Phagocytosis Of Polystyrene Latex Particles Research Articles

Induction of phagocytic activity of M1 cells by an inhibitor of vacuolar FT-ATPase, bafilomycin A 1

Bafilomycin a 1, a selective inhibitor of vacuolar H +-ATPase, time-and dose-dependently induced the differentiation of M1 cells, a murine myeloid leukemic cell line, into macrophage-like cells as revealed by the phagocytosis of polystyrene latex particles. This differentiation was inhibited not only by actinomycin D and cycloheximide but also by ST-638 (an inhibitor of tyrosine kinase). However, it was affected neither by K-252a (an inhibitor of C-kinase) nor by H-89 (an inhibitor of A-kinase), in contrast to the M1 cell differentiation induced by leukemia inhibitory factor (LIF). Okadaic acid inhibited both the bafilomycin A 1-induced and LIF-induced differentiation of M1 cells.

In vitro uptake of polystyrene latex particles and parenteral fat emulsions by human granulocytes

Suspensions of human granulocytes were used to study the in vitro phagocytosis of polystyrene latex particles (1030 nm) as i.v. model drug carriers and of commercial fat emulsions for parenteral nutrition (Lipofundin, Intralipid). Chemiluminescence (CL) was employed to follow the time-dependent uptake of the particle. The total uptake was quantified by the area under the curve of the CL intensity/time profiles and comparitively by a modified fluorimetric assay. The data proved that a linear relationship existed between the AUC of the CL assay and the total mass of internalized polymer. The uptake of fat emulsions could not be followed directly by CL because the CL intensity signal was too weak. Their uptake could be quantified via the impairment of the phagocytic function of granulocytes after pre-incubation with emulsion. The polystyrene particles were used as test colloid. The phagocytic function was reduced up to 50% by 10% fat emulsions.

Effect of azelastine hydrochloride on macrophage chemotaxis and phagocytosis in vitro.

Azelastine, a newly synthesized anti-allergic agent, was tested for its effects on guinea pig macrophage chemotaxis and phagocytosis. As specific macrophage chemo-attractants, we used macrophage chemotactic factors a and c; separated and highly purified from inflamed skin sites. Macrophage chemotaxis induced by skin extract or chemotactic factors was significantly suppressed by a low concentration of the agent (1 microgram/ml); the effect was dose-dependent. The inhibition of chemotaxis was reversible, because chemotactic activity was restored when the agents was removed by washing cells before chemotactic assay. Inactivation of chemotactic factors was not detected by mixing azelastine and factors a and c. Azelastine may directly interact with macrophages to decrease their chemotactic responsiveness. beta-Glucuronidase activity in the medium and macrophages after phagocytosis of polystyrene latex particles was not affected by this agent at concentrations ranging from 1 to 10 micrograms/ml. The phagocytosis of latex particles or sheep red blood cells opsonized with IgG antibodies (EA) and anchoring of macrophages to substrate were not inhibited and azelastine did not damage the macrophages as determined by lactate dehydrogenase (LDH) release assay.

Degradation of endogenous protein by rabbit pulmonary macrophages.

Pulmonary macrophages were preincubated for 1 or 20 h with L-[U-14C]phenylalanine and the degradation of labeled proteins studied by reincubating these cells in the presence of 4 mM L-phenylalanine and measuring rates of [14C]phenylalanine released from the cells into the medium. We found that proteins prelabeled in 1 or 20 h were degraded 8.0 and 3.0%.h-1, respectively. Decreases in cell viability reduced the rate of protein degradation. Lack of exogenous glucose slowed the rate of degradation of proteins labeled in 1 h, but not of those labeled in 20 h. Varying amino acids in the medium from normal to 5 times normal rabbit plasma levels had no effect on the degradation of either group of proteins. Rates of degradation of both fast and slowly turning over proteins were inhibited during phagocytosis of polystyrene latex particles by about 62 and 33%, respectively. The time course of the changes in protein degradation suggests they are related to intracellular events in the phagocytic process, rather than particle attachment or uptake.

Leukocyte function after aortic valve replacement

Leukocyte function was studied in patients with prosthetic heart valves by oxygen consumption measurements during phagocytosis of polystyrene latex particles. The consumption reflects the phagocytotic capacity of the cells. In 38 patients with Starr-Edwards aortic ball valves the mean oxygen consumption was 3.95 nanoatoms per minute per 10 6 leukocytes, as compared to 4.15 in 50 healthy subjects, the difference not being statistically significant. The number of leukocytes per ml. of blood and the distribution of cell types was quite similar in the two groups, although slightly more younger cells were found in the patients. It is concluded that the capacity for phagocytosis is not significantly reduced after aortic ball valve implantation.

