The effect of polyamines on lysosome fusion with phagosomes in mouse peritoneal macrophages

The influence of polyamines on the phagosome-lysosome fusion in murine peritoneal macrophages and on polymerization of G-actin from the rabbit muscle in vitro has been studied. Both natural polyamines (spermin, spermidin, putrescin) and synthetic phenyl derivates of polyamines (3,3'-diaminobensidin, 1,5-naphtalin diamine, 4,4'-diaminodiphenilmetan, dancylcadaverin) were used. Unlike the phenyl derivates of polyamines and putrescin, spermin and spermidin stimulate the phagosome-lysosome fusion to induce G-actin polymerization. Possible mechanisms of action of the above polyamines are discussed.     

The effect of chemical agents on lysosome fusion with phagosomes and on the F-actin content in murine peritoneal macrophages]

The influence of natural and synthetic polyamines, phalloidin, cytochalasin D, vinblastine, colchicine, puromycin, chlorpromazine, urea, glutaraldehyde, and ethanol on the phagosome-lysosome fusion and the content of F-actin in murine peritoneal macrophages has been studied. Fluorescent phallotoxin FITC-phalloidin was used to stain F-actin. Natural polyamines (spermine, spermidine, putrescine), phalloidin, ethanol (0.1 M) stimulated the phagosome-lysosome fusion and increased the mid-content of F-actin in macrophages. Cytochalasin D, vinblastine, colchicine, puromycin, chlorpromazine, urea, glutaraldehyde, ethanol (0.15 and 0.2 M) inhibited this process and decreased the mid-content of F-actin. Possible mechanisms of the interconnection of cytoskeleton and the phagosome-lysosome fusion are discussed.     

Effect of polyamine synthesis inhibitors separately and in combination with epidermal growth factor on fusion of lysosomes with phagosomes and F-actin level in mouse peritoneal macrophages

Effects of polyamine (PA) synthesis inhibitors--alpha-difluoromethylornithinchloride (DFMO) and alpha-methylornithinchloride (MO)--separately or in combination with the epidermal growth factor (EGF)--on lysosome-phagosome fusion (P-LF) and F-actin content in murine peritoneal macrophages were studied using fluorescent dye Acridine orange for lysosome labelling, FITC-phalloidin for F-actin, and yeast cells as a target. DFMO and MO significantly inhibited P-LF and decreased F-actin content in murine peritoneal macrophages. A combination of DFMO and MO with EGF failed to inhibit P-LF or to decrease F-actin content in these cells. The results obtained with DFMO and MO suggested new cellular targets of their effects. These results may be extended to cancer research to provide a rationale for clinical trials using combinations of EGF with DFMO or MO.     

Differential and sequential delivery of fluorescent lysosomal probes into phagosomes in mouse peritoneal macrophages

It has previously been inferred that the fusion of a macrophage secondary lysosome with a phagosome delivers the entire lysosomal contents uniformly to the phagosome. We found, however, that different fluorescent lysosomal probes can enter phagosomes at remarkably different rates, even though they are initially sequestered together in the same organelles. Thus, sulforhodamine is almost exclusively delivered to yeast-containing phagosomes within 2 h of phagocytosis. But fluoresceinated, high molecular weight dextran accumulates in the same phagosomes only over a period of approximately 24 h. We postulate that the delivery of lysosomal contents may involve an intermittent and incremental process in which individual components can be selectively and sequentially transferred.     

Danazol action on 5'-nucleotidase activity in mouse peritoneal macrophages]

The paper reports the results of danazol action in vivo and in vitro on 5'-nucleotidase activity of mouse peritoneal macrophages. The results showed, that danazol at a concentration of 10 M (the pharmacologic level in women taking 600 mg/day) significantly stimulated the enzyme activity.     

The effect of bilirubin on the fc receptor expression and phagocytic activity of mouse peritoneal macrophages

The effect of bilirubin on the phagocytic activity of mouse peritoneal macrophages and on the expression of Fc receptors and receptors for SRBC was studied. Intraperitoneally administered bilirubin influenced the expression of Fc receptors for IgM, IgG2B, IgA and IgE, whereas the expression of other receptors as well as the phagocytic activity of peritoneal macrophages remained unchanged. The possible mechanism of the effect of bilirubin on Fc receptors is discussed.     

