Ingestion of latex beads by filopodia of adherent mouse peritoneal macrophages. A scanning electron microscopical and reflection contrast microscopical study

Scanning electron microscopy (SEM) of mouse peritoneal macrophages attached to glass shows that these cells have filopodia, i.e., cord-like extensions arising from the cell surface. To confirm that these extensions are not the result of the preparative procedure required for SEM or cell-surface material left behind by cells moving on the substrate surface, the cells were studied with a reflection contrast microscope prior to the preparative procedure. The results indicate that reflection-contrast microscopy and SEM both show the same filopodia for a given cell. The filopodia appear to be functional components of the cytoplasm, as shown by their ability to ingest latex beads.     

Definition of fibronectin-mediated uptake of gelatinized latex by liver slices and macrophages

These studies show that both liver slices and macrophages carried out fibronectin concentration-dependent uptake of 125I-labeled gelatin-coated latex (test latex). Lack of phagocytosis of test latex by liver slices was shown directly by electron microscopy and indirectly by trypsin treatment, which caused the release of all test latex taken up in response to fibronectin. Inhibitors of phagocytosis did not alter this uptake. On the other hand, trypsin released only a portion of test latex from macrophages. Inhibitors of phagocytosis did not effect the released radioactive particles from macrophages but greatly reduced the trypsin-resistant radioactivity, taken as representing phagocytized particles. Opsonization of test latex with fibronectin did not require heparin but its association with liver slices occurred only in the presence of heparin. Macrophages, however, readily bound and internalized the opsonized test latex and heparin only potentiated these reactions. Gelatin competed with test latex for fibronectin for opsonization, but did not inhibit binding and phagocytosis of fibronectin-test latex complexes. Finally, soluble fibronectin-gelatin complexes did not compete for binding and phagocytosis of fibronectin-test latex complexes. Thus, fibronectin concentrated on the surface of latex is preferred for interaction with the fibronectin receptor of macrophages. Gelatin, however, was not essential for this reaction, because fibronectin directly coupled to latex was also readily taken up.     

The galactose-recognizing system of rat peritoneal macrophages. Receptor-mediated binding and uptake of glycoproteins

Binding and phagocytosis of sialidase-treated cells by peritoneal macrophages is mediated by a galactose-specific receptor. So far, only cells or particles exposing terminal galactose residues were demonstrated to be ligands. We present results obtained with a newly developed radio-receptor assay, which proves both binding and uptake of glycoproteins mediated by the galactose-recognizing receptor of peritoneal macrophages. Requirement of Ca2+ for binding is used to distinguish between reversibly surface-bound and irreversibly internalized ligands. By using this approach, the uptake of the ligand is followed and its inhibition with phenylglyoxal and N-ethylmaleimide is demonstrated. Evidence was also obtained that internalization is followed by degradation of the ligand. Studies on the specificity show that only galactose is recognized but that the binding strength depends on the arrangement of galactose residues presented by the ligand.     

Ascorbate uptake and antioxidant function in peritoneal macrophages

Since activated macrophages generate potentially deleterious reactive oxygen species, we studied whether ascorbic acid might function as an antioxidant in these cells. Thioglycollate-elicited murine peritoneal macrophages contained about 3 mM ascorbate that was halved by culture in ascorbate-free medium. However, the cells took up added ascorbate to concentrations of 6-8 mM by a high-affinity sodium-dependent transport mechanism. This likely reflected the activity of the SVCT2 ascorbate transporter, since its message and protein were present in the cells. Activation of the cells by phagocytosis of latex particles depleted intracellular ascorbate, although not below the basal levels present in the cells in culture. Glutathione (GSH) was unaffected by phagocytosis, suggesting that ascorbate was more sensitive to the oxidant stress of phagocytosis than GSH. Phagocytosis induced a modest increase in reactive oxygen species as well as a progressive loss of alpha-tocopherol, both of which were prevented in cells loaded with ascorbate. These results suggest that activated macrophages can use ascorbate to lessen self-generated oxidant stress and spare alpha-tocopherol, which may protect these long-lived cells from necrosis or apoptosis.     