Inhibition of macrophage phagocytic activity by group A streptococcal cell walls

Group A streptococcal cell walls inhibited phagocytosis of polystyrene latex particles by rat peritoneal macrophages in vitro. This inhibition was not accompanied by a measurable loss of cell viability. Group D streptococcal cell walls were relatively ineffective in altering phagocytosis. The effectiveness of group A variant streptococcal cell walls and peptidoglycan derived from group A cell walls was increased if cell wall preparations were added to macrophage cultures 12 h before, rather than simultaneously with, latex particles.

Increased Protein Synthesis by Human Platelets during Phagocytosis of Latex Particles in Vitro

A three-fold increase of protein synthesis by human platelets during in vitro phagocytosis of polystyrene latex particles was detected. During the first two hours of incubation, the percentage of phagocytizing platelets and the number of latex particles per platelet increased; by the end of the third hour, the first parameter remained stable, while the number of latex particles per cell had decreased. Vincristine (20 mug/ml of cell suspension) inhibited platelet protein synthesis. This effect was both time- and dose-dependent. The drug also caused a decrease in the number of phagocytizing cells, as well as in their phagocytotic activity.

Cyclic adenosine monophosphate in phagocytizing granulocytes and alveolar macrophages.

The ingestion of particles by phagocytizing cells is accompanied by marked changes in such metabolic parameters as oxygen consumption, glycolysis, glycogenolysis, H202 production, and activity of the hexose-monophosphate shunt, with the extent of these events depending on the particular cell type studied (for references see 1, 4, 6, 7) . In general, these metabolic changes can be observed within a few minutes or less after the addition of particles and are considered to reflect an increased energy demand during phagocytosis . It is not clear, however, in which way the contact of particles with the outer membrane or the initial phase of phagocytosis is signaled to the interior of the cell, where these metabolic changes take place . It is now well established that the interaction of many hormones with specific receptor sites at the outer membrane is signaled to the interior by changes in the intracellular concentration of adenosine 3',5'-monophosphate (Ado-3',5'-P, cyclic AMP) via stimulation of the membrane-located adenylate cyclase (13) . This system is not restricted to hormone effects, e.g., lymphocytic transformation by stimulation of phytohemagglutinins (13) . It was therefore tempting to speculate that cyclic AMP may have a messenger function in the process of phagocytosis . Indeed, results presented by Park et al. (11) suggest that during phagocytosis of polystyrene-latex particles by crude leukocyte preparations, the intracellular concentration of cyclic AMP is increased . Similar results were reported by Manganiello et al . (9) . It was not clear, however, whether the observed increase in cyclic AMP concentration was in fact related to phagocytosis . On the other hand, Stossel and colleagues (14) were unable to detect significant changes of cyclic AMP concentration in phagocytizing guinea pig peritoneal leukocytes . In view of these conflicting observations, it was of interest to reinvestigate the possible participation of cyclic AMP in the phagocytosis of polystyrene-latex particles by two different cell types, pig peripheral leukocytes and rabbit alveolar macrophages .

Plasma Membrane Synthesis in the Macrophage following Phagocytosis of Polystyrene Latex Particles

Phagocytosis of polystyrene latex beads by mouse peritoneal macrophages results in the interiorization of large amounts of plasma membrane to form the phagolysosomal membrane. The isolated membrane of the phagolysosomes is initially similar to the plasma membrane in cholesterol and phospholipid content, and carries 5′-nucleotidase activity with the characteristics of the plasma membrane marker enzyme. Immediately after ingestion, the amount of the plasma membrane has descreased as judged by a reduction in cell volume, spreading, pinocytosis, and phagocytosis. During the next 6 hours, the phagolysosomal 5′-nucleotidase activity decreases with a half-life of about 2 hours. Total cellular 5′-nucleotidase also decreases to a degree accounted for by the decrease in the phagolysosomal enzyme and in proportion to the amount of latex interiorized. After a lag of about 6 hours, cellular levels of the membrane constituents cholesterol and phospholipid begin to increase. The final net increase is linearly proportional to the amount of latex initially ingested and to the amount of plasma membrane interiorized. Only at this time do the macrophages begin to spread out on the glass surface, resume pinocytosis, and regain the ability to phagocytize a new test particle. The cellular levels of 5′-nucleotidase also begin to increase reaching control levels by 10 to 12 hours after phagocytosis. This activity is not associated with the phagolysosomes. These changes suggest the net synthesis of plasma membrane to replace the membrane interiorized during phagocytosis. This synthesis requires the presence of exogenous cholesterol molecules for use in the membrane. In the absence of cholesterol, plasma membrane functions are not restored and 5′-nucleotidase activity does not return to control levels. The inhibition of protein and RNA synthesis after phagocytosis blocks the net increment in cellular cholesterol, phospholipid, and 5′-nucleotidase activity.

