Internalization of monomeric lipopolysaccharide occurs after transfer out of cell surface CD14.

Lipopolysaccharide (LPS) fluorescently labeled with boron dipyrromethane (BODIPY) first binds to the plasma membrane of CD14-expressing cells and is subsequently internalized. Intracellular LPS appears in small vesicles near the cell surface and later in larger, punctate structures identified as the Golgi apparatus. To determine if membrane (m)CD14 directs the movement of LPS to the Golgi apparatus, an mCD14 chimera containing enhanced green fluorescent protein (mCD14-EGFP) was used to follow trafficking of mCD14 and BODIPY-LPS in stable transfectants. The chimera was expressed strongly on the cell surface and also in a Golgi complex-like structure. mCD14-EGFP was functional in mediating binding of and responses to LPS. BODIPY-LPS presented to the transfectants as complexes with soluble CD14 first colocalized with mCD14-EGFP on the cell surface. However, within 5-10 min, the BODIPY-LPS distributed to intracellular vesicles that did not contain mCD14-EGFP, indicating that mCD14 did not accompany LPS during endocytic movement. These results suggest that monomeric LPS is transferred out of mCD14 at the plasma membrane and traffics within the cell independently of mCD14. In contrast, aggregates of LPS were internalized in association with mCD14, suggesting that LPS clearance occurs via a pathway distinct from that which leads to signaling via monomeric LPS.

Interferon-gamma depresses binding of ligand by C3b and C3bi receptors on cultured human monocytes, an effect reversed by fibronectin.

Cultivation of human monocytes with recombinant IFN-gamma causes a 5-10-fold depression in their binding of EC3b or EC3bi. This effect is observed within 18 h and is expressed for 5 d in the presence of 100 U/ml IFN-gamma. The capacity of IFN-gamma-treated phagocytes to bind EC3b and EC3bi is fully restored if the phagocytes are allowed to spread for 45 min on surfaces coated with Fn. IFN-gamma-treated cells express normal levels of cell surface C3b and C3bi receptors as measured with monoclonal anti-receptor antibodies, and spreading on Fn does not alter receptor number. We conclude that cultivation with IFN-gamma causes a change in the nature of these receptors that prevents them from interacting with ligand. Immunoelectron microscopy shows that C3bi receptors are expressed on the apical surface of the IFN-gamma-treated MO and that these receptors exhibit normal capacity to migrate in the plane of the membrane. Thus, the nature of the change caused by IFN-gamma is not related to changes in receptor number, location, or mobility. While spreading of IFN-gamma-treated cells on Fn enables C3 receptors to bind ligand, it does not enable them to promote phagocytosis. Treatment of cells with PMA alone does not affect binding or phagocytosis, but treatment of cells with both Fn and PMA enables cells to phagocytose EC3b and EC3bi. These data indicate that the binding and signaling activities of C3 receptors are separately regulated. Fn enables receptors to bind ligand and PMA enables them to signal phagocytosis.

Transient adhesion of neutrophils to endothelium.

Fluorescently labeled polymorphonuclear leukocytes (PMN) were used to measure adhesion to human umbilical vein endothelial cells (EC) cultured in vitro. Stimulation of PMN with phorbol dibutyrate (PDB), TNF, or C5a caused an increase in adhesion followed by a return to prestimulation levels of adhesion of longer times of incubation. Maximal adhesion of PMN to EC occurred rapidly in response to C5a (5 min) and more slowly with TNF or PDB (15 min). PMN stimulated to adhere with C5a detached from EC by 15 min. PMN from CD11/CD18-deficient patients and PMN incubated with anti-CD18 mAbs failed to bind to EC despite maximal stimulation. Anti-CD11a/CD18 and anti-CD11b/CD18 each partially inhibited adhesion, and a combination of these two reagents completely blocked adhesion. The adhesion we measured was therefore completely dependent on CD11/CD18, and CD11a/CD18 and CD11b/CD18 each contributed to adhesion. Stimuli that enhanced adhesion of PMN to EC also enhanced expression of CD11b/CD18 on the cell surface, but the time course of expression correlated poorly with changes in adhesivity. To determine if changes in the expression of CD11b/CD18 are necessary for the changes in adhesivity, we used enucleate cytoplasts that did not increase expression of CD11b/CD18. Cytoplasts showed a normal rise and fall in adhesivity in response to PDB. We conclude that the transient adhesion of stimulated PMN to naive EC is regulated by changes in the nature of existing CD11/CD18 molecules on the PMN surface. Changes in expression of CD11b/CD18 may contribute to enhancement of adhesivity, but a definite role for this phenomenon has yet to be established.

