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Screening for specific inhibitors of phagocytosis of thioglycollate-elicited macrophages.

Phagocytosis is one of the basic and characteristic properties of macrophages. We screened metabolites of Actinomyces for low molecular weight substances that selectively inhibited phagocytosis of dried yeast but not pinocytosis of neutral red by thioglycollate-elicited peritoneal macrophages. Inhibitors of actin filament organization, protein kinases, respiration, and lipid synthesis selectively inhibited phagocytosis, and blockers of proton gradients selectively inhibited pinocytosis. This suggests that these functions are differently regulated. We applied this system to screening of metabolites of Actinomyces, and identified mycotrienin, piericidin, and genistein as selective inhibitors of phagocytosis.

Actinomyces↗

Intestinal barriers to water-soluble macromolecules.

Neonates of some species of mammals absorb water-soluble macromolecules from the lumen of the gut to the circulation. This is a means for providing the neonate with passive immunological protection. The accepted model for absorption of macromolecules, particularly immunoglobulin G (IgG), has at least three phases: adherence of the macromolecule to the brush border on enterocytes; internalization of the macromolecule within the enterocytes; and egress of the macromolecule into the lamina propria. With regard to the absorption of IgG, adherence is thought to be a specific reaction of ligand (IgG) with a plasmalemma binding site. Pinocytosis is activated and internalization follows. Egress into the lamina propria occurs at the basal-lateral membrane by a process of reverse pinocytosis. Unbound (unprotected) macromolecules that are internalized in the pinocytosic fluid are shunted off to lysosomes and either digested or stored therein. Neonatal rodents fit this model for macromolecular absorption. However, in another group of neonates (e.g., pig, cow, horse), nonselected absorption takes place, in that IgG and other macromolecules are transported from the gut lumen to the blood. In a third group of neonates, (e.g., human, guinea pig) absorption of IgG is either of low order or nonexistent. Since neonatal mammals possess a mechanism for absorbing macromolecules, there is the potential for internalizing toxic macromolecules if the toxin is presented to the neonate's enterocytes in competitive amounts. Adults retain remnants of the neonatal absorptive mechanism.

Adult↗

Formation of reticular fibers in the developing spleen of the chick embryo.

An electron microscope study was made to elucidate the manner in which reticular fibers form in the developing spleen of the chick embryo. On the 7th day of incubation, the splenic reticulum consisted of dendritic reticular cells without reticular fibers. From the 9th day onward, the intercellular gaps between processes of adjacent reticular cells were expanded, becoming occupied by fine flocculent materials. With the growth of the embryos, these materials were aggregated into felt-like bands, exhibiting the characteristics of mature reticular fibers. In the interstices of the reticulum, there appeared microfibrils of 8-30 nm in diameter. The reticular cells possessed numerous free ribosomes and polysomes, well-developed Golgi apparatuses, dilated rough-surfaced endoplasmic reticula, and many coated vesicles. The coated vesicles contained fine flocculent materials as presumable fiber precursors. The vesicles were enlarged as they approached the cell periphery, where they appeared to discharge their contents by reverse pinocytosis. some vesicles close to the cell membrane contained ruthenium red-positive materials which were considered to be glycosaminoglycan in nature and to compose the matrix of fibers. These materials also seemed to be secreted by reverse pinocytosis, being densely accumulated in the intercellular gaps. The differentiation of fibrous proteins into felt-like reticular fibers or striated microfibrils seems, therefore, to be intimately associated with the amount of glycosaminoglycan.

Animals↗

Targeting cells with MR imaging probes based on paramagnetic Gd(III) chelates.

The low sensitivity is the major disadvantage of MRI as compared to PET. Therefore, amplification strategies are necessary for specific pathway labeling. This survey is aimed at exploring different routes to the entrapment of Gd(III) chelates in various type of cells at amounts sufficiently large to allow MRI visualization. Namely, the obtained results have been summarized in terms of internalization via i) pinocytosis; ii) phagocytosis; iii) receptors; iv) receptor mediated endocytosis; v) transporters; vi) transmembrane carrier peptides. MRI visualization of cells appears possible when the number of internalized Gd(III) chelates is of the order of 10(7)-10(8)/cell. Pinocytosis shows to be particularly useful for labeling cells that can be incubated for several hours in the presence of high concentrations of Gd-agent. This approach appears very effective for labeling stem cells. Nanoparticles filled with Gd-chelates can be used for an efficient loading of cells endowed with a good phagocytic activity. Entrapment via receptors most often results in receptor mediated endocytosis. Suitably functionalized monomeric and multimeric Gd-chelates can be considered for being internalized by this route as well as supramolecular systems such as those formed between Avidin and biotinylated Gd-complexes. Exploitation of up-regulated transporters of nutrients in tumor cells appears to be a promising route for their differentiation from healthy cells. Finally, properly designed systems entering the cells by means of penetrin-like peptides deserve great attention.

