Leishmania mexicana: inhibition and stimulation of phagosome-lysosome fusion in infected macrophages

The polyanionic compound poly-d-glutamic acid was found to inhibit significantly the fusion of secondary lysosomes to phagosomes containing Leishmania mexicana mexicana amastigotes for at least 96 hr. This process was viewed both by dark-field vital fluorescence microscopy and transmission electron microscopy. In poly-d-glutamic acid-treated macrophages parasites multiplied at a significantly greater rate than in untreated macrophages. Conversely, the secondary amine chloroquine caused a marked reduction in parasite growth. When L. m. mexicana promastigotes were substituted for amastigotes these results were strikingly more pronounced.     

Effects of protein deficiency on selective elicitation of lysosomal enzymes in rat peritoneal macrophages

Young albino rats were fedad libitum 4, 8 or 20 % (control) protein diet for 1–4 weeks. Total activities of some of the lysosomal enzymes, namely, acid phosphatase, aryl sulphatase, Β-glucuronidase and cathepsin D, were determined in resident and protease-peptone elicited peritoneal macrophages. Total cell number, protein content and the lysosomal enzyme activities were increased significantly in protease-peptone elicited macrophages; though at a lower rate in 4 % protein-fed group compared to control ones. However, the rate of induction of the tested hydrolases was selective and their response to the stimulant varied widely. Similarly, response of each enzyme to low protein diet also varied. Thus, at 4 weeks, cathepsin D and Β-glucuronidase activities, expressed per total number of elicited macrophages were reduced by 45 and 60 %, respectively, in 4 % protein-fed animals. These results indicate that the metabolic events related to lysosomal function in macrophages, are affected by dietary restriction of proteins     

Effects of cationic vectors complexed with plasmid DNA on the phagosome-lysosome fusion in murine peritoneal macrophages and J744 macrophages

Polyethylenimine (PEI) and cationic polypeptides complexed with plasmid DNA are the most efficient nonviral vectors for gene therapy. It is believed that endocytosis is the major pathway for cell entering by PEI/DNA or cationic peptides/DNA complexes. Effects of plasmid DNA complexed with PEI, poly-L-lysine (PLL), poly-D-lysine (PDL) and polyarginine (PA) on the phagosome-lysosome fusion (P-LF) were studied in murine peritoneal macrophages and J774 macrophages. Cationic polypeptide PLL can be hydrolysed by cellular peptidases, but its stereoisomer, PDL, cannot be split by these enzymes. PEI, PDL, and PA have been shown to inhibit P-LF. PLL showed a low effect on the P-LF. On the basis of these studies, we assume that lysosomotropic agents able to change functions of lysosomes in the cell may affect transfection efficiency and thus be used for gene therapy.     

Effect of γ-interferon on phagosome-lysosome fusion in Salmonella typhimurium-infected murine macrophages

Abstract Effect of recombinant γ-interferon (rIFN-γ) on phagosome-lysosome fusion in Salmonella typhimurium -infected murine macrophages was examined. rIFN-γ enhanced phagosome-lysosome fusion in macrophages infected with S. typhimurium in a dose-dependent manner, and over a range of 10² to 10³ U/ml of rIFN-γ exhibited maximum phagosome-lysosome fusion, although phagocytosis was slightly decreased. The enhancement of phagosome-lysosome fusion occured > 3 h post-treatment with rIFN-γ. Furthermore, the macrophage activation for phagosome-lysosome fusion was found to persist for 4 days even when rIFN-γ had been removed. These results demonstrate that INF-γ may serve as a mediator for the activation of phagosome-lysosome fusion in murine macrophages.     

Influence of phagosomal contents on the apparent inhibition of phagosome-lysosome fusion mediated by polyanionic substances in mouse peritoneal macrophages.

