Role of active forms of oxygen in inducing luminol-dependent chemiluminescence in macrophages

The luminol-dependent chemiluminescence of mouse peritoneal macrophages during phagocytosis of opsonized zymosan was studied by using specific active oxygen scavengers and metabolic inhibitors. Extracellular hydrogen peroxide and superoxide anion were shown to contribute immensely to the induction of the chemiluminescence. The role of the hydroxyl radical was rather insignificant, whereas singlet oxygen was not involved in this process. The interaction between luminol and peroxide was shown to be peroxidase-dependent. An inhibitory analysis revealed that the interaction between luminol, peroxide and superoxide anion obeyed a hybrid enzyme-free radical mechanism.     

Carnosine as a stimulator of cytotoxic and phagocytic function of peritoneal macrophages]

Biochemical changes in peritoneal macrophages and their relatedness to the cytostatic and phagocytotic function in C3HA mice injected with a single intraperitoneal dose of 0.45 mM carnosine and 4-methyluracil or stimulated with peptone have been studied. During the first 24 hours after injection both carnosine and 4-methyluracil increase the activity of adenosine deaminase and purine nucleoside phosphorylase, the key enzymes of purine catabolism which is the main source of O2-. radicals in macrophages. In carnosine-stimulated macrophages the activity of membrane 5'-AMP nucleotidase decreases on days 1-3 after injection which points to alleviation of adenosine-induced inhibition as well as to macrophage activation. Carnosine increases the cytostatic and phagocytotic activities of macrophage coupled to O2-. production. The mechanism of the stimulating effect of carnosine on macrophages seems to consist in the dipeptide interaction with specific receptors localized on the plasma membrane of macrophagal cells.     

Post-receptor formation of active forms of oxygen in nonphagocytic cells]

Generation of reactive oxygen species (ROS) in A431 cells, NIH3T3 fibroblasts expressing normal epidermal growth factor (EGF) receptor, L929 fibroblasts, and in mouse peritoneal macrophages (professionally phagocytic cells) upon the effect of different activators has been studied. It has been shown that ROS formation in A431 and NIH3T3 cells upon the effect of EGF is time- and dose-dependent process. A variety of stimuli were used to stimulate macrophage ROS production. However, the effect of only phorbol ester, opsonized zymozan, peptide fMLP, and platelet activating factor led to ROS generation, whereas tumor necrosis factor alpha, interferon gamma, and lipopolysaccharide did not stimulate macrophage oxidative burst. The literature data on ROS generation in a variety of cell types are presented. ROS formed in cells acted upon certain agents are considered as the molecules participating in intracellular signaling.     

Generation of active oxygen forms in rat thymocytes under action of hydrogen peroxide and fullerene C60

The dynamics of active oxygen forms (AOF) generation in rat thymocytes 50 min after treatment with 0.1 and 0.5 mM H2O2 was estimated with the use of fluorescent probe DCFDA. Both enhanced AOF generation, which was dependent on H2O2 concentration, and glutathione peroxidase and superoxide dismutase activation, followed by a decrease of thymocytes viability were demonstrated. Preincubation of cells with 10(-5) M fullerene C60 was shown not only to prevent H2O2--induced AOF generation but to increase viability of H2O2-treated thymocytes at more prolonged time period. The data obtained indicate to fullerene C60 ability to prevent oxidative stress in thymocytes.     

The cytotoxic effect of macrophages studied by time-lapse microcinematography

Cytotoxic action of murine peritoneal macrophages is demonstrated in vitro by time-lapse phase contrast microcinematography. The process was found to be contact-dependent. Morphological observations of effector/target cells interaction may support the idea that some sort of osmotic shock is involved in the red cell lysis. It was demonstrated that the lysing process occurred in the course of less than 4 sec.     

Carbohydrate metabolism in the rat peritoneal macrophages

Rat peritoneal macrophages derive energy differently from other tissues. Resting rat peritoneal macrophages have been taken for the present investigation. Lactate produced by extracellular glycolysis in the peritoneal lavage fluid, is readily converted into pyruvate by resting peritoneal macrophages and is oxidised in mitochondria. Glycolytic enzymes other than phosphoglucoisomerase and lactate dehydrogenase could not be substantially demonstrated. Glucose-6-phosphate dehydrogenase was detected. The presence of glucose-6-phosphate dehydrogenase along with phosphoglucoisomerase indicates the operation of the hexose monophosphate shunt as a pathway supplementary to glycolysis. Resting rat peritoneal macrophages thus appear to utilize extracellular lactate as their main energy source instead of glucose, bypass glycolysis and have active hexose monophosphate shunt.     

Generation of reactive forms of oxygen by polymorphonuclear leukocytes during hepatoma growth in the peritoneal cavity of animals]

In the present work, an attempt was made to analyse generation of reactive oxygen species (ROS) by polymorphonuclear leucocytes (PMN) in the course of tumour growth, using chemiluminescence (CL). A multiple increase in the capacity of polymorphonuclear leucocytes of generating active forms of oxygen in the course of tumor growth was discovered. Two causes of this process were found. 1) the increase in specific activity of leucocytes; 2) the increase in the total quantity of PMN circulating in the blood. Leucocytes were also found in the ascite liquid. PMN leucocytes were shown to participate in the antitumor defence of the organism.     