Animals↗

Pulmonary surfactant protein A up-regulates activity of the mannose receptor, a pattern recognition receptor expressed on human macrophages.

Inhaled particulates and microbes are continually cleared by a complex array of lung innate immune determinants, including alveolar macrophages (AMs). AMs are unique cells with an enhanced capacity for phagocytosis that is due, in part, to increased activity of the macrophage mannose receptor (MR), a pattern recognition receptor for various microorganisms. The local factors that "shape" AM function are not well understood. Surfactant protein A (SP-A), a major component of lung surfactant, participates in the innate immune response and can enhance phagocytosis. Here we show that SP-A selectively enhances MR expression on human monocyte-derived macrophages, a process involving both the attached sugars and collagen-like domain of SP-A. The newly expressed MR is functional. Monocyte-derived macrophages on an SP-A substrate demonstrated enhanced pinocytosis of mannose BSA and phagocytosis of Mycobacterium tuberculosis lipoarabinomannan-coated microspheres. The newly expressed MR likely came from intracellular pools because: 1) up-regulation of the MR by SP-A occurred by 1 h, 2) new protein synthesis was not necessary for MR up-regulation, and 3) pinocytosis of mannose BSA via MR recycling was increased. AMs from SP-A(-/-) mice have reduced MR expression relative to SP-A(+/+). SP-A up-regulation of MR activity provides a mechanism for enhanced phagocytosis of microbes by AMs, thereby enhancing lung host defense against extracellular pathogens or, paradoxically, enhancing the potential for intracellular pathogens to enter their intracellular niche. SP-A contributes to the alternative activation state of the AM in the lung.

Adjuvants, Immunologic↗

Type 1 and type 2 cytokine regulation of macrophage endocytosis: differential activation by IL-4/IL-13 as opposed to IFN-gamma or IL-10.

Cytokine regulation of endocytic activity in primary human macrophages was studied to define ultrastructural changes and mechanisms of pinocytic regulation associated with cytokines secreted by activated T cells. The effects of IFN-gamma (type 1) and IL-4/IL-13 and IL-10 (type 2) cytokines on fluid phase and mannose receptor-mediated endocytosis were assessed by horseradish peroxidase and colloidal gold-BSA uptake and computer-assisted morphometric analysis. IL-4 and IL-13 enhanced fluid phase pinocytosis and mannose receptor-mediated uptake by activation of phosphatidylinositol 3-kinase. Inhibition of actin assembly showed that both cytokines exerted actin-dependent and -independent effects. Ultrastructurally, IL-4 and IL-13 increased tubular vesicle formation underneath the plasma membrane and at pericentriolar sites, concurrent with decreased particle sorting to lysosomes. By contrast, IL-10 or IFN-gamma decreased both fluid phase pinocytosis and mannose receptor-mediated uptake. IFN-gamma stimulated increased particle sorting to perinuclear lysosomes, while IL-10 decreased this activity. In summary, our data document differential effects on macrophage endocytic functions by type 1 or type 2 cytokines associated with induction and effector pathways in immunity.

Actins↗

[Participation of alveolar macrophages in the catabolism of fibrinogen and fibrin].

The penetration of FITC-fibrinogen in rabbit alveolar macrophages depends on Ca2+ and Mg2+. Therefore there is reason to believe the process is not restricted only by diffusion on concentration gradient, but pinocytosis takes place, which depends Ca2+. The absorption of FITC-label from fibrin by alveolar macrophages is more slow than from fibrinogen. Soluble high-molecular products of fibrin degradation can be trapped by pinocytosis. On the basis of such studies it was assumed that degradation of fibrin proceeds parietally.

Animals↗

Similarities in function between pancreatic tumor cells and macrophages and their inhibition by murine monoclonal antibodies.