Activation of the adhesive capacity of CR3 on neutrophils by endotoxin: dependence on lipopolysaccharide binding protein and CD14.

Tumor necrosis factor alpha, granulocyte colony-stimulating factor, granulocyte/macrophage colony-stimulating factor, and formyl peptide were each found to cause a twofold increase in expression of CD14 on the surface of polymorphonuclear leukocytes (PMN). Upregulation of CD14 was complete by 20 min and thus appeared to result from expression of preformed stores of protein. The CD14 on the surface of PMN was shown to serve two biological functions. It bound particles coated with complexes of lipopolysaccharide (LPS) and LPS binding protein (LBP). This binding activity was enhanced by agonists that upregulated CD14 expression and may serve in the clearance of Gram-negative bacteria opsonized with LBP. Interaction of CD14 with LPS in the presence of LBP or serum also caused a dramatic, transient increase in the adhesive activity of CR3 (CD11b/CD18) on PMN. Enhanced activity of CR3 and other members of the CD11/CD18 family underlies many of the known physiological responses of PMN to LPS and may be a central feature of the in vivo responses of PMN to endotoxin.

Neutrophil-activating protein 1/interleukin 8 stimulates the binding activity of the leukocyte adhesion receptor CD11b/CD18 on human neutrophils.

The cytokine NAP-1/IL-8 is produced by a variety of different cells in response to inflammatory stimuli and elicits several biological responses from PMN. Experiments presented here demonstrate that PMN exposed to NAP-1/IL-8 expressed increased amounts of CD11b/CD18, as well as CD11c/CD18 and CR1, on their cell surface, while expression of Fc gamma RIII and HLA-A,B,C remained essentially unchanged. Increased CD11b/CD18 and CD11c/CD18 appears to correspond with the release of specific granules by NAP-1/IL-8. NAP-1/IL-8 was also a potent stimulator of several of the binding activities of CD11b/CD18. Ligation of EC3bi by CD11b/CD18 was rapidly enhanced by NAP-1/IL-8, but phagocytosis of the ligated particles was not induced by the agonist. In addition, enhanced binding of EC3bi was observed in the absence of an increase in receptor expression as shown with PMN cytoplasts. NAP-1/IL-8 promoted additional adhesive interactions between CD11b/CD18 and the biosynthetic precursor of LPS, lipid IVa, fibrinogen, and endothelial cells, suggesting that NAP-1/IL-8 may promote leukocyte adhesion in vivo that could lead to recruitment of PMN to sites of tissue inflammation.

Infectious agents are not necessary for murine atherogenesis.

Recent work has revealed correlations between bacterial or viral infections and atherosclerotic disease. One particular bacterium, Chlamydia pneumoniae, has been observed at high frequency in human atherosclerotic lesions, prompting the hypothesis that infectious agents may be necessary for the initiation or progression of atherosclerosis. To determine if responses to gram-negative bacteria are necessary for atherogenesis, we first bred atherosclerosis-prone apolipoprotein (apo) E(-/)- (deficient) mice with animals incapable of responding to bacterial lipopolysaccharide. Atherogenesis was unaffected in doubly deficient animals. We further tested the role of infectious agents by creating a colony of germ-free apo E(-/)- mice. These animals are free of all microbial agents (bacterial, viral, and fungal). Atherosclerosis in germ-free animals was not measurably different from that in animals raised with ambient levels of microbial challenge. These studies show that infection is not necessary for murine atherosclerosis and that, unlike peptic ulcer, Koch's postulates cannot be fulfilled for any infectious agent in atherosclerosis.