Anti-inflammatory effect of lysozyme from hen egg white on mouse peritoneal macrophages

Lysozyme from hen egg has been reported to possess an anti-inflammatory effect. However, little is known about its detailed mechanism. The mechanism of anti-inflammatory effect of lysozyme was examined in this study. When mouse macrophage-like cell line RAW264.7 cells and mouse peritoneal macrophages were activated with lipopolysaccharide (LPS) and then treated with lysozyme, the production of tumor necrosis factor-α and interleukin-6 was significantly suppressed. The effect was induced by suppressing the gene expression levels of both cytokines. Phagocytosis activity of peritoneal macrophages was not altered by the treatment with lysozyme, suggesting that lysozyme shows the anti-inflammatory effect without inhibiting the phagocytotic response of macrophages. In addition, lysozyme inhibited phosphorylation of c-jun N-terminal kinase (JNK) and was taken up by macrophages within 1 h after treatment of the cells with lysozyme. Overall results suggest that lysozyme is taken up intracellularly and suppresses LPS-induced inflammatory responses by inhibiting JNK phosphorylation.     

The effect of cytochalasin B and colchicine on concanavalin A induced vacuolation in mouse peritoneal macrophages

Macrophages bind concanavalin A (ConA) and interiorize the ConA-receptor conjugates in minute ConA-bearing pinosomes that undergo intracellular fusion processes to yield medium size (2–5 μ) and large size (>5 μ) vacuoles. The number and size of the vacuoles depends on ConA concentration and on the composition of the incubation medium. Cytochalasin B (CB) reduces ConA internalization by 30% and the surface bound lectin redistributes to form numerous clumps of fluorescence. Macrophage ConA-induced vacuolation is inhibited by CB both when the incubation is carried out in Dulbecco's modified Eagle's medium (90% inhibition) or in phosphate buffered saline (80% inhibition). Colchicine reduces [³H]ConA internalization by 20% and has no detectable effect on the surface distribution of fluorescein-ConA conjugates. Incubation of macrophages with colchicine prior to or during ConA induction of vacuole formation results in an enhancement of cell vacuolation; the number of the developing large size vacuoles exceeds 4–6-fold that obtained in the absence of the alkaloid. The results suggest the involvement of elements of the cytoskeleton (microfilaments-microtubuli) in regulation of the sequence of events leading to ConA-induced vacuolation and that CB and colchicine have opposite effects on the process.     

The effect of the immunization of animals with a live plague vaccine on the lysosome count in organ-specific macrophages

Vital fluorochromatic lysosomes of peritoneal, liver, splenic and lung macrophages of white mice, white rates, and guinea-pigs are studied. Reliable differences in a quantity of lysosomal granules of macrophages from various tissues as well as differences between macrophages from the same tissues of different experimental animals are found. At 21 days after animal immunization with live plague vaccine EV the most significant changes in the number of lysosomal granules are revealed in white mice. The number of lysosomes in guinea pigs increased in 1 and 7 days after vaccination, in 14 days their number became normal.     

The effect of endotoxin on the lipoxygenase-mediated conversion of exogenous and endogenous arachidonic acid in mouse peritoneal macrophages

The influence of lipopolysaccharide (LPS, endotoxin) or its lipid A component (bacterial and synthetic) on the synthesis of zymosan induced leukotriene C4, prostaglandin E2 and prostacyclin and on the conversion of exogenous arachidonic acid was studied in mouse peritoneal macrophages. It was found that following preincubation with LPS the amount of leukotriene C4 released during phagocytosis of zymosan was substantially decreased. The levels of prostaglandin E2 and prostacyclin, however, were the same in LPS-treated cells and controls. Likewise, pretreatment with LPS impaired the capacity to convert exogenously added arachidonic acid to mono- and di-HETE's. Lipid A (bacterial and synthetic) exhibited the same activity as LPS. LPS had no effect on macrophages of the endotoxin low responder mouse strain (C3H/HeJ). Several explanations could be possible for the observed LPS effect. The finding that low doses of alpha-tocopheryl acetate prevented the LPS-induced decrease of LTC4 synthesis indicates a protective role of this agent. We would, therefore, favour the idea that lipoxygenases undergo oxidative selfinactivation during LPS action.     