Fine structural studies of human pancreatic adenocarcinomas have demonstrated their composition of one major secretory cell type, a mucin secreting cell. It was also demonstrated that pancreatic tumor cells have a high endocytotic activity via the regular endocytotic pathway with uptake via coated pits and vesicles. These findings indicate a striking similarity between pancreatic tumor cells and human mononuclear phagocytes. Therefore monocyte-related functions like endocytosis, lysosomal enzyme secretion and superoxide anion production were measured in various cell lines of human pancreatic adenocarcinomas in comparison to human mononuclear phagocytes. These functions were measured under basal conditions and after exposure to zymosan or immune complexes. All pancreatic tumor cells tested demonstrated increased endocytotic, secretory and oxidative burst activity to a similar extent as human mononuclear phagocytes after exposure to the stimuli (zymosan or immune complexes). In a further attempt murine monoclonal antibodies produced against pancreatic tumor cells were characterized in relation to their tissue specificity, the epitope recognized in in vitro and in vivo radiolocalization and their potential to mediate biological functions (e.g. antibody dependent cellular cytotoxicity or complement mediated cytolysis). Mouse monoclonal IgG1 antibodies which specifically bind to the majority of well differentiated adenocarcinomas of the human pancreas as well as to human mononuclear phagocytes have been shown to inhibit several cellular functions (lysosomal enzyme release, pinocytosis of colloidal gold 198Au, and chemiluminescence) of those cells. Incubation of 10 micrograms/ml of monoclonal IgG1 antibody with pancreatic tumor cell lines or human monocytes either simultaneous or followed by the stimulation with zymosan (50 micrograms/ml) or immune complexes (100 micrograms/ml) resulted in the inhibition of lysosomal enzyme release and uptake of colloidal radioactive gold of both human mononuclear phagocytes and pancreatic tumor cells responding to zymosan or immune complexes. In addition those monoclonal antibodies have been found to inhibit luminol-dependent chemiluminescence. Kinetic measurements of chemiluminescence showed a quick inhibition of this activity on both cell types within 1 to 3 minutes. Antibody F(ab')2 fragments behaved analogously to the whole antibodies, whereas monovalent Fab fragments were not able to inhibit any of the mentioned functions. When used alone the monoclonal antibodies were not cytotoxic and did not theirselves trigger release of acid hydrolases, did not alter pinocytosis and did not induce oxidative metabolism.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenocarcinoma↗

Metabolic and functional alterations in macrophages induced by essential fatty acid deficiency.

Essential fatty acid (EFA) deficiency has been shown to protect against the glomerulonephritis in a murine model of systemic lupus erythematosus. Since macrophages are an important cellular constituent of the inflammatory lesion, the effects of EFA deficiency on the eicosanoid metabolism and function of these cells were determined. EFA-deficient macrophages exhibited a depletion of phospholipid arachidonate and an accumulation of 20:3(n-9); phosphatidylinositol was the phospholipid most affected. When these macrophages were stimulated with unopsonized zymosan, they produced markedly less leukotriene C4 and B4 than control macrophages. EFA-deficient macrophages also synthesized leukotriene C3 from endogenous 20:3(n-9). No leukotriene B3 was detected. In contrast to the effects on leukotriene production, prostaglandin and thromboxane production were only minimally affected by EFA deficiency. When challenged with zymosan, EFA-deficient macrophages released less arachidonate relative to control macrophages and released half again as much 20:3(n-9) as arachidonate. Release of arachidonate from phosphatidylcholine in the EFA-deficient cells was highly selective for arachidonate; however, release of arachidonate from phosphatidylinositol was depressed relative to control and was not selective. Incubation of macrophages with exogenous arachidonate and 20:3(n-9) established that 20:3(n-9) decreased leukotriene C4 and B4 synthesis from arachidonate but did not affect prostaglandin production. To determine the functional effects of the deficiency state, receptor-mediated pinocytosis and phagocytosis were also examined in EFA-deficient cells. EFA-deficient macrophages exhibited a marked reduction in receptor-mediated pinocytosis. Phagocytosis, however, was unaffected by the deficiency state. These effects on macrophage eicosanoid metabolism and function may comprise a significant component of the anti-inflammatory effect of EFA deficiency.

Animals↗

Macrophage-activating factors from different T cell clones induce distinct macrophage functions.