The study of fusion of phagosomes with secondary lysosomes in macrophages is facilitated by assessing transfer of fluorescent or electron-opaque markers (or both) from the lysosomes to the phagosomes. When certain virulent viable pathogens are phagocytosed by mouse peritoneal macrophages, phagosome-lysosome fusion (P-LF) is inhibited. Nonviable counterparts ordinarily cannot impose this block. A similar, but spurious, block to P-LF seems to be mediated from the lysosomal domain following sequestration of certain polyanionic substances. This block has been judged to be relieved by, for example, heat-killed yeasts and various viable bacteria designated as fusion-inducing microorganisms, acting from the phagosome. In this study we tested this concept and believed it to be unfounded. Macrophages labeled with Thorotrast and incubated with dextran sulfate were offered a variety of viable and heat-killed microorganisms for phagocytosis: Saccharomyces cerevisiae, Mycobacterium lepraemurium, Streptococcus faecalis, and Escherichia coli. By electron microscopy, a transfer of Thorotrast to phagosomes up to 18 h was seen to be highly suppressed as compared with controls, but was not notably different for any of the targets, whether viable or not. Instead, inert 0.45-micron carboxylated polystyrene beads (the smallest target) showed the most delivery of marker. If polyanionic agents truly inhibited fusion, then "fusiogenic" microorganisms should free the marker for delivery. If polyanions do not inhibit P-LF and only trap the marker, the behavior of the various targets would correspond to what we found.     

Influence of the galactosyl ligand structure on the interaction of galactosylated liposomes with mouse peritoneal macrophages

Liposomes bearing at their surface mono- and triantennary galactosyl ligands were prepared and their interaction with the galactose receptor of mouse peritoneal macrophages studied. Triantennary structures were synthesized by coupling derivatives of 1-thio--d-galactose to the amino groups of lysyl-lysine dipeptide. Galactosylated liposomes were obtained either by synthesis of neo-galactolipids followed by their incorporation into the vesicles or by neo-galactosylation of preformed liposomes by reaction between thiol-functionalized galactosyl ligands and vesicles bearing maleimido groups. The interaction of the galactosylated liposomes with the macrophage lectin was remarkably sensitive to the topology of the ligands, i.e., a spacer-arm length about 3 nm was necessary and, in contrast to results obtained with the galactose receptor of other cells, the triantennary structure did not provide additional binding. Related to the strategy of drug delivery with targeted liposomes, these results indicate that lectins from different cells might possibly be distinguished by using multiantennary ligands having optimal geometries.Abbreviations Gal d-galactose - GalNAc 2-acetamido-2-deoxy-d-galactose - PC l--phosphatidylcholine - PE l--phosphatidylethanolamine - DPPE dipalmitoyl-l--phosphatidylethanolamine - PG l--phosphatidylglycerol - SPDP N-succinimidyl-3-(2-pyridyldithio)propionate - SMPB succinimidyl-4-(p-maleimidophenyl)butyrate - MPB-PE 4-(p-maleimidophenyl)butyryl-PE - Succ-DPPE N-succinyl-DPPE - NHS N-hydroxysuccinimide - DCC N,N-dicyclohexylcarbodiimide - EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide - NHS-Succ-DPPE NHS ester ofN-succinyl-DPPE - REV vesicles obtained by reversed phase evaporation - Hepes 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid - DMEM Dulbecco's modified Eagle's medium.     

Biochemical characterization of mouse microsomal prostaglandin E synthase-1 and its colocalization with cyclooxygenase-2 in peritoneal macrophages.

We cloned the cDNA for mouse microsomal prostaglandin (PG) E synthase-1 (mPGES-1) and expressed the recombinant enzyme in Escherichia coli. The membrane fraction containing recombinant mPGES-1 catalyzed the isomerization of PGH2 to PGE2 in the presence of GSH with K(m) values of 130 microM for PGH2 and 37 microM for GSH, a turnover number of 600 min(-1), and a k(cat)/K(m) ratio of 4.6 min(-1) microM(-1). Recombinant mPGES-1 was purified and used to generate a polyclonal antibody highly specific for mPGES-1. The antibody showed a single band on Western blotting of microsomal fractions from lipopolysaccharide-treated mouse peritoneal macrophages. Northern and Western blotting analyses revealed that mPGES-1 was induced together with cyclooxygenase-2 in mouse macrophages after treatment of the cells with lipopolysaccharide. Confocal immunofluorescence microscopy revealed that both mPGES-1 and cyclooxygenase-2 were colocalized in the lipopolysaccharide-treated macrophages. Taken together, these results demonstrate that mPGES-1 is an efficient downstream enzyme for the production of PGE2 in the activated macrophages treated by lipopolysaccharide.     