Elongation of the fertilization tubule in Chlamydomonas: new observations on the core microfilaments and the effect of transient intracellular signals on their structural integrity.

Experimental manipulations of gametes of Chlamydomonas reinhardi and ultrastructural observation were used to examine the composition of the microfilaments in the fertilization tubule, their probable mode of formation, and their interaction with intracellular signals. Decoration with myosin subfragment-1 was used to demonstrate that the microfilaments in the fertilization tubule were actin filaments having uniform polarity: Myosin subfragment-1 arrowheads pointed away from the membrane at the tip of the process. Filaments were attached to the cone-shaped "doublet zone" at the base of the process by their pointed ends. Discrete attachment sites for filaments on the surface of the doublet zone were seen in stereo view. To test whether actin polymerization might accompany elongation of the fertilization tubule, mating gametes were exposed to cytochalasin D in an attempt to block actin polymerization. Treatment of mating type "plus" gametes with cytochalasin D prior to and during mating inhibited the appearance of actin filaments in fertilization tubules, suppressed fertilization tubule outgrowth, and lowered mating efficiency from 90 to 15%. The role of signals generated by flagellar adhesion in maintaining the structural integrity of the microfilament-doublet zone complex was examined by correlating flagellar disadhesion with the kinetics of breakdown of the complex. In zygotes, where flagellar disadhesion occurred after cell fusion, the complex disassembled within 3 h after mating. In gametes that had been agglutinated by isolated mating type "minus" flagella, microfilaments and fertilization tubules progressively disassembled over a 3-h time course following flagellar disadhesion. Disassembly of microfilaments was inhibited by maintaining flagellar agglutination, suggesting that signals generated by flagellar adhesion were necessary to maintain microfilaments intact.

Activation for cell fusion in Chlamydomonas: analysis of wild-type gametes and nonfusing mutants.

Gametes of Chlamydomonas reinhardi become activated for cell fusion as the consequence of sexual adhesion between membranes of mating-type plus and minus flagella. By using tannic acid plus en bloc uranyl acetate staining, and by fixing at very early stages in the mating reaction, we have demonstrated the following. (a) Activation of the minus mating structure entails major modifications in the structure of the organelle, causing it to double in size and to concentrate surface coat material, termed fringe, into a central zone. (b) The unactivated plus mating structure is endowed with fringe that moves with the tip of the actin-filled fertilization tubule during activation. Pre-fusion images suggest the occurrence of a specific recognition event between the plus and minus fringes. (c) Gametes carrying the imp-1 mutation fail to form a fringe and are unable to fuse. The imp-1 mutation is linked to the mating-type plus (mt+) locus, suggesting that the gene specifying the synthesis or insertion of fringe is encoded in this sector of the genome. (d) Gametes carrying the imp-11 mutation fail to form both a normal fringe and a normal submembranous density beneath the fringe, and are also unable to fuse. The imp-11 mutation converted a wild-type minus cell into a pseudo-plus strain; a model to explain this conversion proposes that the normal imp-11 gene product represses plus-specific genes concerned with Chlamydomonas gametogenesis.

Aggregation of complement receptors on human neutrophils in the absence of ligand.

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.

Multiplexed assays by high-content imaging for assessment of GPCR activity.