The data reported in this paper are the first demonstration that different T cell clones (PC-AKR-CI 96, clone 96; PK 7.1.2 E8, clone 7.1.2 E8) secrete different macrophage-activating factors (MAF) that induce distinct macrophage activities. Incubation of resident murine macrophages with MAF 7.1.2 E8 increased RNA, protein, and glycoprotein synthesis, hexosemonophosphate shunt (HMPS) activity, release of oxygen metabolites (O-2, H2O2), pinocytosis, phagocytosis, and tumor cytostasis, whereas no effect on prostaglandin E (PGE) release, schistosomula killing, and tumor cytolysis could be observed. In contrast, MAF 96 increased glycoprotein synthesis, HMPS activity, release of oxygen metabolites and PGE, schistosomula killing, and tumor cytostasis and cytolysis, whereas RNA and protein synthesis and pinocytosis were decreased and phagocytosis remained unaffected. Thus, MAF from both T cell clones share some macrophage-activating properties but differ in others. Most importantly, both MAF could be differentiated serologically by a rabbit anti-lymphokine antiserum that selectively inhibited MAF 96 but not MAF 7.1.2 E8 activity. At optimal concentrations, MAF 96 and 7.1.2 E8 were active in the absence of lipopolysaccharide (LPS) whereas LPS enhanced the activity of suboptimal doses of MAF 96 but not of MAF 7.1.2 E8. These data are discussed with respect to the possibility that the functional dichotomy of T cell clones might reflect different activities of normal T cell subpopulations.

Animals↗

Peritoneal mesothelium and ovarian surface cells--shared characteristics.

This is a transmission electron microscopy study of peritoneal mesothelium and surface cells covering the ovary. One thousand two hundred cells derived from 22 samples of peritoneum and 1,200 derived from 22 samples of ovary were compared for cell size, length-to-diameter ratio, and distribution and complexity of surface villi. Cilia were looked for. Other factors studied included mitochondria, pinocytosis, secretory vacuoles, tonofilaments, desmosome-tonofilament complexes, lysosomes, and mucin. In general, peritoneal mesothelial cells tend to be flat, and surface cells of the ovary, cuboidal. The villous surfaces are essentially similar. Two ciliated cells were found in 1,200 surface cells of the ovary; one blunted cilium was found in 1,200 peritoneal cells. Mitochondria, pinocytosis, secretory vacuoles, and lysosomes were essentially similar in both cell types. Desmosome-tonofilament complexes were rare in surface cells and not found in mesothelial cells. Tonofilaments were present in 340 of 1,200 peritoneal mesothelial cells (28.30%). They were present in only 16 of 1,200 surface cells of the ovary (1.33%). It appeared that peritoneal mesothelium and surface cells of the ovary are, in most respects, structurally similar.

Epithelium↗

Endocytosis, membrane recycling and Fc receptor function.

We have studied the composition and fate of plasma membrane internalized during both fluid-phase and receptor-mediated endocytosis in mouse macrophages. Particular attention has been paid to the macrophage Fc receptor, an intrinsic membrane glycoprotein that we have isolated and characterized biochemically and immunologically. Monoclonal and polyclonal antibodies directed against the receptor and against a series of other unrelated plasma membrane proteins have been used. In addition, we have used radioiodination techniques to label selectively the polypeptides of pinocytic vesicle membrane from within intact cells. Our results indicate that fluid pinocytosis in macrophages involves the internalization of a largely representative sample of plasma membrane polypeptides. Significantly, the Fc receptor seems to be internalized at a rate similar to that of most other membrane proteins. However, selective internalization of the receptor is induced during the endocytosis of certain ligands. The phagocytosis of immunoglobulin G (IgG)-coated erythrocyte ghosts results in the selective and largely irreversible removal of Fc receptors from the macrophage surface. Selectively internalized receptors do not recycle but are rapidly degraded. Fc receptors also appear to be preferentially interiorized during the rapid pinocytosis of IgG-containing soluble immune complexes. Uptake is accompanied by a sharp decrease in the number of surface receptors, which is partially reversed after the removal of ligand.

Animals↗

The effect of growth-promoting alpha-globulin (GPAG) on the incorporation of exogenous DNA into L-cells. I. Incorporation of isologous and heterologous 3H-DNA.

Isologous and heterologous 3H-DNA (optimum concentration 25 microgram/ml of medium) are incorporated into L-cells from the medium during short-term incubation (up to 60 min). The incorporation of DNA is stimulated by a protein complex from calf serum--the growth-promoting alpha-globulin (GPAG) in the concentration 0.8 mg/ml of medium), which is rapidly taken into cells by pinocytosis. GPAG increases the frequency of incorporation of exogenous 3H-DNA into L-cells and the quantity of DNA incorporated. The quantity of 3H-DNA incorporated is further increased on the incubation of L-cells in a medium containing the complex 3H-DNA + GPAG, which is formed by the joint preincubation of the two components at 37 degrees C (20 hr); under these conditions the quantity of DNA incorporated is 150% greater than when 3H-DNA is used alone. GPAG acts as an activator of exogenous DNA transfer, stimulates its pinocytosis by the cells, and at the same time accelerates its intracellular transfer to the cell nuclei.

