Pinocytosis and phagocytosis: the effect of size of a particulate substrate on its mode of capture by rat peritoneal macrophages cultured in vitro

Both phagocytosis (of particles) and pinocytosis (of solutes) occur in macrophages. It is not known, however, whether particles, if they are small enough, can enter by pinocytosis, nor whether there is a minimum size of particle capable of triggering phagocytic uptake. These questions have been investigated by studying, in vitro, the uptake by rat peritoneal macrophages of particles ranging in diameter from 30 nm to 1100 nm. Percoll (30 nm diameter) and polystyrene beads (100, 300, 600, 800 or 1100 nm diameter) were 125I-iodinated and their uptake by macrophages was measured in the absence or presence of metabolic and cytoskeletal inhibitors. Since uptake, expressed as an Endocytic Index (microliter/10(6) cells per h), increased steadily with the duration of incubation and was inhibited by low temperature or metabolic inhibitors, it was concluded that true endocytosis, and not a superficial cell-association, was being measured. Rates of clearance increased with increasing particle diameter. The rate of uptake of Percoll was 10-times, and of 100 nm polystyrene beads 100-times, the rate of fluid-phase pinocytosis, as measured by the uptake of 125I-labelled polyvinylpyrrolidone. Polystyrene beads of 1100 nm diameter were captured at 700-times this rate. The differential effects of colchicine and cytochalasin B on the uptake of 125I-labelled polyvinylpyrrolidone and of 1100 nm polystyrene beads were taken as indicators of their effects on pinocytosis and phagocytosis respectively. It is concluded that Percoll, although particulate, is captured by pinocytosis. The pattern of inhibition of uptake of polystyrene particles suggests that there is no radical discontinuity between pinocytic and phagocytic uptake, but that the contribution of phagocytosis steadily increases with increasing particle diameter. The results are discussed.     

Effect of suramin on pinocytosis by resident rat peritoneal macrophages: an analysis using four different substrates

The effect of suramin on pinocytosis and intralysosomal proteolysis by resident rat peritoneal macrophages cultured in vitro has been studied. Suramin had little effect on the rate of pinocytic uptake of two non-adsorptive substrates [14C]sucrose and [3H]dextran, but unexpectedly enhanced uptake of a third, 125I-labelled polyvinylpyrrolidone (PVP). Since this enhanced uptake was completely abolished by NaF at a concentration known to inhibit pinocytosis, it clearly represented an increased internalization of substrate and not merely a superficial binding to the cell surface. It was concluded that suramin (i) does not affect the rate of formation of pinocytic vesicles but (ii) acts as a bivalent ligand, binding to both the macrophage surface and the 125I-labelled polyvinylpyrrolidone, thus converting a non-adsorptive into an adsorptive substrate. Suramin (500 micrograms/ml) decreased both the rate of uptake of formaldehyde-denatured 125I-labelled bovine serum albumin (BSA) (an adsorptive substrate) and the rate of its subsequent intracellular degradation. Thus, depending on the substrate chosen to measure pinocytosis, the same modifier may stimulate or inhibit uptake or be without effect.     

Dependence of fluid-phase pinocytosis in arterial smooth-muscle cells on temperature, cellular ATP concentration and the cytoskeletal system.

125I-labelled poly(vinylpyrrolidone) was used as a marker of fluid-phase pinocytosis in cultured pig arterial smooth-muscle cells. The rate of pinocytosis was temperature-dependent. A decrease in cellular ATP concentrations as a result of inhibition of either glycolysis or oxidative phosphorylation was associated with a similar decrease in pinocytosis. A microfibrillar-disruptive agent, cytochalasin B, caused a concentration-dependent stimulation of pinocytosis, whereas the microtubular-disruptive agents colchicine and vinblastine decreased pinocytosis to approximately half of control values at all concentrations used. These results indicate that fluid-phase pinocytosis in smooth-muscle cells is dependent on a continuing supply of energy and the integrity of the microtubules. Furthermore, microfilaments appear to exert a certain degree of constraint on pinocytosis, possibly by restricting invagination of the plasma membrane.     