Immunostimulatory effect of spinach aqueous extract on mouse macrophage-like J774.1 cells and mouse primary peritoneal macrophages

We herein report the immunostimulatory effect of spinach aqueous extract (SAE) on mouse macrophage-like J774.1 cells and mouse primary peritoneal macrophages. SAE significantly enhanced the production of interleukin (IL)-6 and tumor necrosis factor-α by both J774.1 cells and peritoneal macrophages by enhancing the expression levels of these cytokine genes. In addition, the phagocytosis activity of J774.1 cells was facilitated by SAE. Immunoblot analysis revealed that SAE activates mitogen-activated protein kinase and nuclear factor-κB cascades. It was found that SAE activates macrophages through not only TLR4, but also other receptors. The production of IL-6 was significantly enhanced by peritoneal macrophages from SAE-administered BALB/c mice, suggesting that SAE has a potential to stimulate macrophage activity in vivo. Taken together, these data indicate that SAE would be a beneficial functional food with immunostimulatory effects on macrophages.     

Effects of sulphydryl compounds on the radiation damage in biologically active DNA

The effect of sulphydryl compounds on the induction of alkali-labile sites and on the contribution of such sites to the inactivation of single-stranded phi X174 DNA was studied. Cysteamine is capable of reacting with DNA radicals, thereby modifying the radiation damage in such a way that the induction of immediate and latent breaks is reduced. This depends on the pH of the solution. With cysteine only, a pH dependent protection, against lethal alkali-labile potential breaks could be observed. The damage other than breaks is not influenced by the presence of sulphydryl compounds.     

Effects of microwave exposure on the hamster immune system. III. Macrophage resistance to vesicular stomatitis virus infection

Exposure of hamsters to microwave (MW) energy (2.45 GHz, 25 mW/cm2, 1 h) resulted in activation of peritoneal macrophages (PM) to a viricidal state restricting the replication of vesicular stomatitis virus (VSV). The PM from MW-exposed hamsters were viricidal as early as 1 day after exposure and remained active for 5 days. Immunization of hamsters with vaccinia virus induced viricidal PM by 3 to 4 days and they remained active for 7 days. To test the hypothesis that thermogenic MW exposure results in the release of endotoxin across the intestinal epithelium which subsequently activates PM, hamsters were injected with lipopolysaccharide (LPS) and their viricidal activity was studied. Lipopolysaccharide in vitro (0.2 microgram) and in vivo (0.5 microgram) activated macrophages to a viricidal state. When administered in vivo, LPS (0.5 microgram) activated macrophages as early as 1 day and the activity remained for 3 days. While MW exposure of PM in vitro failed to induce viricidal activity, exposure of PM to LPS in vitro induced strong viricidal activity. This suggests that the in vivo response of PM to MW is an indirect one, which is consistent with the hypothesis that MW-induced PM viricidal activity may be mediated via LPS. In preliminary experiments, MW exposure resulted in extended survival time for hamsters challenged with a lethal dose of vesicular stomatitis virus, supporting the concept that MW-activated PM may be a useful therapeutic modality.     

Increased cyclooxygenase-2 and 5-lipoxygenase activating protein expression in peritoneal macrophages during ovalbumin immunization of mice and cytosolic phospholipase A2 activation after antigen challenge

