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The enzyme 11β–hydroxysteroid dehydrogenase (HSD) type 1 converts inactive cortisone into active cortisol in cells, thereby raising the effective glucocorticoid (GC) tone above serum levels. We report that pharmacologic inhibition of 11β-HSD1 has a therapeutic effect in mouse models of metabolic syndrome. Administration of a selective, potent 11β-HSD1 inhibitor lowered body weight, insulin, fasting glucose, triglycerides, and cholesterol in diet-induced obese mice and lowered fasting glucose, insulin, glucagon, triglycerides, and free fatty acids, as well as improved glucose tolerance, in a mouse model of type 2 diabetes. Most importantly, inhibition of 11β-HSD1 slowed plaque progression in a murine model of atherosclerosis, the key clinical sequela of metabolic syndrome. Mice with a targeted deletion of apolipoprotein E exhibited 84% less accumulation of aortic total cholesterol, as well as lower serum cholesterol and triglycerides, when treated with an 11β-HSD1 inhibitor. These data provide the first evidence that pharmacologic inhibition of intracellular GC activation can effectively treat atherosclerosis, the key clinical consequence of metabolic syndrome, in addition to its salutary effect on multiple aspects of the metabolic syndrome itself.

Addition of lipopolysaccharide (LPS) to cells in the form of LPS–soluble (s)CD14 complexes induces strong cellular responses. During this process, LPS is delivered from sCD14 to the plasma membrane, and the cell-associated LPS is then rapidly transported to an intracellular site. This transport appears to be important for certain cellular responses to LPS, as drugs that block transport also inhibit signaling and cells from LPS-hyporesponsive C3H/HeJ mice fail to exhibit this transport. To identify the intracellular destination of fluorescently labeled LPS after its delivery from sCD14 into cells, we have made simultaneous observations of different organelles using fluorescent vital dyes or probes. Endosomes, lysosomes, the endoplasmic reticulum, and the Golgi apparatus were labeled using Texas red (TR)–dextran, LysoTracker™ Red DND-99, DiOC6(3), and boron dipyrromethane (BODIPY)–ceramide, respectively. After 30 min, LPS did not colocalize with endosomes, lysosomes, or endoplasmic reticulum in polymorphonuclear leukocytes, although some LPS-positive vesicles overlapped with the endosomal marker, fluorescent dextran. On the other hand, LPS did appear to colocalize with two markers of the Golgi apparatus, BODIPY–ceramide and TRITC (tetramethylrhodamine isothiocyanate)–labeled cholera toxin B subunit. We further confirmed the localization of LPS in the Golgi apparatus using an epithelial cell line, HeLa, which responds to LPS–sCD14 complexes in a CD14-dependent fashion: BODIPY–LPS was internalized and colocalized with fluorescently labeled Golgi apparatus probes in live HeLa cells. Morphological disruption of the Golgi apparatus in brefeldin A–treated HeLa cells caused intracellular redistribution of fluorescent LPS. These results are consistent with the Golgi apparatus being the primary delivery site of monomeric LPS.

We have previously reported that lipopolysaccharide (LPS) binding protein (LBP) opsonizes endotoxin (LPS) for recognition by CD14 on phagocytes. Here we show that normal human plasma contains high titers of an activity that also binds LPS (Re, 595) and mediates recognition by CD14. Opsonization of LPS-coated particles with plasma enables the particles to be bound by phagocytes. Further, opsonization with plasma also enables subnanogram-per-milliliter concentrations of LPS to induce dramatic alterations in the function of leukocyte integrins on polymorphonuclear leukocytes and to induce secretion of tumor necrosis factor by monocytes, suggesting that opsonization by factors in plasma may be important in responses of cells to endotoxin. The opsonic activity in plasma appears distinct from LBP since it is not blocked by neutralizing antibodies against LBP. Surprisingly, the opsonic activity of plasma is not present in a single protein species, but at least two species must be combined to observe activity. Further, the opsonic activity of plasma for LPS is blocked by addition of protease inhibitors, suggesting that proteolytic activity or activities are required for opsonization. These properties are suggestive of the action of a protease cascade, but opsonic activity of plasma is not affected by blockade or depletion of either the complement or clotting cascades. We propose the name "septin" to describe this novel LPS-opsonizing activity in plasma.