Pinocytic uptake and intracellular degradation of N-(2-hydroxypropyl)methacrylamide copolymers a potential drug delivery system

Synthetic ¹²⁵I-labelled N-(2-hydroxypropyl)methacrylamide copolymers containing four different, potentially degradable peptidyl side chains were incubated with rat visceral yolk sacs cultured in vitro. All copolymers were captured by fluid-phase pinocytosis and three of the side chains were susceptible to lysosomal hydrolysis, resulting in release of [¹²⁵I]iodotyrosine back into the culture medium. Uptake and degradation was completely inhibited by 2,4-dinitrophenol. The thiol-proteinase inhibitor leupeptin did not affect the rate of pinocytosis, but caused different degrees of inhibition of hydrolysis depending on side chain composition.     

Murine bone marrow-derived macrophages differentiated with GM-CSF become foam cells by PI3Kγ-dependent fluid-phase pinocytosis of native LDL

Accumulation of cholesterol by macrophage uptake of LDL is a key event in the formation of atherosclerotic plaques. Previous research has shown that granulocyte-macrophage colony-stimulating factor (GM-CSF) is present in atherosclerotic plaques and promotes aortic lipid accumulation. However, it has not been determined whether murine GM-CSF-differentiated macrophages take up LDL to become foam cells. GM-CSF-differentiated macrophages from LDL receptor-null mice were incubated with LDL, resulting in massive macrophage cholesterol accumulation. Incubation of LDL receptor-null or wild-type macrophages with increasing concentrations of ¹²⁵I-LDL showed nonsaturable macrophage LDL uptake that was linearly related to the amount of LDL added, indicating that LDL uptake was mediated by fluid-phase pinocytosis. Previous studies suggest that phosphoinositide 3-kinases (PI3K) mediate macrophage fluid-phase pinocytosis, although the isoform mediating this process has not been determined. Because PI3Kγ is known to promote aortic lipid accumulation, we investigated its role in mediating macrophage fluid-phase pinocytosis of LDL. Wild-type macrophages incubated with LDL and the PI3Kγ inhibitor AS605240 or PI3Kγ-null macrophages incubated with LDL showed an ∼50% reduction in LDL uptake and cholesterol accumulation compared with wild-type macrophages incubated with LDL only. These results show that GM-CSF-differentiated murine macrophages become foam cells by fluid-phase pinocytosis of LDL and identify PI3Kγ as contributing to this process.     

Degradation of insulin by human fibroblasts: effects of inhibitors of pinocytosis and lysosomal activity

The role of the pinosome-lysosome pathway in the degradation of 125I-labelled bovine insulin by cultured human fibroblasts was examined by comparing the effects of various known inhibitors of pinocytosis and lysosomal degradation on the uptake and degradation of 125I-labelled polyvinylpyrrolidone, formaldehyde-denatured bovine serum albumin and bovine insulin by these cells. Fibroblasts incubated with polyvinylpyrrolidone steadily accumulate this substrate, whereas incubations with insulin or denatured albumin led to the progressive appearance in the culture medium of [125I]iodotyrosine. Inhibitors of pinocytosis (bacitracin, colchicine and monensin), metabolic inhibitors (2,4-dinitrophenol and NaF), lysosomotropic agents (chloroquine and NH4Cl) and an inhibitor of cysteine-proteinases (leupeptin) decreased the rate of uptake of polyvinylpyrrolidone and denatured albumin very similarly, but only bacitracin had an effect on the processing of insulin. Chloroquine, NH4Cl and leupeptin strongly inhibited the digestion of denatured albumin, but not of insulin. The different responses to the modifiers, with polyvinylpyrrolidone and denatured albumin on the one hand and insulin on the other, suggest that insulin degradation can occur by a non-lysosomal pathway. The very strong inhibitory effect of bacitracin on insulin processing by fibroblasts may point to an important role of plasma membrane proteinases in insulin degradation.     