Fate of ecto-NAD+ glycohydrolase during phagocytosis of normal and mannosylated latex beads by murine macrophages

In order to gain a better understanding of the role of ecto-NAD+ glycohydrolase, an enzyme predominantly associated with phagocytic cells, we have studied its fate in murine macrophages (splenic, resident peritoneal and Kupffer cells) during phagocytosis of opsonized on mannosylated latex beads. In parallel, we have also monitored nucleotide pyrophosphatase, another ecto-enzyme of macrophages. Phagosomes were isolated by flotation in a discontinuous sucrose gradient and the enzyme activities were determined with fluorometric methods. Low levels of NAD+ glycohydrolase and nucleotide pyrophosphatase could be measured associated with the phagosomal fractions, eg, respectively less than 4.5% and 10% in spleen macrophages. The phagosomal activities originate from the plasma membrane, ie they were latent and inactivation of ecto-NAD+ glycohydrolase with the diazonium salt of sulfanilic acid resulted in a marked decrease of this enzyme activity in the phagosomal fractions. Pre-labelling of the cell surface by [3H]-galactosylation indicated that NAD+ glycohydrolase is internalized to a lesser extent than an average surface-membrane unit. These results indicate that if ecto-NAD+ glycohydrolase of macrophages can be internalized to a limited extent during phagocytosis of opsonized or mannosylated latex beads, this enzyme appears to be predominantly excluded from the surface area involved in the uptake of such particles.     

Fate of ecto-NAD+ glycohydrolase during phagocytosis of normal and mannosylated latex beads by murine macrophages

In order to gain a better understanding of the role of ecto-NAD⁺ glycohydrolase, an enzyme predominantly associated with phagocytic cells, we have studied its fate in murine macrophages (splenic, resident peritoneal and Kupffer cells) during phagocytosis of opsonized or mannosylated latex beads. In parallel, we have also monitored nucleotide pyrophosphatase, another ectoenzyme of macrophages. Phagosomes were isolated by flotation in a discontinuous sucrose gradient and the enzyme activities were determined with fluorometric methods. Low levels of NAD⁺ glycohydrolase and nucleotide pyrophosphatase could be measured associated with the phagosomal fractions, eg, respectively less than 4.5% and 10% in spleen macrophages. The phagosomal activities originate from the plasma membrane, ie they were latent and inactivation of ecto-NAD⁺ glycohydrolase with the diazonium salt of sulfanilic acid resulted in a marked decrease of this enzyme activity in the phagosomal fractions. Pre-labelling of the cell surface by [³H]-galactosylation indicated that NAD⁺ glycohydrolase is internalized to a lesser extent than an average surface-membrane unit. These results indicate that if ecto-NAD⁺ glycohydrolase of macrophages can be internalized to a limited extent during phagocytosis of opsonized or mannosylated latex beads, this enzyme appears to be predominantly excluded from the surface area involved in the uptake of such particles.     

Nitroblue tetrazolium-dye reduction by rat peritoneal macrophages during the uptake of Diplococcus pneumoniae,type VI

The relationship between the rate of particle ingestion and the rate of Nitroblue Tetrazolium (NBT)-dye reduction by macrophages was studied after incubation of peritoneal exudate macrophages with heat-killed type VI pneumococci. The adherence to a polyethylene surface of the macrophages during the uptake of the pneumococci was determined as well. In some experiments pneumococci opsonized with heat-stable opsonins were used as material to beingested. The NBT-dye reduction and the surface adherence of the macrophages was enhanced when ingesting normal heat-killed pneumococci. During the uptake of opsonized, heat-killed pneumococci the macrophages showed an unaltered NBT-dye reduction and less adherence to a polyethylene surface as compared with macrophages incubated with normal heat-killed pneumococci. This implies that using opsonized pneumococci the quantitative NBT-dye reduction assay is not reliable as a parameter for macrophage phagocytosis, because the uptake was in fact enhanced. The surface adherence of the macrophages did not reflect the enhanced ingestion of opsonized bacteria either.     

Comparison of rat blood preparation methods for acetaldehyde assay

A comparison is made of four previously described methods for the preparation of blood for acetaldehyde (AcH) assay in the rat. The spontaneous formation of AcH which occurs during the treatment of blood containing ethanol and the recovery of known amounts of AcH added to the blood were studied. The methods using sodium nitrite-sulfosalicylic acid or perchloric acid (PCA) in saline gave low levels of spontaneous formation (1 to 2 microM AcH for 48 mM ethanol). In the recovery studies it was seen that semicarbazide does not allow displacement of all the AcH; treatment of the blood with the reactant sodium nitrite-sulfosalycilic acid and use of the hemolysis method gave levels of recovery lower than 50%. Only treatment of the blood with perchloric acid in NaCl allowed all the AcH added to the blood to be recovered. In vivo, PCA in saline releases the AcH which was seen to remain bound in the red blood cells with the semicarbazide method. So the recommended procedure for accurate assay of blood AcH in the rat is cold deproteinization in PCA/saline before head-space gas chromatography. The levels of in vivo blood AcH (4.1 +/- 0.33 microM) obtained in the rat using this method for a blood alcohol concentration of 52 mM are lower than those previously described in the literature.     