We report here that human macrophages bind Escherichia coli by recognizing bacterial lipopolysaccharide (LPS). Purified LPS was inserted into erythrocyte membranes, and the resulting LPS-coated red cells were bound by macrophages with the same temperature and cation dependence as observed for E. coli. When receptors for LPS were withdrawn from the plasma membrane by spreading the macrophages on LPS-coated surfaces, the binding of E. coli was blocked. We have also identified the receptors on macrophages that recognize LPS. Macrophages express three structurally homologous cell surface proteins, CR3, lymphocyte function-associated antigen (LFA-1), and p150,95. We used surface-bound monoclonal antireceptor antibodies to selectively remove these proteins from the apical surface of macrophages. We found that each of these proteins mediated the binding of E. coli to macrophages.

The calcium ion has been implicated as a cytosolic signal or regulator in phagocytosis. Using the Ca++-sensitive photoprotein aequorin we have measured intracellular free Ca++ ion concentration ([Ca++]i) in thioglycolate-elicited mouse peritoneal macrophages during phagocytosis and IgG-induced spreading. Macrophages plated on glass were loaded with aequorin and [Ca++]i was then measured from cell populations, both as previously described (McNeil, P. L., and D. L. Taylor, 1985, Cell Calcium, 6:83-92). Aequorin indicated a resting [Ca++]i in adherent macrophages of 84 nM and was responsive to changes in [Ca++]i induced by the addition of Mg-ATP (0.1 mM) or serum to medium. However, during the 15 min required for phagocytosis of seven or eight IgG-coated erythrocytes per macrophage loaded with aequorin, we measured no change in [Ca++]i. Similarly, the ligation of Fc-receptors that occurs when macrophages spread on immune complex-coated coverslips did not change macrophage [Ca++]i. In contrast, a rise in [Ca++]i of macrophages was measured during phagocytosis occurring in a serum-free saline of pH 7.85, and as a consequence of incubation with quin2 A/M. We estimate that had a change in [Ca++]i occurred during phagocytosis, aequorin would have detected a rise from 0.1 to 1.0 microM taking place in as little as 2% of the macrophage's cytoplasmic volume. We therefore suggest that either Ca++ is not involved as a cytoplasmic signal for phagocytosis or that increases in [Ca++]i during phagocytosis are confined to such small regions of cytoplasm as to be below the limits of detection by our cellular averaging method. Our data emphasizes, moreover, the need for well-defined, nonperturbing conditions in such measurements of [Ca++]i.

C3bi receptors (CR3) on human polymorphonuclear leukocytes (PMN) bind ligand-coated particles and promote their ingestion. The binding activity of CR3 is not constitutive but is transiently enabled by phorbol esters (Wright, S. D., and B. D. Meyer, 1986, J. Immunol. 136:1759-1764). Our observations indicate that the capacity of CR3 to bind ligand is tightly correlated with the degree of ligand-independent aggregation of the receptor in the plane of the membrane. Fixed PMN were labeled with anti-CR3 monoclonal antibodies and streptavidin colloidal gold before viewing in the electron microscope either en face or in thin section. On unstimulated PMN, gold particles marking CR3 were dispersed randomly. Stimulation of PMN for 25 min with phorbol myristate acetate (PMA) dramatically enhances binding of C3bi-coated particles, and the CR3 on such stimulated cells was observed in clusters containing more than six gold particles. CR3 was not aggregated over coated pits. After 50 min in PMA, the binding activity of CR3 falls, and the distribution of CR3 was again observed to be disperse. If a hydrophilic phorbol ester was washed away after a 20-min stimulation, binding activity remains elevated for at least 50 min, and CR3 remained aggregated. Thus, clustering of CR3 was temporally correlated with its ability to bind ligand and initiate phagocytosis. Unlike CR3, Fc receptors and HLA did not exhibit changes in their aggregation state in response to PMA. Treating PMN with formyl-methionyl-leucyl-phenylalanine, which enhances expression of CR3 but not its function, did not lead to aggregation of CR3. These observations suggest that a clustered configuration is a precondition necessary for binding ligand and signaling phagocytosis.