Adsorptive pinocytosis of polycationic copolymers of vinylpyrrolidone with vinylamine by rat yolk sac and rat peritoneal macrophage

Polycationic copolymers of vinylpyrrolidone and vinylamine (10:0.77) were prepared, and ¹²⁵I-labelled with either Bolton-Hunter reagent or methyl 3,5-di-[¹²⁵I]iodohydroxybenzimidate. The rate of pinocytic capture of the copolymer was compared with that of ¹²⁵I-labelled polyvinylpyrrolidone, using rat visceral yolk sacs and rat macrophages cultured in vitro as test systems. Whereas polyvinylpyrrolidone was captured entirely by non-adsorptive pinocytosis, the cationic derivative was captured more efficiently, probably because it adsorbs to the cell surface. Copolymer of M_r 120 000 was internalized by macrophages somewhat more rapidly than copolymer of M_r 46 000, but was excluded from the yolk sac.     

Uptake and degradation of 125I-labelled high density lipoproteins in rat liver cells in vivo and in vitro.

1. The uptake of 125I-labelled high density lipoproteins (HDL) in various organs of the rat was determined after an intravenous injection. The uptake of 125I-labelled polyvinylpyrrolidone in the same organs was determined in order to assess uptake by fluid endocytosis. The uptake/organ was highest for the liver. The adrenals showed the highest uptake/unit weight of the organs studied. The liver, the kidneys and the spleen showed comparable values for uptake/g of tissue. The uptake of 125I-labelled HDL exceeded by far that of 125I-labelled polyvinylpyrrolidone in the liver, the kidneys, the spleen and the adrenals, indicating that the uptake of 125I-labelled HDL was mediated by adsorptive endocytosis. 2. The in vivo uptake of 125I-labelled HDL was determined in purified hepatocytes and non-parenchymal cells prepared by collagenase perfusion of livers from animals after intravenous injections of 125I-labelled HDL. When expressed per cell, the hepatocytes and the non-parenchymal liver cells took up about the same amount of 125I-labelled HDL. 3. The in vitro uptake and degradation of 125I-labelled HDL in isolated rat hepatocytes was studied. The uptake at increasing concentrations of 125I-labelled HDL was saturable indicating uptake mediated through binding sites. 125I-labelled HDL were easily degraded by contaminating proteases from the perfusate. 4. Subcellular fractionation by isopycnic centrifugation indicated that the accumulation of 125I-labelled HDL did not take place in the lysosomes, but rather on the plasma membrane and possibly in the endosomes (phagosomes). 5. 125I-labelled HDL were internalized into the cells and degraded in the lysosomes. Leupetin and chloroquine, inhibitors of the lysosomal function effectively inhibited the formation of 125I-labelled acid-soluble radioactivity by the cells. Chloroquine, but not the protease inhibitor leupeptin, reduced the hydrolysis of the cholesteryl ester moiety of HDL.     

Phorbol esters and horseradish peroxidase stimulate pinocytosis and redirect the flow of pinocytosed fluid in macrophages

Lucifer Yellow CH (LY) is an excellent probe for fluid-phase pinocytosis. It accumulates within the macrophage vacuolar system, is not degraded, and is not toxic at concentrations of 6.0 mg/ml. Its uptake is inhibited at 0 degree C. Thioglycollate-elicited mouse peritoneal macrophages were found to exhibit curvilinear uptake kinetics of LY. Upon addition of LY to the medium, there was a brief period of very rapid cellular accumulation of the dye (1,400 ng of LY/mg protein per h at 1 mg/ml LY). This rate of accumulation most closely approximates the rate of fluid influx by pinocytosis. Within 60 min, the rate of LY accumulation slowed to a steady-state rate of 250 ng/mg protein per h which then continued for up to 18 h. Pulse-chase experiments revealed that the reduced rate of accumulation under steady-state conditions was due to efflux of LY. Only 20% of LY taken into the cells was retained; the remainder was released back into the medium. Efflux has two components, rapid and slow; each can be characterized kinetically as a first-order reaction. The kinetics are similar to those described by Besterman et al. (Besterman, J. M., J. A. Airhart, R. C. Woodworth, and R. B. Low, 1981, J. Cell Biol. 91:716-727) who interpret fluid-phase pinocytosis as involving at least two compartments, one small, rapidly turning over compartment and another apparently larger one which fills and empties slowly. To search for processes that control intracellular fluid traffic, we studied pinocytosis after treatment of macrophages with horseradish peroxidase (HRP) or with the tumor promoter phorbol myristate acetate (PMA). HRP, often used as a marker for fluid-phase pinocytosis, was observed to stimulate the rate of LY accumulation in macrophages. PMA caused an immediate four- to sevenfold increase in the rate of LY accumulation. Both HRP and PMA increased LY accumulation by stimulating influx and reducing the percentage of internalized fluid that is rapidly recycled. A greater proportion of endocytosed fluid passes into the slowly emptying compartment (presumed lysosomes). These experiments demonstrate that because of the considerable efflux by cells, measurement of marker accumulation inaccurately estimates the rate of fluid pinocytosis. Moreover, pinocytic flow of water and solutes through cytoplasm is subject to regulation at points beyond the formation of pinosomes.     