The present study investigates phenotypic and functional differentiation of peritoneal macrophages during ovalbumin-induced subcutaneous immunization of mice. For the first time we show that, in mouse peritoneal macrophages, ovalbumin immunization induces an increase in cyclooxygenase-2 (COX-2) and 5-lipoxygenase activating protein (FLAP) expression whereas it inhibits cytosolic phospholipase A₂ (cPLA2) expression. The study of arachidonic acid (AA) metabolism in peritoneal macrophages from control (cPM) and ovalbumin-immunized (iPM) mice shows that the reduced cPLA2 expression is correlated to a reduced basal AA metabolism, but is not a limiting factor for the opsonized zymosan-, PMA-, or A23187-triggered AA metabolism. We also show that in vitro ovalbumin challenge induces, only in iPM, cPLA2 activation through phosphorylation of serine residues, via a mechanism involving MAP kinases, and through increased intracellular calcium concentrations, leading to eicosanoid production. In parallel, we report that, in peritoneal macrophages, ovalbumin immunization induces the expression of CD23, the low affinity receptor for IgEs known for its involvement in allergic diseases. Thus, the modified expression of the enzymes involved in AA metabolism and the difference of response of cPM and iPM toward the antigen are important elements to understand the underlying mechanisms of ovalbumin-induced allergic responses.     

Optical resolution of some biologically active compounds by chiral liquid chromatography on BSA-silica (resolvosil) columns

Optical resolution on the analytical scale of a number of racemic pharmaceuticals and some other biologically active compounds has been studied using immobilized bovine serum albumin (BSA) as the stationary phase. For some of the compounds the elution order was determined by the use of optically enriched fractions obtained from a preceding passage of a sample through a preparative column containing microcrystalline triacetylcellulose (MCTA). The reversal in the sign of optical rotation shown in the polarimetric elution profile from the latter, combined with the integrated peak area ratio obtained on resolution on the analytical column, gave directly the order of elution. For one of the benzothiadiazines studied (bendroflumethiazide), increasing the pH of the mobile phase produced opposite effects on the retention of the two enantiomers, leading to a large effect on the separation factor. For many of the compounds studied, high separation factors (α > 2) could be achieved.     

Chemiluminescent analysis of the antioxidant and immunomodulation effects of several psychotropic drugs on peritoneal macrophages.

The present study describes the application of several chemiluminescent (CL) methods for evaluation of antioxidant and immunomodulation effects of psychotropic drugs upon phagocytes: KO2-induced luminal-dependent CL for detection of superoxide anion radicals in a pure chemical system; PMA- and A23187-induced CL of peritoneal macrophages for detection of free radicals in cell suspension; and CL, produced by the luciferase-catalyzed luciferin + ATP reaction, for evaluation of cell viability before and after drug application. These methods provide also a way to investigate the location of drug action. It was found that the psychotropic drugs in fluence the 'oxidative burst' of macrophages through two mechanisms: by expression of drug antioxidant properties and/or by a direct immunomodulation effect.     

Effect of polar glycopeptidolipids from Mycobacterium Chelonae (GPLp-Mc) on phagocytosis and superoxide anion production of macrophages from mice. Influence of physical activity

It is not clear how macrophages respond to exercise when the immune system is previously activated. The aim of the present work was to determine the response of macrophages to exercise in already immunostimulated animals with polar glycopeptidolipids extracted from Mycobacterium chelonae (GPLp-Mc). Results showed an increased phagocytosis and O₂ ^- production in murine macrophages induced by the intraperitoneal administration of 25 mg/kg body weight of GPLp-Mc. In addition exercise stimulated phagocytic activity and decreased the O₂ ^- production of these cells. Unexpectedly, exercise did not potentiate the immunostimulatory effect of GPLp-Mc. However, we can conclude that the effect of exercise is not detrimental to immunostimulated animals.     

The effect of oxidized glutathione and its pharmacological analogue glutoxim on intracellular Ca<Superscript>2+</Superscript> concentration in macrophages Ca<Superscript>2+</Superscript>

Using Fura-2AM microfluorimetry, the effect of oxidized glutathione (GSSG) and its pharmacological analogue glutoxim on the intracellular Ca²⁺ concentration in rat peritoneal macrophages was investigated. It was shown that GSSG or glutoxim increase the intracellular Ca²⁺ concentration by inducing Ca²⁺ mobilization from thapsigargin-sensitive Ca²⁺ stores and subsequent Ca²⁺ entry from external medium. Dithiothreitol, which reduces S-S-bonds in proteins, completely prevents or reverses the increase of intracellular Ca²⁺ concentration induced by GSSG or glutoxim. This suggests that the increase of intracellular Ca²⁺ concentration induced by GSSG or glutoxim can be mediated by their interactions with functionally important SH-groups of proteins involved in Ca²⁺-signaling.Two structurally different tyrosine kinase inhibitors genistein and methyl-2,5-dihydroxycinnamate prevent or completely reverse the increase in the intracellular Ca²⁺ concentration induced by GSSG or glutoxim. On the contrary, tyrosine phosphatase inhibitor Na orthovanadate enhances the increase of intracellular Ca²⁺ concentration evoked by oxidizing agents. The data suggest that tyrosine kinases and tyrosine phosphatases are involved in the regulatory effect of GSSG and glutoxim on the intracellular Ca²⁺ concentration in macrophages.     

