Quantitation of pinocytosis in human monocytes during in vitro maturation into macrophages

Journal oflmmunological Methods, 132 (1990) 111-117

111

Elsevier JIM 05663

Quantitation of pinocytosis in human monocytes during in vitro maturation into macrophages D i a n e E. D a v i e s a n d J o h n B. L l o y d Cellular Pharmacology Research Group, Department of Biological Sciences, University of Keele, Staffordshire ST5 5BG, U.K.

(Received25 January 1990, revisedreceived 1 May 1990, accepted2 May 1990) Methods are reported for the quantitative measurement of pinocytosis in human monocytes isolated from peripheral blood. The cells, in adherent culture in plastic wells, were exposed for periods of up to 48 h to culture medium containing 125I-labelled polyvinylpyrrolidone (50 /~g/ml) and the pinocytosis enhancer suramin (500/~g/ml). Uptake of radiolabel was linear with time and was inhibited by colchicine (100 ~tg/ml), results that are consistent with uptake of radiolabelled substrate by pinocytosis but not with superficial adsorption of radiolabel. Similar results were obtained using a a25I-labelled vinylamine-vinylpyrrolidone copolymer as radiolabelled substrate. The rates of pinocytotic uptake of 125I-labelled polyvinylpyrrolidone (in the presence of suramin) and of 125I-labelled copolymer were measured at various stages of in vitro monocyte-to-macrophage maturation. In contrast to an earlier report, we found no consistent differences in pinocytotic activity between cells at different stages of differentiation. Key words: Pinocytosis;Monocyte, human; Macrophage, human; Differentiation

Introduction

It was reported by Knight and Soutar (1982) that the rate of fluid-phase pinocytosis in cultured blood monocytes increases five-fold as the cells mature into macrophages. We regarded this as an important finding, as it would permit an investigation to be made of the accompanying metabolic events, with the consequent possibility of elucidating the proximate stimuli that maintain pinocytosis as a constitutive activity of most cells. We have previously studied the apparent stimulation of pinocytosis by modifiers such as the polycations (Pratten et al., 1978; Duncan et al., 1979), trypan blue (Roberts et al., 1980) and suramin (Pratten and Lloyd, 1983), and shown that, although the rate of substrate uptake was increased, there was no stimulation of fluid or membrane internalization: the modifiers caused the substrate to enter Correspondence to: D.E. Davies, Cellular PharmacologyResearch Group, Department of Biological Sciences, University of Keele, StaffordshireST5 5BG, U.K.

the pinocytotic vesicles in the adsorptive mode, a phenomenon that has been termed piggy-back pinocytosis. These results suggested that cells display an intrinsic rate of pinocytosis that is not easily modified by either substrates or potential agonists, and comparisons with an escalator (Steinman et al., 1983) or a paternoster-type elevator (Lloyd, 1980) were drawn. Furthermore, a survey of published data (Pratten et al., 1980) had shown that the pinocytotic activities of different cell types were not as disparate as had been supposed. In order to exploit the maturing monocyte as a model system in which to study the regulation of pinocytosis, it was first necessary to establish the validity of the original observation of Knight and Soutar (1982). We therefore developed experimental procedures for the isolation of human monocytes and for their reliable maturation into macrophages in vitro. These procedures have recently been published (Davies and Lloyd, 1989). We now report measurements of pinocytosis in these cells.

0022-1759/90/$03.50 © 1990 Elsevier SciencePublishers B.V. (Biomedical Division)

112 To our disappointment, we have been unable to confirm the earlier report (Knight and Soutar, 1982) of a dramatic increase in pinocytosis as monocytes mature. Nevertheless the methods we have developed for measuring pinocytosis quantitatively in maturing monocytes in culture are of potential value to other investigators.

Materials and methods

Materials 125I-labelled polyvinylpyrrolidone was from Amersham International and colchicine from Sigma. Suramin BP was a generous gift from ICI Pharmaceuticals, and vinylpyrrolidone-vinylamine copolymer (100:7 molar ratio) was synthesized and radiolabelled with a25I as described by Pratten et al. (1982). Monocyte isolation and culture Mononuclear cells were obtained from human peripheral blood (180 ml, defibrinated) using the scaled-up Percoll method recently reported (Davies and Lloyd, 1989). The cells were seeded (3-4 × 1 0 6 cells and 1 ml culture medium per well) into 24-well Linbro plates, and lymphocyte-free monocyte monolayers established and cultured as described (Davies and Lloyd, 1989). The culture medium employed throughout was medium 199 (Gibco Europe, Uxbridge, U.K.) containing penicillin-streptomycin (200 U/ml), fungizone (2.5 /~g/ml) and gentamycin (200 /~g/ml), supplemented with fetal calf serum and horse serum (Flow Laboratories, Hertfordshire, U.K., heat-inactivated, 10% by volume of each). Incubation was at 37 o C in an atmosphere of air : CO 2 (95 : 5). As reported in detail in our previous paper (Davies and Lloyd, 1989), a range of morphological, cytochemical and biochemical criteria were used to confirm the transition of monocytes to macrophages in culture. Cell viability was greater than 95% in the adherent cell populations throughout the culture period. Uptake experiments Cells, after various times in culture, were incubated at 37°C in culture medium (1 ml) containing either 125I-labelled polyvinylpyrrolidone