A quantitative study of pinocytosis and lysosome function in experimentally induced lysosomal storage.

The highly pinocytic epithelial cells of the visceral yolk sac from 17.5-day rat conceptuses were used as a model in which to induce engorgement of the vacuolar system by direct accumulation of substances that are not hydrolysed by lysosomal enzymes. The ultra-structural appearances of these cells in pregnant animals that 24-48h before had received intraperitoneal injections of Triton WR-1339, polyvinylpyrrolidone, dextran or sucrose revealed gross abnormalities that were confined to the vacuolar system; in comparison with normal tissue the number, and in some cases the size, of vacuoles was increased, leading to close packing within the apical cytoplasm and distortion of the normal rounded shape. By culturing yolk sacs in vitro, rates of ingestion of 125I-labelled polyvinylpyrrolidone and of 125I-labelled bovine serum albumin were determined, together with the rate of digestion of the labelled protein. The rates of exocytosis of 125I-labelled polyvinylpyrrolidone and of lysosomal enzymes were also determined. No significant differences between normal and highly vacuolated tissues were found. Apparently marked vacuolation of these cells by these agents is without significant effect on pinocytosis, exocytosis or intralysosomal proteolysis.     

Analysis of the effect of amines on inhibition of receptor-mediated and fluid-phase pinocytosis in rabbit alveolar macrophages

Incubation of rabbit alveolar macrophages in vitro with methyl amine led to a time- and concentration-dependent inhibition of uptake of alpha macroglobulin-125I-trypsin complexes (alpha M-125I-T). Upon addition of methyl amine (50 mM) to cells prelabeled with alpha M-125I-T there was a rapid inhibition of lysosomal catabolism of internalized ligand. In the absence of ligand, incubation of cells with 50 mM methyl amine led to a 40-70% decrease in surface-receptor number. The methyl amine-induced decrease in surface-receptor number only occurred in metabolically active cells since cells incubated at 0 degrees C, or treated with N-ethyl maleimide and incubated at 37 degrees C, did not show the effect. Incubation of cells at 37 degrees C with methyl amine also effected a 40-70% decrease in fluid-phase pinocytosis. Although there was a decline in surface-receptor number, the remaining population of receptors were capable of mediating (at least) one round of ligand internalization. However, further ligand uptake was prevented. Data demonstrate that although receptors were present on cell surfaces, they were incapable of mediating ligand internalization. Incubation of macrophages with chloroquine at 37 degrees C for 60 min also led to a disappearance of receptors, and a concomitant reduction in fluid-phase pinocytosis.     

The fate of homologous 125I-labelled immunoglobulin G within rat visceral yolk sacs incubated in vitro.