We have used mice selectively tolerized to antigens of human lymphocytes by treatment with cyclophosphamide to raise an mAb, BH2-C6, that reacts with a plasma membrane antigen specific for human neutrophils. This specificity is demonstrated by indirect immunofluorescence microscopy, cytochemical analysis of fluorescence-positive and -negative cell populations separated by flow cytometry, and by the selective, complement-mediated killing of mAb BH2-C6-treated neutrophils. Additional evidence for the neutrophil specificity of mAb BH2-C6 is shown by immunoelectron microscopy, which demonstrates a lack of reactivity with human eosinophils. Immunoblotting of SDS-PAGE-separated proteins of polymorphonuclear leukocytes with 125I-labeled BH2-C6 identifies protein with an average molecular mass of 157 kD. Binding studies show that, at saturation, neutrophils bind 214,000 molecules of 125I-BH2-C6 per cell. Addition of mAb BH2-C6 to neutrophils significantly reduces the number of C3bi-opsonized sheep erythrocytes (EIgMC3bi) bound by these cells. This reduction is partly reversed by the presence of soybean trypsin inhibitor (SBTI), indicating that at least one part of this inhibition is due to BH2-C6-stimulated secretion of a serine protease that may affect ligand binding. Cytochemical analysis of normal human bone marrow cells sorted by cytofluorimetry identifies the promyelocyte as the precursor cell that first expresses BH2-Ag on the plasma membrane. Using the leukemic cell line HL-60, we demonstrate that only inducers of granulocytic differentiation, cis-retinoic acid, and dimethyloxazolidine stimulate the expression of BH2-Ag. These results show that the expression of BH2-Ag during myelomonocytic differentiation is a property uniquely possessed by cells committed to the neutrophilic lineage.

Receptors for IgG stimulate the release of approximately 20% of cellular arachidonic acid (20:4) from murine resident peritoneal macrophages. In contrast, C3 receptors do not trigger the secretion of any 20:4 in excess of that released constitutively from the cells. Since the ability of C3 receptors to promote phagocytosis is regulated, we compared resting macrophages, whose C3 receptors do not promote phagocytosis of C3-coated particles, and lymphokine-treated cells, whose receptors do promote ingestion. Despite their ability to promote phagocytosis, the C3 receptor of lymphokine-treated macrophages remain unable to initiate release of 20:4. We speculate that the intracellular signals that initiate phagocytosis are distinct from those that initiate release of 20:4.

Monocytes were isolated in high yield (approximately 80%) and purity (greater than 90%) by Percoll gradient centrifugation and incubated in Teflon culture vessels. Using this culture method, we routinely recovered 80% of the cells originally placed into culture. Studies of the C3b and C3b' receptors of these monocytes showed that the function of both receptors could be dramatically altered by treating the cells with tumor-promoting phorbol esters. Both C3b and C3b' receptors of human monocytes efficiently mediate attachment of erythrocytes coated with the corresponding ligands, but do not promote their ingestion. However, monocytes treated with phorbol myristate acetate (PMA) or phorbol didecanoate ingest C3b- and C3b'-coated erythrocytes. Phorbol esters that are inactive as tumor promoters do not stimulate C3 receptor-mediated phagocytosis. The ability of monocytes to respond to PMA by activation of C3 receptors is developmentally regulated. Freshly isolated monocytes do not take up C3b- or C3b'-coated erythrocytes in response to PMA, but after 3 d of culture they show strong PMA-stimulated uptake. The stimulatory effect of PMA on monocyte C3b and C3b' receptor function occurs within minutes, is stable for hours, is cycloheximide insensitive, and can be inhibited with colchicine. Several lines of evidence indicates that phagocytosis of C3b or C3b'-coated erythrocytes is specifically mediated by the monocytes' C3b and C3b' receptors. First, erythrocytes attached to monocytes with concanavalin A are not ingested when the monocytes are treated with PMA. Second, monocytes plated on IgG-bearing substrates lose Fc receptor activity on their nonadherent surfaces but retain the capacity to ingest C3b- or C3b'-coated erythrocytes after PMA treatment. Third, PMA-treated monocytes plated on C3b-coated surfaces lose C3b receptor activity on their nonadherent surfaces but retain the capacity to ingest C3b'-coated erythrocytes. Conversely, PMA-treated monocytes plated on C3b'-coated surfaces show reduced C3b' receptors activity on their nonadherent surfaces but retain the capacity to ingest C3b-coated erythrocytes.