification of endotoxin in animals.

Animals↗
PubMed1991

Aggregation behavior of lipid IVA in aqueous solutions at physiological pH. 1: Simple buffer solutions.

We have investigated the aggregation behaviour of lipid IVA (a bioactive precursor of lipid A and the lipid anchor of lipopolysaccharide) in aqueous solutions in the physiological pH range using dynamic light scattering, nuclear magnetic resonance, fluorescence, surface pressure, electron microscopy and force field simulation studies. The sonication of lipid IVA in PBS, Tris and Hepes produces vesicles which are stable in the concentration range of 10(-3) - 10(-7) M, possibly even at lower concentrations. The vesicle size is not sensitive to the nature of the buffer, only to the pH and to some extent to the ionic strength. The long time stability of the small unilamellar vesicles as well as the structureless 1H-NMR spectra might be attributed to a rigid surface structure. This structure is also supported by the simulation studies. We have tentatively proposed a coexistence of micelles and/or other aggregates with the bilayered vesicles at higher lipid concentrations in order to explain some of the experimental observations.

Buffers↗
PubMed1990

Human phagocytes have multiple lipid A-binding sites.

Bacterial lipopolysaccharide (LPS) is a potent stimulus of cells, yet a target protein for LPS has not been defined. We used two approaches to define LPS-binding sites on cell surfaces: one assay measured binding of LPS-coated erythrocytes (ELPS) to cultured human cells, and a second measured binding of a radiolabeled probe, [32P]lipid IVA, to intact leukocytes. The first approach identified the CD11-CD18 family of integrins as lipid A-binding sites in human phagocytes, and the latter approach demonstrated saturable lipid A binding to intact murine macrophages, as well as to an approximately 95-kDa binding protein in purified membrane preparations. Because CD18 has a known molecular mass of 95 kDa, we sought to determine whether the [32P]lipid IVA-binding site was CD18. Binding of ELPS and [32P]lipid IVA to human macrophages was found to differ with respect to temperature, divalent cation dependence, cellular specificity, and susceptibility to competition by polyanions. To determine whether the previously described 95-kDa lipid A-binding protein was CD18, nitrocellulose-immobilized RAW264.7 membrane proteins were probed with [32P]lipid IVA and subsequently immunoblotted with a monoclonal antibody to murine CD18. The lipid A-binding protein has an electrophoretic mobility slightly different from that of CD18. Moreover, monoclonal antibodies and polyclonal antiserum to the CD11-CD18 family of proteins did not inhibit lipid IVA binding to intact human macrophages. Finally, mononuclear cells from two patients with CD18 deficiency failed to form rosettes with ELPS but bound [32P]lipid IVA normally. Thus, different LPS preparations may bind to cells in a CD18-dependent or -independent manner. Since ELPS is particulate and lipid IVA is a fine dispersion, the identity of the binding site may depend on the physical state of the LPS.

Antigens, Differentiation↗
PubMed1988

Lipid A binding sites in membranes of macrophage tumor cells.

Lipopolysaccharide affects a variety of eukaryotic cells and mammalian organisms. These actions are involved in the pathogenesis of Gram-negative septicemia. Many of the actions of lipopolysaccharide are believed to be caused by its active moiety, lipid A. Our laboratory has previously identified a bioactive lipid A precursor, termed lipid IVA (Raetz, C. R. H., Purcell, S., Meyer, M. V., Qureshi, N., and Takayama, K. (1985) J. Biol. Chem. 260, 16080-16888), which can be labeled with 32P of high specific activity and purified. In this work we have used the labeled probe, 4'-32P-lipid IVA, to develop a novel assay for the specific binding of lipid IVA to whole cells. We have also demonstrated its use in a ligand blotting assay of immobilized cellular proteins. Using the whole cell assay, we show that 4'-32P-lipid IVA specifically binds to RAW 264.7 macrophage-like cultured cells. The binding is saturable, is inhibited with excess unlabeled lipid IVA, and is proteinase K-sensitive. It displays cellular and pharmacological specificity. Using the ligand blotting assay, we show that several RAW 264.7 cell proteins can bind 4'-32P-lipid IVA. The two principal binding proteins have Mr values of 31 and 95 kDa, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fractionation studies indicate that the 31-kDa protein is enriched in the nuclear fraction and may be a histone, whereas the 95-kDa protein is enriched in the membrane fraction. The binding assays that we have developed should lead to a clearer understanding of lipid A/animal cell interactions.