Effects of physical exercise and aging on ascorbic acid and superoxide anion levels in peritoneal macrophages from mice and guinea pigs

The relationship of physical activity and aging, two processes with a high production of oxygen-free radicals to the ascorbate and superoxide anion (O₂^-) contents of peritoneal macrophages was studied in two animal species: guinea-pig (in which ascorbic acid is a vitamin) and mouse (in which ascorbic acid is not a vitamin). The effects of exhaustive exercise were examined in young and old animals. The results show that macrophages from old animals have a lower ascorbate content than those from young ones, whereas with exercise the ascorbate content increased in both old and young animals. This increase was higher in young than in old animals, and more evident in mice than in guinea-pigs. Aging also resulted in an increase in the O₂^-levels of macrophages. With exercise these levels decreased in young mice but increased in young guinea-pigs. In old animals the exhaustive exercise did not change the O₂^-levels. The results suggest in general a lack of correlation between the intracellular ascorbate and O₂^-levels in relation to both physical exercise and aging.Abbreviations PBS phosphate buffered saline - NBT nitroblue tetrazolium - PEC peritoneal exudate cells - PMN polymorphonuclear     

Effects of immune lymphocyte products and serum antibody on the multiplication of Toxoplasma in murine peritoneal macrophages.

In vitro studies on cell-mediated immunity against Toxoplasma infection were carried out by estimating the ability of antigenically stimulated lymphocytes. Splenic lymphocytes from normal mice and from hyper-immunized mice were cultured in the presence or absence of Toxoplasma lysate antigen. The cell-free supernatant fluids from the lymphocyte cultures were assassed for their ability to alter the functional capacities of normal macrophages. When glycogen-induced peritoneal macrophages were incubated with the culture supernatant from immune lymphocytes reacting with specific angigen, the intracellular multiplication of the organisms was inhibited remarkably. In contrast, the addition of antitoxoplasma antibody to culture medium had no effect on the enhancement of phagocytic activity of culture macrophages. However, when extra-cellular organisms were exposed to fresh or heat-inactivated immune serum just before infection of the monolayers, the intracellular multiplication of Toxoplasmas was inhibited significantly by either activated or normal macrophages.     

Effects of the afferent stimulation on neurons in the medial septal region slices and their modulation by biologically active compounds in hibernating and waking ground squirrels

Responses of the medial septal (MS-DB) neurons to electrical stimulation of the medial forebrain bundle (MFB) and their modulation by some neuropeptides and monoamines were investigated in brain slices taken from two groups of ground squirrels-hibernating (HGS) and waking (WGS). Electrical stimulation evoked mostly inhibitory effects of various duration. Besides, responses by phase reset of the background rhythmic bursts and short-latency single spike responses were observed. The neuropeptides identified in the brain of hibernators differentially and reversibly modulated responses even in those neurons where they did not influence the level and pattern of the background activity. Effects of the peptides were state-dependent. E.g., the peptide TSKYR increased the duration of inhibitory effects in the HGS but shortened them in the WGS, while TSKY which had low efficacy in the HGS, increased the duration of inhibition in the WGS. Dipeptide DY depressed inhibitory components and augmented excitatory components of responses in the HGS but was much less effective in the WGS. Effects of noradrenaline and serotonin had stronger correlation with their influence on spontaneous activity. It is suggested that endogenous substances provide for the increased latent excitability and reactivity of the MS-DB neurons during seasonal hibernation. Thus, the MS-DB may function as a "sentry post" participating in signal detection and urgent arousal during hibernation.