Effect of aflatoxins on rat peritoneal macrophages

Phagocytosis, intracellular killing of Candida albicans, and superoxide production by rat peritoneal macrophages exposed to aflatoxins B1, B2, G1, G2, B2a, and M1 at several times and concentrations were analyzed to evaluate the intensity of a depressive effect for each mycotoxin. All aflatoxins used at very low concentrations had a depressive effect on the functions of macrophages. The biggest impairment of phagocytosis, intracellular killing, and spontaneous superoxide production was observed in macrophages exposed to aflatoxins B1 and M1.     

Effect of aflatoxins on rat peritoneal macrophages.

Phagocytosis, intracellular killing of Candida albicans, and superoxide production by rat peritoneal macrophages exposed to aflatoxins B1, B2, G1, G2, B2a, and M1 at several times and concentrations were analyzed to evaluate the intensity of a depressive effect for each mycotoxin. All aflatoxins used at very low concentrations had a depressive effect on the functions of macrophages. The biggest impairment of phagocytosis, intracellular killing, and spontaneous superoxide production was observed in macrophages exposed to aflatoxins B1 and M1.     

Hydrolysis of cyclic GMP in rat peritoneal macrophages

Intact rat peritoneal macrophages (rPM) treated with 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterases (PDEs), accumulated more cGMP than untreated cells. A PDE activity toward [(3)H]cGMP was detected in the soluble and particulate fractions of rPM. The hydrolysis of cGMP was Ca(2+)/calmodulin-independent but increased in the presence of cGMP excess. Similar results were obtained when [(3)H]cAMP was used as a substrate. The hydrolytic activity towards both nucleotides was inhibited in the presence of IBMX. Therefore, the PDEs of families 2, 5, 10 and 11 are potential candidates for cGMP hydrolysis in the rPM. They may not only regulate the cGMP level in a feedback-controlled way but also link cGMP-dependent pathways with those regulated by cAMP.     

Susceptibility of peritoneal macrophages to rat cytomegalovirus infection

Abstract Peritoneal macrophages from Lewis (Lew) and Brown Norway (BN) rats did not support rat cytomegalovirus (RCMV) replication. Intraperitoneal (i.p.) inoculation of virus into the rats resulted in a rapid clearance of virus from the peritoneal lavage fluid and an uptake of virus in the macrophages. The virus did not persist in the peritoneal macrophages of the rats.     

Carbohydrate specificity of the galactose-recognizing receptor of rat peritoneal macrophages

The galactose-recognizing system of rat peritoneal macrophages mediates the binding and uptake of desialylated blood cells and glycoproteins. To characterize the specificity of this receptor, binding studies were performed with various galactose derivatives as competitive inhibitors and sialidase-treated erythrocytes or asialoorosomucoid as ligands for receptors, which were either membrane-bound or isolated after solubilization. From the results obtained it can be concluded that galactose is recognized via its hydrophobic and/or hydrophilic regions, formed by the accumulation of OH-functions on one side and of H-atoms on the other ("side effect"), whereas the binding partner or the anomeric configuration of galactose has no significant influence. Although it became apparent that not a single hydroxyl group of the sugar is responsible for binding, the hydroxyl at C-4 seems to be most important, followed by the OH-group at C-3. Those at C-1, C-2 and C-6 do not play a great role. This order of importance ("position effect") was found with galactose, derivatized by methylation or otherwise, and with diastereomers of galactose. Whereas the recognition of a single galactose residue leads to weak binding only, an appropriate arrangement of several of these ligands in one molecule results in an enormous increase in the binding strength of each galactose residue. This "cluster effect" was observed not only with membrane-bound but also with solubilized receptor. However, the binding of asialoorosomucoid by the latter was better inhibited with free galactose, when compared with the membrane-bound receptor.     

Redistribution of subpopulations of peritoneal macrophages with Fc gamma receptors under the effect of an immunomodulator

The heterogeneity of resident peritoneal macrophages and peritoneal macrophages was studied in different periods following oral administration of sodium nucleinate according to their ability to bind and phagocytize sheep erythrocytes opsonized by means of specific rabbit IgG. Using a mathematical method developed earlier, it has been possible to demonstrate that resident peritoneal macrophages can be divided into two subpopulations--actively and poorly binding macrophages but, after activation by sodium nucleinate--into three subpopulations. Fractionation of the macrophages according to their ability to adhesion within a temperature gradient has shown that the same peaks are traced in the fractions as in the overall pool of cells, but in different quantitative ratios. It has also been demonstrated that phagocytic activity is reduced in macrophages capable of adhesion to plastic at lower temperatures.     