(up to 220 /~g/ml) or 125I-labelled vinylpyrrolidone-vinylamine copolymer (10 /~g/ml). In most experiments using labelled polyvinylpyrrolidone, suramin (usually 500/~g/ml) was also present. In some experiments colchicine was added with the radiolabelled substrate to give a final concentration of 100/~g/ml. All additions were achieved by removing 100/~1 of culture medium from the wells and replacing it with 100 #1 of the relevant substance dissolved in prewarmed culture medium. Radiolabelled substrates were also incubated in cell-free wells containing 1.0 ml of culture medium only, in order to determine the extent of adsorption to the well itself. At the end of each incubation with radiolabelled substrate, duplicate 200 t~l samples of the post-culture medium were removed for the determination of radioactivity. Cells were then washed gently in warm culture medium (6 × 1 ml changes) and digested in sodium hydroxide (500 /zl of 500 mM) for 1 h at 37 o C. Cell digests were assayed for protein (Lowry et al., 1951) and radioactivity. The data relating to each well were used to calculate a clearance value, i.e., the volume of culture medium whose content of radioactivity had become cell-associated, expressed per mg of cell protein. Where clearance was found to be linear with time, uptake rates were calculated and expressed as an endocytic index (Williams et al., 1975), with units of/~l/mg protein per h.

Results

Our initial experiment attempted to repeat that of Knight and Soutar (1982). Cells at 2, 5, 7, 9 and 12 days of maturation were incubated for 3 h in the presence of 125I-labelled polyvinylpyrrolidone. Uptake of radioactivity, expressed as a clearance, showed no apparent relationship with the stage of cell maturation. However, the amounts of cell-associated radioactivity, although higher in absolute value than those reported by Knight and Soutar (1982), were very close to those found in incubations in cell-free wells. Moreover, the amount of 125I-labelled polyvinylpyrrolidone (5 #Ci/well) was prohibitively expensive for an extensive series of experiments. For subsequent experiments, therefore, we increased the duration of incubation

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Fig. 1. Accumulationof radioactivity(expressed as a clearance) by day 9 cells cultured in the presence of 125I-labelled polyvinylpyrrolidone (50/~g/ml), and suramin at 0 (o), 500 (11)or 1000 ( x )/tg/ml. from 3 to 48 h, in the hope of obtaining uptake values well above those seen in cell-free wells. In order to increase still further the pinocytotic uptake of ]25I-labelled polyvinylpyrrolidone, two different approaches were tested. The first approach was to include in the incubation medium the polysulphonated trypanocide suramin. Suramin had been shown to stimulate pinocytosis of 125I-labelled polyvinylpyrrolidone in macrophages (Pratten and Lloyd, 1983); it apparently acts as a bivalent ligand between the cell surface and the substrate, thus converting a fluid-phase substrate into an adsorptive one. The second approach was to use a copolymer of vinylpyrrolidone and vinylamine, in place of the homopolymer, as the ]25I-labelled substrate. This copolymer had been shown to enter rat macrophages by adsorptive pinocytosis (Pratten et al., 1982).

Uptake of 1251-labelled polyoinylpyrrolidone in the presence of suramin Fig. 1 shows a typical experiment in which day 9 cells were incubated for 6, 24 or 48 h in the

presence of 125I-labelled polyvinylpyrrolidone (50 /~g/ml) and 0, 500 or 1000 /~g suramin/ml. Uptake of radiolabel was approximately linear and, although the higher concentration proved inhibitory, the rate of uptake was significantly stimulated by suramin at 500 /zg/ml. The extent of stimulation varied between experiments from 2- to 6-fold. The rate of uptake of 125I-labelled polyvinylpyrrolidone in these experiments was directly proportional to its concentration in the culture medium. This is indicated by the constancy of the Endocytic Index over a ten-fold range of radiolabel concentrations (Fig. 2, Table I). Table I also illustrates another regular feature of the experiments, namely that each blood donation yielded monocytes with a characteristic rate of uptake. TABLE I UPTAKE OF 125I-LABELLED POLYVINYLPYRROLIDONE BY DAY 7 CELLS The results of three experiments are shown. Each experiment used cells derived from a different blood donor. Uptake is expressed as an endocytic index (see text). Cells were cultured for periods up to 48 h in the presence of 125I-labelled polyvinylpyrrolidone and of suramin (500/~g/ml). Concentration of 125I-labelled polyvinylpyrrolidone (/~g/ml)

Endocytic index (#1 mg protein/h) Donor 1

Donor 2

Donor 3

10 50 100

0.154 0.128 0.151

0.068 0.064 0.067

0.201 0.194 0.198

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Fig. 3. Radioactivity accumulated by cells at different stages of maturation, after incubation for 24 h i n t h e presence of 125I-labelled polyvinylpyrrolidone (50/~g/ml) and suramin (500/~g/ml). Each graph represents the uptake by cells derived from a single donor.