When 125I-labelled rat IgG (immunoglobulin G) is incubated in vitro with visceral yolk sacs from 17.5-day-pregnant rats, the protein is readily degraded. The major radioactive digestion product that accumulates in the medium is [125I]iodo-L-tyrosine. When rotenone (10 microM) is also present in the incubation medium, the rate of digestion of IgG is inhibited to the same extent as the rate of pinocytosis of 125I-labelled polyvinylpyrrolidone. Proteolysis is likewise inhibited when either NH4Cl (30 mM) or leupeptin (30 micrograms/ml) is present in the medium. The above findings strongly suggest that the observed proteolysis occurs within lysosomes. Normally, yolk sacs that have been exposed in vitro to radiolabelled substrates release radioactivity slowly when the tissue is re-incubated, unless the substrate can be degraded within lysosomes and released in the form of low-molecular-weight hydrolysis products. However, in such experiments 125I-labelled rat IgG shows quite exceptional behaviour in being rapidly released in an apparently intact form (as well as being degraded). If an agent that inhibits pinocytosis (e.g. rotenone or 2,4-dinitrophenol) is present in the incubation medium during exposure of the tissue to 125I-labelled rat IgG, it abolishes release of macromolecular radioactivity on re-incubation of the tissue. Enhanced tissue accumulation of 125I-labelled rat IgG, induced by the presence of leupeptin in the medium during the uptake phase, resulted in no concomitant increase in the amount of 125I-labelled IgG released in macromolecular form on re-incubation of the tissue. These findings indicate that the observed rapid release of 125I-labelled IgG is unlikely to represent release from lysosomes and is more compatible with release from a separate class of vesicle that does not fuse with lysosomes.     

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.     

Fluid-Phase Pinocytosis of Native Low Density Lipoprotein Promotes Murine M-CSF Differentiated Macrophage Foam Cell Formation

During atherosclerosis, low-density lipoprotein (LDL)-derived cholesterol accumulates in macrophages to form foam cells. Macrophage uptake of LDL promotes foam cell formation but the mechanism mediating this process is not clear. The present study investigates the mechanism of LDL uptake for macrophage colony-stimulating factor (M-CSF)-differentiated murine bone marrow-derived macrophages. LDL receptor-null (LDLR−/−) macrophages incubated with LDL showed non-saturable accumulation of cholesterol that did not down-regulate for the 24 h examined. Incubation of LDLR−/− macrophages with increasing concentrations of ¹²⁵I-LDL showed non-saturable macrophage LDL uptake. A 20-fold excess of unlabeled LDL had no effect on ¹²⁵I-LDL uptake by wild-type macrophages and genetic deletion of the macrophage scavenger receptors CD36 and SRA did not affect ¹²⁵I-LDL uptake, showing that LDL uptake occurred by fluid-phase pinocytosis independently of receptors. Cholesterol accumulation was inhibited approximately 50% in wild-type and LDLR−/− mice treated with {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 or wortmannin, inhibitors of all classes of phosphoinositide 3-kinases (PI3K). Time-lapse, phase-contrast microscopy showed that macropinocytosis, an important fluid-phase uptake pathway in macrophages, was blocked almost completely by PI3K inhibition with wortmannin. Pharmacological inhibition of the class I PI3K isoforms alpha, beta, gamma or delta did not affect macrophage LDL-derived cholesterol accumulation or macropinocytosis. Furthermore, macrophages from mice expressing kinase-dead class I PI3K beta, gamma or delta isoforms showed no decrease in cholesterol accumulation or macropinocytosis when compared with wild-type macrophages. Thus, non-class I PI3K isoforms mediated macropinocytosis in these macrophages. Further characterization of the components necessary for LDL uptake, cholesterol accumulation, and macropinocytosis identified dynamin, microtubules, actin, and vacuolar type H(+)-ATPase as contributing to uptake. However, Pak1, Rac1, and Src-family kinases, which mediate fluid-phase pinocytosis in certain other cell types, were unnecessary. In conclusion, our findings provide evidence that targeting those components mediating macrophage macropinocytosis with inhibitors may be an effective strategy to limit macrophage accumulation of LDL-derived cholesterol in arteries.     

Mechanism of stimulation of pinocytosis by trypan blue

Trypan blue at 50 microgram/ml stimulates the pinocytic uptake of 125I-labelled PVP, but not of colloidal 198Au or formaldehyde-denatured 125I-labelled bovine serum albumin, by the 17.5-day rat visceral yolk sac incubated in vitro. Neither Trypan blue nor a combination of the dye with 125I-labelled PVP stimulated the rate of pinocytosis of liquid by the tissue. Trypan blue itself was shown to enter the yolk-sac cells by adsorptive pinocytosis. It is proposed that an interaction between Trypan blue and 125I-labelled PVP enables the latter substrate to enter the cells adsorptively by so-called 'piggy-back' pinocytosis.     