G-protein-coupled receptors (GPCR) participate in many disease pathways and represent the largest family of therapeutic targets. Thus, great investments are made to discover drugs modulating GPCR-mediated events. Among functional assays for screening GPCRs, the Transfluor imaging assay is based on redistribution of cytosolic beta-arrestin to an activated GPCR and has become widely used in high-content screening. However, assessing Transfluor alone has limitations: relying on a single mechanistic step of beta-arrestin redistribution during GPCR activation, providing no information on the stimulated GPCR's intracellular fate, and using only a single fluorescent color (green fluorescent protein). Taking full advantage of high-content imaging to screen approximately 2000 compounds, the authors multiplexed the Transfluor assay with an immunofluorescence-based quantification of GPCR internalization. This approach identified and classified 377 compounds interfering with agonist-induced activation of the Transfluor assay, receptor internalization, or both. In addition, a subset of compounds was analyzed for their performance across imaging, cell-based calcium release (fluorometric imaging plate reader [FLIPR]), and biochemical receptor binding assays (scintillation proximity assay). This indicated that the imaging assays have even better predictive power for direct inhibition of receptor binding than the FLIPR assay. In conclusion, compounds inducing unique responses can suggest novel mechanisms of action and be used as tools to study GPCR activation and internalization.

Azurocidin and a homologous serine protease from neutrophils. Differential antimicrobial and proteolytic properties.

Two 29-kD polypeptides, azurocidin and p29b, were purified to homogeneity from human neutrophils by acid extraction of azurophil granule membrane-associated material followed by gel filtration and reverse-phase chromatography. Azurocidin and p29b share NH2-terminal sequence homology with each other as well as with elastase, cathepsin G, and other serine proteases. p29b bound [3H]diisopropyl fluorophosphate and hydrolyzed elastin, casein, and hemoglobin. A peptide substrate for p29b could not be identified. Azurocidin neither bound [3H]diisopropyl fluorophosphate nor hydrolyzed any of the proteins, peptides, or esters tested. In microbicidal assays, purified azurocidin was comparable to p29b in activity against Escherichia coli, Streptococcus faecalis, and Candida albicans. The antimicrobial activity of azurocidin was enhanced under mildly acidic conditions, but was inhibited in a dose-dependent manner by NaCl, CaCl2, or serum. Immunoblot analysis with monospecific antibodies localized greater than 90% of the azurocidin and greater than 75% of the p29b to azurophil granule-rich fractions of PMN lysates. Immunoelectron microscopy confirmed the localization of azurocidin to the azurophil granules. Azurocidin associated with the azurophil granule membrane, but did not appear to be an integral membrane protein. Thus, azurocidin and p29b are members of a family of serine protease homologs stored in azurophil granules and may play a role in inflammatory and antimicrobial processes involving PMN.

Intestinal absorption of cholesterol is mediated by a saturable, inhibitable transporter.

Although the mechanism by which dietary cholesterol is absorbed from the intestine is poorly understood, it is generally accepted that cholesterol is absorbed from bile acid micelles in the jejunum. Once inside the enterocytes, cholesterol is esterified by the action of acyl-coenzyme A:cholesterol acyltransferase (ACAT), assembled into chylomicrons, and secreted into the lymph. In this work, mechanistic aspects of cholesterol absorption were probed using compounds that block cholesterol absorption in hamsters. Sterol glycoside cholesterol absorption inhibitors, exemplified by L-166,143, (3 beta, 5 alpha,25R)-3-[(4", 6"-bis[2-fluoro-phenylcarbamoyl]-B-D-cellobiosyl)oxy]-spirostan -11-on e, potently blocked absorption of radioactive cholesterol, and the potencies of several analogs correlated with their ability to lower plasma cholesterol. Each molecule of L-166,143 blocked the uptake of 500 molecules of cholesterol, rendering it unlikely that the inhibitor interacts directly with the cholesterol or bile acid. Radiolabeled L-166,143 bound to the mucosa and binding was blocked by active, but not inactive, cholesterol absorption inhibitors. Subtle changes in the structure of sterol glycosides yielded large changes in their ability to block both cholesterol absorption and binding of radiolabeled L-166,143. Large species-to-species variation in potency was also observed. These lines of evidence support the interpretation that dietary cholesterol is absorbed via a specific transporter found in the intestinal mucosa.