Animals↗
PubMed1983

Effects of changes in osmolality on the stability and function of cultured chromaffin cells and the possible role of osmotic forces in exocytosis.

Recent evidence indicates that osmotic forces may play a role in exocytosis. To examine this possibility and to investigate the osmotic properties of storage granules within cells, we investigated the effects of changes of osmolality on stability and function of cultured bovine chromaffin cells. Cell volume measurements indicated that the cells behaved as osmometers and that the intracellular osmolality rapidly equilibrated with the osmolality of the extracellular medium. Hyperosmotic solutions strongly inhibited nicotinic agonist-stimulated secretion but did not alter nicotinic agonist-stimulated Ca(2+) uptake. Hyperosmotic solutions also strongly inhibited elevated potassium- stimulated secretion but only weakly inhibited elevated K(+)-stimulated Ca(2+) uptake. Thus, hyperosmotic solutions inhibited secretion at a step after calcium entry. Cells exposed to 165 mOs(1) solutions did not lyse and retained their capacity to store and secrete catecholamine upon stimulation. Significant intracellular lysis of chromaffin granules occurred within cells exposed to lower osmolalities. In contrast, 75 percent of the catecholamine was released from granules from cultured cells or from fresh adrenal medulla incubated in vitro at 210 mOs. The data provide evidence for a role for osmotic forces in exocytosis and suggest that if osmotic stress of the granule occurs during exocytosis, then water influx into chromaffin granules increases granule volume by at least 70 percent. The results also indicate that the osmotic properties of the granules are altered upon homogenization and subcellular fractionation of the cells.

Adrenal Medulla↗
PubMed1982

Stereospecific binding of 3H-phencyclidine in brain membranes.

Phencyclidine (PCP) displaceable binding of 3H-PCP to glass-fiber filters was eliminated and total binding markedly reduced by initial treatment of the discs with 0.05% polyethyleneimine. Assessed with treated filters, unlabeled PCP displaced 3H-PCP in both rat and pigeon brain membranes with an EC50 of 1 microM. Of similar high inhibitory potency were dextrorphan, levorphanol, SKF 10047 and ketamine, while morphine, naloxone and etorphine had EC50 values higher then 1 mM. Using the dissociative anesthetic dexoxadrol and its inactive isomer levoxadrol as displacing agents, stereospecific binding of 3H-PCP was obtained in rat and pigeon brain membranes. The markedly higher potency of dexoxadrol, relative to levoxadrol, in displacing bound 3H-PCP is compatible with behavioral data for these enantiomers. However, they were equipotent in displacing 3H-PCP bound to glass-fiber filters in the absence of tissue. Heat denaturation, but not freezing, abolished stereospecific binding of 3H-PCP, which was also absent in rat liver membranes. The stereospecific binding component in brain displayed biphasic saturability at 60-70 nM and 300-400 nM, respectively.

Animals↗
PubMed1982

A procedure for rapid evaluation of the discriminative stimulus effects of drugs.

Rhesus monkeys were trained in a two-lever drug-discrimination procedure with several discrete trials per session. During training sessions, either a sham injection or a subcutaneous injection of the training drug was administered ten minutes prior to each trial. During each trial, completion of 100 consecutive responses on the lever appropriate for the animal's pharmacological condition (e.g., left for sham, right for drug) resulted in the delivery of three grams of food. Training sessions consisted of from zero to four sham trials that preceded two consecutive drug trials. The number of sham trials varied unsystematically to preclude discrimination of the drug trials on the basis of the number of preceding trials. Discriminations were established with each of the training drugs employed (codeine, methohexital, and ketamine). During dose-effect evaluations of the training drug or other drugs (test sessions), a progressively large dose of drug was injected prior to each trial and 100 consecutive responses on either the sham- or drug-appropriate lever resulted in the delivery of food. Test sessions continued until either drug-lever responding or a marked suppression in the rate of responding occurred. Thus, a cumulative dose-effect curve for each drug was generated within a single session. Preliminary findings suggest that the pattern of cross-drug generalization generated by this cumulative-dosing procedure is similar to that obtained with procedures that evaluate only a single dose of drug per session.

Animals↗