Alpha-Globulins↗

NTBN, a free radical spin trap induces programmed cell death in human pancreatic cancer (PANC-1) cells.

N-tertiary butyl-a-phenylnitrone, a free radical spin trap at > or = 10 mM concentration, inhibited proliferation and reduced the viability of human pancreatic cancer (Panc-1) cells. The drug concentration determined the extent of inhibition, and with continued culture a proportion of the cells detached, most of which stained with trypan blue. Although hypodiploid cells were detected by flow cytometry of cells cultured with 20 mM NTBN, DNA laddering was absent and the TUNEL reaction negative. "Dark" cells present in samples cultured with 10 mM NTBN exhibited decreased cytoplasmic volume and increased staining with methylene blue and azure II, but lacked characteristic nuclear changes of type 1 programmed cell death. Cells cultured with > 10 mM of the spin trap exhibited nuclear and cytoplasmic changes more consistent with a non-type 1, type 2 variant of PCD with extensive cytoplasmic vacuolization. Careful analysis revealed evidence of marked pinocytosis in some cells. In view of the spin-trap associated pinocytosis, augmented uptake of chemotherapy in affected cells might be anticipated, but additive, synergistic or antagonistic interactions between NTBN and 5-fluorouracil were not observed.

Antimetabolites, Antineoplastic↗

AGE VARIATIONS IN CORTICAL MEMBRANES OF ROTIFERS.

Electron micrographs were made of rotifers of known ages embedded in Vestopal and stained with lead hydroxide. In epithelial cells lining the gut of the rotifer, anatomical continuity is seen between the cell membrane, the cortical membranes, and the rough endoplasmic reticulum at the basal region of the cells. At their luminal surface, these cells possess a terminal web, and colloidal gold is taken up via food vacuoles at this surface. Such pinocytosis does not occur on the peripheral or distal surface (pseudocoelomic) of these cells. Exposure of rotifers to adenine inhibits the formation of cortical infoldings of the cell membrane and the formation of the rough reticulum, whereas the ribosomes and the Golgi apparatus appear to be unaffected. The suggestion is made that the rough reticulum may be derived from modifications of the cell membrane and that the rate of formation of the rough reticulum in adult and elderly rotifers is lower than in the actively growing animal.

Aging↗

Membrane ruffling, macropinocytosis and antigen presentation in the absence of gelsolin in murine dendritic cells.

Previous studies have shown that mice lacking the actin-severing and capping protein gelsolin have defects in leukocyte and platelet function. Moreover, dermal fibroblasts from gelsolin knockout (Gsn(-)) mice showed substantially reduced motility, membrane ruffling and pinocytosis. We have generated dendritic cells (DC) from spleens of Gsn(-) mice to investigate the importance of gelsolin in antigen endocytosis and processing. We show here that Gsn(-) DC produce apparently normal membrane ruffles which can resolve to form large macropinosomes. Moreover, presentation of exogenous antigens on both MHC class II and class I molecules was equivalent in Gsn(-) and wild-type DC. Thus the major rearrangements of the actin cytoskeleton needed for DC antigen uptake and presentation can proceed in the absence of a major actin filament regulatory protein.

Amino Acid Sequence↗

The mechanism of entry of viruses into plant protoplasts.

Plant protoplasts may be efficiently infected with viruses. Attachment to the plasmalemma is a critical step in infection; positively charged viruses readily adhere and infect but negatively charged viruses require the presence of a polycation (e.g. poly-L-ornithine) before infection can occur. The role of the polycation appears to be twofold: to modify the charge of the virus and to damage the surface of the protoplast. Virus probably enters the protoplast during repair of damaged regions of the plasmalemma. Pinocytosis does not appear to play a significant part in inoculation.

Adhesiveness↗

Recent advances in understanding brain capillary function.

The endothelial cells in brain capillaries form a blood-brain barrier which limits and controls the movement of solutes between blood and brain. These cells contain continuous tight junctions and exhibit a low rate of pinocytosis, resulting in formation of a permeability barrier to macromolecules and many polar compounds. However, brain capillary endothelial cells also contain specialized transport systems that facilitate blood-to-brain transfer of some solutes and actively pump other solutes from brian to blood. Several investigators have developed methods to isolate microvessels from brain or to grow brain capillary endothelial cells in tissue culture. This review summarizes progress made with these model systems and discusses their usefulness in increasing our knowledge of brain capillary function.

Amino Acids↗