The negative effect of magnetic nanoparticles with ascorbic acid on peritoneal macrophages

Neurochemical Research - Superparamagnetic iron oxide nanoparticles (SPIOn) are widely used as a contrast agent for cell labeling. Macrophages are the first line of defense of organisms in contact...     

Suppressive effect of streptomycin on the phagocytic activity of mouse peritoneal macrophages for Histoplasma capsulatum

Streptomycin inhibited the phagocytic activity of mouse peritoneal macrophages forHistoplasma capsulatum. The inhibitory effect was demonstrable following both in vitro and in vivo administration of drug. The observations from examination by direct smear were confirmed by culturing for viable phagocytized organisms. A simple and reproducible technique for the counting of viable phagocytized organisms was developed. Forty-eight hours in vitro treatment of macrophage cultures with 10 to 200 µg/ml of streptomycin produced a graded inhibition of phagocytic activity, minimal at 10 µg/ml and maximal at 200 µg/ml of streptomycin. The parenteral administration of streptomycin significantly reduced phagocytic activity of mouse peritoneal macrophages forH. capsulatum. Mice were treated daily with the subcutaneous injections of 5, 2.5 or 1 mg streptomycin or saline. At 7, 14, 21 and 28 days post-treatment phagocytic activity of macrophages obtained from these mice was tested. There was a progressive, dose-dependent decrease in the phagocytic activity of macrophages derived from streptomycin-treated mice.     

The effect of inhibitors of glucose utilization on the chemiluminescence and fluorescence of murine peritoneal macrophages]

Using the inhibitors of glucose utilization and mitochondrial oxidation we found that the blue autofluorescence (AF) of murine peritoneal macrophages (MP) originates predominantly from the cytosolic NAD(P)H. AF intensity correlates with the changes in glycolytic enzymes activity. The luminol-dependent chemiluminescence (CL) intensity which characterizes the amount of O2- generating by MP upon the addition of formyl-methionyl-leucyl-phenylalanine (FMLP) is inhibited in the case of maximum activity of glycolytic enzymes. Incubation of MP with non-convertible analog of glucose, such as 2-deoxyglucose increases the CL intensity under the influence of FMLP. A conclusion is drawn that amount of O2- generating by MP depends on the intracellular concentration of sugars.     

Association of the actin cytoskeleton with glass-adherent proteins in mouse peritoneal macrophages

Summary— When mouse peritoneal macrophages adherent to glass surface were removed by treatment with triethanolamine and Nonidet P-40, fine thread structures of unique loops were left behind on glass at the sites of cell adhesion. To examine the ultrastructural relationship between such looped threads and cytoskeletal components in glass-adherent macrophages, we successfully used the ‘zinc method’ to remove most of the cytoplasm including nuclei and to expose the cytoskeleton associated with the ventral plasma membrane. The cytoskeleton was seen to be mainly composed of actin filaments forming dense networks. The network contained scattered star-like foci from which actin filaments radiated. When the ventral plasma membrane-cytoskeleton complex was further treated with Nonidet P-40, the membrane was dissolved to expose the glass surface with actin foci persisting on glass. When the complex was removed by further treatment with Nonidet P-40 and DNase I, the looped threads became visible. Confocal laser microscopy of glass-adherent macrophages stained with fluorescent phalloidin showed the preferential distribution of F-actin in the ventral cytoplasm along the plasma membrane, where intense fluorescent spots were also scattered. Confocal interference reflection microscopy revealed densely populated dark dots and striae of focal contact, which corresponded in overall distribution to actin foci and looped threads. These observations suggest that actin cytoskeleton is closely associated with looped threads to reinforce cell adhesion to glass.     

Stimulation of DNA synthesis in cultured mouse peritoneal macrophages after fusion injection of macrophage growth factor

Macrophage growth factor (MGF), when injected into cultivated mouse peritoneal macrophages by Sendai virus-fusion with loaded human erythrocyte ghosts, induced macrophage DNA synthesis within 40 h. DNA synthesis was observed in approx. 10% of the injected cells. Fusion products between macrophages and red cell ghosts were identified by co-injection of ¹²⁵I-labelled human serum albumin. These studies suggest that the mechanism of the physiological effect of MGF may depend on the endocytosis of at least part of the MGF molecule.