A target for cholesterol absorption inhibitors in the enterocyte brush border membrane.

Uptake of cholesterol by the intestinal absorptive epithelium can be selectively blocked by specific small molecules, like the sterol glycoside, L-166,143. Furthermore, (3)H-labeled L-166,143 administered orally to hamsters binds specifically to the intestinal mucosa, suggesting the existence of a cholesterol transporter. Using autoradiography, the binding site of (3)H-L-166,143 in the hamster small intestine was localized to the very apical aspect of the absorptive epithelial cells. Label was competed by non-radioactive L-166,143 and two structurally distinct cholesterol absorption inhibitors, suggesting a common site of action for these compounds. L-166,143 blocked uptake of (3)H-cholesterol into enterocytes in vivo, as demonstrated by autoradiography, suggesting that it inhibits a very early step of cholesterol absorption, incorporation into the brush border membrane. This conclusion was confirmed by studies in which intestinal brush borders were isolated from hamsters dosed with (3)H-cholesterol in the presence or absence of L-166,143. Uptake of (3)H-cholesterol into the membranes was substantially inhibited by the compound. In contrast, an inhibitor of acyl CoA:cholesterol acyltransferase, did not affect uptake of (3)H-cholesterol into the brush border membranes. These results strongly support the existence of a specific transporter that facilitates the movement of cholesterol from bile acid micelles into the brush border membranes of enterocytes.

Simvastatin has anti-inflammatory and antiatherosclerotic activities independent of plasma cholesterol lowering.

Inhibitors of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, such as simvastatin, lower circulating cholesterol levels and prevent myocardial infarction. Several studies have shown an unexpected effect of HMG-CoA reductase inhibitors on inflammation. Here, we confirm that simvastatin is anti-inflammatory by using a classic model of inflammation: carrageenan-induced foot pad edema. Simvastatin administered orally to mice 1 hour before carrageenan injection significantly reduced the extent of edema. Simvastatin was comparable to indomethacin in this model. To determine whether the anti-inflammatory activity of simvastatin might affect atherogenesis, simvastatin was tested in mice deficient in apoE. Mice were dosed daily for 6 weeks with simvastatin (100 mg/kg body wt). Simvastatin did not alter plasma lipids. Atherosclerosis was quantified through the measurement of aortic cholesterol content. Aortas from control mice (n=20) contained 56+/-4 nmol total cholesterol/mg wet wt tissue, 38+/-2 nmol free cholesterol/mg, and 17+/-2 nmol cholesteryl ester/mg. Simvastatin (n=22) significantly (P<0.02) decreased these 3 parameters by 23%, 19%, and 34%, respectively. Histology of the atherosclerotic lesions showed that simvastatin did not dramatically alter lesion morphology. These data support the hypothesis that simvastatin has antiatherosclerotic activity beyond its plasma cholesterol-lowering activity.

Cytoplasts from cytotoxic T lymphocytes are resistant to perforin-mediated lysis.

Cytotoxic T lymphocytes (CTL) contain a potent cytolytic pore-forming protein (PFP, perforin or cytolysin) localized in their cytoplasmic granules. In the presence of calcium, perforin lyses a variety of target cells (TC) non-specifically. CTL, however, are generally resistant to the lytic effect of perforin. In this work, cytoplasts from CTL and susceptible TC were made by centrifuging cells on a Ficoll density gradient in the presence of cytochalasin B. Characterization by electron microscopy and a serine esterase assay established that both CTL and TC cytoplasts were completely devoid of nuclei and CTL cytoplasts contained essentially no granules. CTL cytoplasts are just as resistant to perforin-mediated lysis as the intact CTL, and both TC and their corresponding cytoplasts are very sensitive to lysis. Furthermore, CTL cytoplasts are less effective than TC cytoplasts in inhibiting hemolysis, a property shared by the respective intact cells. These results indicate that soluble granular components do not confer resistance on CTL, and suggest that the protective agent(s) acts by impeding perforin binding to the CTL membrane.