Enhancement of cholesteryl ester metabolism in cultured human monocyte-derived macrophages by verapamil

The effect of the Ca2+ entry blocker, verapamil, on the biosynthesis of cholesterol and the metabolism of low-density lipoprotein (LDL) was studied in cultured human monocyte-derived macrophages. Addition of verapamil (50 microM) of monocyte-derived macrophages enhanced 125I-LDL and 125I-labelled acetyl-LDL binding and internalization, and increased [2-14C]acetate incorporation into cholesterol. Since higher levels of LDL and modified lipoproteins may be implicated in atherogenesis, the more efficient processing of these lipoproteins by monocyte-derived macrophages in the presence of Ca2+ blocker warrants further assessment for its potential as an antiatherogenic agent.     

Uptake and degradation of human very-low-density lipoproteins by rat liver endothelial cells in culture

Rat liver endothelial cells in primary cultures take up and degrade 125I-labelled human very-low-density lipoproteins (VLDL) in a saturable fashion at physiological triacylglycerol concentrations. The iodinated VLDL are readily taken up by the freshly isolated endothelial cells and degradation products appear in the medium about 30 min after the addition of VLDL to the cultures. Uptake and degradation at 37 degrees C are effectively inhibited by unlabelled human VLDL, low-density lipoproteins (LDL), high-density lipoproteins and lymph chylomicrons, but only modestly by acetylated LDL. Purified apolipoproteins E and C-III:1 also compete with the uptake of iodinated VLDL, but when degradation was studied for longer periods of time, such a competition could not be demonstrated. This may be due to the fact that the added apolipoproteins become associated with the lipoproteins. In binding experiments at 7 degrees C, iodinated apolipoprotein C III:1 bound to the liver endothelial cells in a manner characteristic of receptor binding with a dissociation constant of 0.5 microM. This binding could not only be inhibited by unlabelled apolipoprotein C-III:1 but also by unlabelled apolipoprotein E. The results indicate that rat liver endothelial cells carry receptors for VLDL and that these recognize the apolipoproteins E, C-III and B on the lipoprotein surface. Considering the large endothelial surface and high blood flow through the liver, significant quantities of lipoproteins can be taken up and degraded, thus influencing the levels of circulating lipoproteins in the in vivo situation.     

Quantitative studies of pinocytosis. I. Kinetics of uptake of (125I)polyvinylpyrrolidone by rat yolk sac cultured in vitro

A method is described for the in vitro culture of 17.5-day rat visceral yolk sac. Tissue survival was good as judged by light and electron microscopy. The rate of pinocytic uptake of 125I-labeled polyvinylpyrrolidone by the tissue was constant both within and between experiments. Within the concentration range 0.15-24 mug/ml, the 125I- labeled polyvinylpyrrolidone neither stimulated nor inhibited pinocytosis. The system offers many advantages in the quantitative study of the physical basis of pinocytosis.     

Pinocytosis in the rat visceral yolk sac. Effects of temperature, metabolic inhibitors and some other modifiers.

Low temperature,2,4-dinitrophenol and moniodoacetate could each completely abolish the pinocytic uptake of 125I-labelled polyvinylpyrrolidone, 125I-labelled bovine serum albumin or colloidal 198 Au by 17.5-day rat visceral yolk sac cultured in vitro. Cytochalasin B and colchicine caused a partial and dose-dependent inhibition. It is concluded that the mechanism of pinocytic uptake of these substrates is not micropinocytosis as conventionally defined. Removal of extracellular calcium or the presence of theophylline inhibited liquid-phase pinocytosis by the rat yolk sac, whereas addition of ouabain caused a biphasic response: a slight stimulation of pinosome formation at a low concentration, and an inhibitory effect at a higher concentration.