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.     

Scavenger receptor-mediated uptake and metabolism of lipid vesicles containing acidic phospholipids by mouse peritoneal macrophages

We studied the mechanism of uptake and metabolism of exogenous phospholipids in mouse peritoneal macrophages using vesicles composed of various phospholipids and cholesterol. Macrophages in culture were found to actively incorporate and metabolize phosphatidylcholine/cholesterol vesicles containing small amounts of acidic phospholipids such as phosphatidylserine, phosphatidylinositol, or phosphatidic acid and to store the fatty acyl chains and cholesterol in triacylglycerol and cholesteryl ester form in their cytosol. These cells exhibited massive amounts of oil red O-positive lipid droplets, a typical feature of foam cells. The metabolism of exogenous phospholipid vesicles was completely inhibited by chloroquine and cytochalasin B, suggesting that vesicle uptake occurs by endocytosis. A similar type of metabolism was observed in guinea pig peritoneal macrophages, macrophage cell line J774.1, but not in Swiss 3T3 fibroblasts. Competition studies using various ligands for the scavenger receptor showed that acetylated low density lipoprotein (acetyl-LDL), dextran sulfate, or fucoidan was able to compete for up to 60% of the binding of phosphatidylserine-containing vesicles, and that copper-oxidized LDL (oxidized LDL) competed for more than 90% of the vesicle binding. On the other hand, phosphatidylserine-containing vesicles was able to compete for more than 90% of the binding of acetyl-LDL. These results indicate that acidic phospholipids are recognized by the scavenger receptors on the surface of macrophages and that more than one scavenger receptor exists on mouse peritoneal macrophages, i.e. one capable of recognizing acetyl-LDL, oxidized LDL, and an array of acidic phospholipids on membranes, and the other recognizing both acidic phospholipids and oxidized LDL but not acetyl-LDL.     

Kinetics of uptake and distribution of arachidonic acid by rat alveolar macrophages

The time course of uptake and distribution of ³H-arachidoni acid (³H-AA) into rat alveolar macrophage phospholipid pools was examined. Macrophages incubated with exogenous ³H-AA in RPMI-1640 containing 0.1% bovine serum albumin (BSA), incorporated this radiolabel into phosphatidylcholine and phosphatidylinositol (PI) with plateau reached within 2 to 4 hours, which remained relatively constant for up to 18 hours. Incorporation of ³H-AA into phospholipid pools revealed that treatment with exogenous 5 nM arachidonic acid had no effect upon pool sizes, but there was a selective incorporation if ³H-AA into PI. Cells were incubated with ³H-AA in RPMI alone or medium containing either 0.2% lactalbumin, fetal calf serum at variable concentrations, 10% Nu, Serum, or 0.1% BSA. Incubation of macrophages with ³H-AA in RPMI alone or containing 0.2% lactalbumin, resulted in approximately 70% of the radiolabel taken up by the cells being incorporated into triglyceride. The addition of BSA to RPMI-1640 medium was found to facilitate selective uptake of ³H-AA into phospholipids. Approximately 70% of incorporated ³H-AA was releseable through the action of exogenous phospholipase A₂.     

Kinetics of uptake and distribution of arachidonic acid by rat alveolar macrophages

The time course of uptake and distribution of 3H-arachidonic acid (3H-AA) into rat alveolar macrophage phospholipid pools was examined. Macrophages incubated with exogenous 3H-AA in RPMI-1640 containing 0.1% bovine serum albumin (BSA), incorporated this radiolabel into phosphatidylcholine and phosphatidylinositol (PI) with plateaus reached within 2 to 4 hours, which remained relatively constant for up to 18 hours. Incorporation of 3H-AA into phosphatidylethanolamine was small, but continued to increase for 14 hours. Analysis of phosphate content in phospholipid pools revealed that treatment with exogenous 5 nM arachidonic acid had no effect upon pool sizes, but there was a selective incorporation of 3H-AA into PI. Cells were incubated with 3H-AA in RPMI alone or medium containing either 0.2% lactalbumin, fetal calf serum at variable concentrations, 10% Nu Serum, or 0.1% BSA. Incubation of macrophages with 3H-AA in RPMI alone or containing 0.2% lactalbumin, resulted in approximately 70% of the radiolabel taken up by the cells being incorporated into triglyceride. The addition of BSA to RPMI-1640 medium was found to facilitate selective uptake of 3H-AA into phospholipids. Approximately 70% of incorporated 3H-AA was releasable through the action of exogenous phospholipase A2.     

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.     

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.