Heterogeneity of granules of murine cytolytic T lymphocytes. Isolation of a homogeneous population of dense granules.

A separation procedure based on Percoll gradient centrifugation is described here that allows enrichment for two main populations of granules, with average densities of 1.05 and 1.1 g/ml. The heavy-density granules, which comprise 5-20% of the total amount of membrane-lytic activity, are morphologically and biochemically more homogeneous than the light-density granules, which are contaminated with other subcellular organelles. A trypsin-like serine esterase activity, used in the past as a granule marker, was mainly found in the free cytosol and in the light-density fractions, while a small portion of this activity was associated with the heavy-density fractions. This enzyme marker is therefore not exclusively associated with granules and should not be used as the only granule marker.

Elongation of cytoplasmic processes during gametic mating: models for actin-based motility.

The acrosomal processes of Thyone and Limulus sperm and the fertilization tubule of mt+ gametes of Chlamydomonas are interesting models for actin-based motility. Each is a long thin process that elongates rapidly and contains a core of actin filaments having uniform polarity: arrowheads formed by myosin subfragments point toward the base of the processes. In Limulus, directed outgrowth of the acrosomal process is achieved by a rearrangement in the packing of superhelically coiled actin filaments that form during spermatogenesis. In contrast, outgrowth of the acrosomal process in Thyone and the fertilization tubule in Chlamydomonas is accompanied by actin polymerization. Both Thyone and Chlamydomonas possess structures, the actomere and the doublet zone, respectively, that serve as microfilament organizing centers, nucleating actin polymerization and defining the polarity of the growing filaments. Alkalinization of the cytoplasm may promote polymerization of actin in Thyone, whereas an apparent rise in the intracellular Ca2+ concentration is associated with the transmission of intracellular signals during mating in Chlamydomonas. Further examination of these three actin-based motile systems should provide new insights into the assembly of the actin cytoskeleton, a process critical for many forms of nonmuscle cellular motility.

Trifluoperazine and W-7 inhibit mating in Chlamydomonas at an early stage of gametic interaction.

Gametic mating by Chlamydomonas reinhardi is inhibited in a dose-dependent and reversible manner by the calmodulin antagonists trifluoperazine (TFP) and W-7, but not by W-5, an analog of W-7 having lower affinity for calmodulin. Quantitation of the sequential steps of mating showed that TFP and W-7 both allow normal levels of flagellar agglutination but prevent all subsequent steps. Gametes agglutinate aberrantly and do not form mating pairs. Further, both of these drugs prevent the translocation of latex beads along the flagellar surface. Our observations suggest that calmodulin may play an integral role in the translocation of flagellar adhesion sites during the tip-locking stage of the Chlamydomonas mating reaction. Flagellar surface motility may be crucial to the transduction of signals during mating and may share regulatory mechanisms with other forms of surface motility.

Adhesion-promoting receptors on phagocytes.

Phagocytes express a family of structurally related receptors, LFA-1, CR3, and p150,95, that mediate adhesion of leukocytes to a variety of cells and surfaces. LFA-1 mediates the binding of killer T cells to targets, CR3 mediates binding of phagocytes to iC3b-coated surfaces and to endothelial cells, and LFA-1, CR3, and p150,95 each mediate the binding of bacterial lipopolysaccharide. Here we review the structure and function of each of these receptors and present evidence that they are related to a larger class of adhesion-promoting receptors called integrins. Of particular emphasis are observations that the capacity of these receptors to promote adhesion is strongly and reversibly modulated by both soluble and surface-bound stimuli. We review this form of regulation and present evidence that changes in the binding activity of adhesion-promoting receptors is accomplished by changes in the two-dimensional distribution of receptors in the plane of the membrane. Inactive receptors are randomly distributed in the membrane, and their ability to bind a ligand-coated surface is enabled by a ligand-independent movement into small clusters. The implications of these structural features are discussed.