The relationship between phagocytosis of polystyrene latex particles by polymorphonuclear leucocytes (PML) and aggregation of PML.

A methodological study of the adhesive stage of phagocytosis and its relation to aggregation is presented. Phagocytosis was measured by phase‐contrast microscopy, aggregation by microscopy and the irreversible aggregation by an electronic particle counter. Cinemicrographic observation revealed that polystyrene latex particles (Plx) of 1.0 μm in diameter were mostly attached to the periphery of polymorphonuclear leucocytes (PML) and not engulfed. The highest phagocytic capacity of PML was obtained in serum, and was decreased by addition of EDTA in concentrations greater than 1.5 mM, complete inhibition occurring at 6.0 mM EDTA, whereas 10–30 IU/ml heparin did not influence phagocytosis. Plasma proteins decreased phagocytosis. Fibrinogen had a marked influence whereas gamma globulin and albumin had only a slight effect. Phagocytosis was almost completely restored when PML were isolated from the medium containing the inhibitors EDTA and fibrinogen and resuspended in fresh serum. There seems to be a close correlation between phagocytosis and aggregation. The adhesiveness of Plx shows quite different characteristics to that occurring between individual PML.

Decrease in platelet surface charge during phagocytosis of polystyrene latex particles or thorium dioxide.

Incubation of platelet‐rich citrated plasma with polystyrene particles induced surface contact between platelets and particles, uptake of particles, and changes in platelet morphology. EDTA inhibited both the surface contact between platelets and particles and the uptake of particles. Polystyrene particles and thorium dioxide reduced the electrophoretic mobility of human platelets in vitro. Injected into rabbits both types of particles induced reduction in platelet electrophoretic mobility, platelet aggregation and thrombocytopenia. The reduction in platelet surface charge is suggested to render the platelets more susceptible to aggregation and phagocyosis in the reticuloendothelial system.

Influence of acetylsalicylic acid, butazolidine, colchicine, hydrocortisone, chlorpromazine and imipramine on the phagocytosis of polystyrene latex particles by human leucocytes

It has been demonstrated that acetylsalicylic acid, butazolidine, colchicine, hydrocortisone, chlorpromazine and imipramine inhibit the uptake of polystyrene latex particles and oxygen consumption during phagocytosis by human leucocytes.

Oxygen consumption during the initial stage of human leucocyte phagocytosis of polystyrene latex particles.

A polarographic method of studying oxygen consumption during phago cytosis by human leucocytes has been evaluated. Oxygen consumption during phagocytosis is markedly increased by raising the temperature from 20°C to 37°C, and inhibited by EDTA. Further, 2-deoxy-D-glucose inhibited the increase, but inhibition was stronger in combination with oligomycin. This indicates that the energy for phagocytosis is mainly derived from glycolysis, oxidative phosphorylation playing a minor role. The increase of oxygen consumption by washed human leucocytes during phagocytosis was greater in heparinized plasma than in serum. The difference was due to heparin. Heat-inactivated serum had a higher stimulatory effect than untreated serum. The combination of both calcium and magnesium, added to dialyzed plasma, showed a greater stimulatory effect than did magnesium alone. Calcium alone had no stimulatory effect.

The effect of temperature, mabolic inhibitors, andEDTA on pgocytosis of polystyrene latex particles by human leucocytes.

A modified method of Roberts & Quastel has been used to study the influence of temperature, metabolic inhibitors and EDTA on human leucocyte phagocytosis. The phagocytosis was increased by raising the temperature from 4° to 37° C. 2-deoxy-D-glucose inhibited phagocytosis. Antimycin or oligomycin had no influence. The greatest inhibition of phagocytosis was obtained by a combination of 2-deoxy-D-glucose and antimycin or oligomycin, demonstrating that the energy may derive from both glycolysis and respiration, the main source being glycolytic energy. Increasing concentrations of EDTA from 2 to 4 mM as final concentrations decreased phagocytosis.