The role of peritoneal macrophages in realizing the growth inhibiting effect of glucocorticoids on reinoculated murine hepatoma 22 cells: the effect of hydrocortisone on the cytotoxic activity and metabolism of phospholipids by macrophages

A single injection of hydrocortisone to rats with ascite hepatoma 22 had practically no effect on tumour growth. Inhibition of tumour growth was observed only after reinoculation of ascite hepatoma to mice that had received no less than 8 daily injections of the hormone. A single injection of hydrocortisone induced inhibition of the cytotoxic activity and decreased phospholipid metabolism in peritoneal macrophages. Contrariwise, long-term administration of the hormone caused marked activation of macrophage cytotoxicity. In this case incorporation of 32P into macrophage phospholipids was restored up to the control level. It is concluded that one of mechanisms underlying the inhibitory effect of glucocorticoids on macrophages is inhibition of phospholipid turnover. Presumably, long-term administration of the hormone promotes the formation of a new population of macrophages insensitive to the inhibitory effect of glucocorticoids and possessing a high cytostatic activity. The appearance of such activated macrophages may account for the enhancement of hydrocortisone effect on tumour cells upon prolonged administration of the hormone.     

Effect of oxygen on induction of the cystine transporter by bacterial lipopolysaccharide in mouse peritoneal macrophages

Amino acid transport in mouse peritoneal macrophages is mediated by several membrane carriers with different substrate specificity and sensitivity to environmental stimuli. We reported previously that transport activities of cystine and arginine in the macrophages were induced markedly by low concentrations of bacterial lipopolysaccharide (LPS). It is known that a variety of macrophage functions are affected by ambient oxygen tension. In this study, we have investigated the effects of oxygen on the induction of amino acid transport activity by LPS and found that the induction of cystine, but not arginine, transport activity was dependent on the ambient oxygen tension. When the macrophages were cultured with 2% O(2) in the presence of 1 ng/ml LPS, induction of cystine transport activity was reduced by approximately 70% compared with cells cultured under normoxic conditions. In macrophages, transport of cystine is mediated by a Na(+)-independent anionic amino acid transporter named system x(c)(-). System x(c)(-) is composed of two protein components, xCT and 4F2hc, and the expression of xCT was closely correlated with system x(c)(-) activity. A putative NF-kappaB binding site was found in the 5'-flanking region of the xCT gene, but the enhanced expression of xCT by LPS and oxygen was not mediated by NF-kappaB binding. An increase in intracellular GSH in macrophages paralleled induction of xCT, but not gamma-glutamylcysteine synthetase. These results suggest the importance of system x(c)(-) in antioxidant defense in macrophages exposed to LPS and oxidative stress.     

Uptake by rat peritoneal macrophages of125I-labelled polyvinylpyrrolidone entrapped within liposomes

Rat peritoneal macrophages in vitro capture¹²⁵I-labelled polyvinylpyrrolidone entrapped within either negatively or positively charged liposomes more rapidly than they do the free macromolecule. The uptake of negatively charged liposomes was linear with time over l0 h, whilst the uptake of positively charged ones, although more rapid, was more transient. Neither type of liposome was taken up in the presence of 2,4-dinitrophenol (100 g/ml), and 5 mM calcium chloride increased the uptake of negatively charged liposomes. The enhanced uptake of 125I-labelled polyvinylpyrrolidone when presented in liposomes must have been a consequence of entrapment rather than of a simple interaction between lipid and polyvinylpyrrolidone, since the presence of the lipids employed or of empty liposomes had no effect on the uptake of unentrapped¹²⁵I-labelled polyvinylpyrrolidone.     

Effect of some metabolites on the synthesis of nitric oxide by rat peritoneal macrophages

The peculiarities of some purine and energetic metabolites effect on the nitric oxide synthesis by vaccine BCG activated rat peritoneal macrophages has been studied. It was shown, that the glutamine and hypoxanthine caused essential increased of the nitrite level in the cell culture medium, but glutamic acid, adenosine, inosine and ATP did change these parameter. The mechanisms of studied compounds effect on the macrophages Ca(2+)-independent inducible NO-synthase activity are being discussed.     

Activation of peritoneal macrophages by lysophosphatidylcholine

Lysophosphatidylcholine (lyso-PC), a product of inflammation induced by infectious and other agents, is able to stimulate mouse peritoneal macrophages to ingest target cells coated with IgG but not IgM regardless of the presence of complement. In vitro treatment of mouse resident peritoneal macrophages (adherent cells) alone with lyso-PC stimulated spreading activity but did not enhance ingestion activity of macrophages. However, when mixed cultures of adherent and nonadherent (lymphocytes) cells were treated with lyso-PC, macrophage ingestion activity of IgG-coated target cells (i.e., via Fc-mediated ingestion) was markedly enhanced. Analysis of lyso-PC activation process of macrophages for ingestion activity suggests that nonadherent (lymphocytes) cells are required for the induction of the manifestation of ingestion capacity. This requirement was also met by addition of untreated nonadherent cells to treated adherent cells. Thus, the activation mechanism of macrophages by lyso-PC for ingestion requires contribution of lymphocytes to promote enhanced ingestion activity. Since lyso-PC is a metabolite of a representative membrane phospholipid, we propose that lyso-PC and other lysophospholipids are mediators for activation of macrophages regardless of the type of inflammation-causative agent.