Colchicine is probably the most specific known inhibitor of fluid-phase pinocytosis. At 100/xg/ml it significantly inhibits the pinocytotic uptake of XESI-labelled polyvinylpyrrolidone by rat peritoneal macrophages (Pratten and Lloyd, 1979). Table II shows that colchicine inhibits pinocytosis by about 50% in both day 0 and day 7 human monocytes. Again the wide variation in absolute values between cells from different blood donations is apparent. A further confirmation that the radiolabel accumulated by the cells was truly internalized and not merely adherent was provided by experiments in which cells were exposed to 125I-labelled polyvinylpyrrolidone (50 /~g/ml) and suramin (500 /~g/ml) for 24 h and then washed and re-incubated for a further hour in the presence of a

TABLE II E F F E C T OF C O L C H I C I N E ON U P T A K E LABELLED P O L Y V I N Y L P Y R R O L I D O N E

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1251-

The results of six experiments are shown. Each experiment used cells derived from a different donor. Uptake is expressed as an endocytic index (see text) a n d t h e value measured i n t h e presence of colchicine expressed as a percentage of t h a t in its absence. Cells were cultured for periods up to 24 h i n t h e presence of 125I-labelled polyvinylpyrrolidone (50 ~ g / m l ) a n d of suramin (500/.tg/ml). Concentration

Endocytic

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0.215

0.247

0.168

0.515

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0.126 (58%)

0.126 (51%)

0.075 (45%)

0.272 (53%)

0.011 (19%)

115 high concentration of unlabelled polyvinylpyrrolidone (500 /~g/ml) and also suramin (500 /~g/ml). There was little or no loss of radioactivity in the re-incubation period (results not shown). In order to measure the pinocytotic capacity of monocytes at different stages of maturation, cells were incubated for 24 h in the presence of 125Ilabelled polyvinylpyrrolidone (50 /xg/ml) and suramin (500 /~g/ml). A single time-point was deemed to be adequate, since the previous experiments had shown that uptake was consistently linear with time up to 48 h. This approach also permitted measurements to be m~de on the same batch of cells at several points in their maturation. Six experiments were performed, using cells from a diffe:'ent donor on each occasion. Because of the donor-dependent variations observed, the results of all six experiments are presented in full (Fig. 3). Although the experiments showed changes in clearance rate as the cells matured, there was no consistent pattern. Certainly the five-fold increase between day 2 and day 5 reported by Knight and Soutar (1982) could not be substantiated.

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Fig. 4. Accumulation of radioactivity (expressed as a clearance) by day 0 cells cultured in the presence of 1251-labelled vinylamine-vinylpyrrolidonecopolymer (10 #g/ml).

Uptake of t e51-1abelled vinylpyrrolidone-vinylamine copolymer Fig. 4 shows a typical experiment in which day 0 cells were incubated for periods of up to 24 h in the presence of a25I-labelled copolymer. Uptake of radiolabel was progressive and linear with time. Similar results were given by day 7 cells. Table III shows the outcome of three experiments on day 0 cells and three on day 7 cells. The effects of colchicine (100 /~g/ml) are also shown. Interdonation variation was again evident, but colchicine always inhibited uptake. When washed cells (day 0 or day 7), previously exposed to the radiolabelled copolymer (10 # g / m l , 24 h), were re-incubated with a ten-fold excess of the radiolabelled polymer, there was very little release of the (previously) captured copolymer into the medium (results not shown). This suggests that the copolymer was being internalized by the cells. As in the corresponding experiments with 1251labelled polyvinylpyrrolidone in the presence of suramin, pinocytotic uptake was measured at several different stages of maturation. Again a single sampling after 24 h was performed and the

results for three donors are shown in Fig. 5. It is apparent that, apart from the day 0 cells generally having the highest clearance values, there was no

TABLE III EFFECT OF COLCHICINE ON UPTAKE OF 1251LABELLED VINYLAMINE-VINYLPYRROLIDONE COPOLYMER The results of six experiments are shown. Each experiment used cells derived from a different donor. Uptake is expressed as an endocytic index (see text), and the value measured in the presence of colchicine expressed as a percentage of that in its absence. Cells were cultured for periods up to 24 h in the presence of 125I-labelledcopolymer (10 #g/ml). Concentration Endocytic index (#1 per mg protein/h) of colchicine Day 0 cells Day 7 cells O,g/ml) 0 100

3.87

3.42

6 . 5 0 2.36 2.64 2.09

2.94 2.45 2.82 1.44 1.73 0.73 (75%) (72%) (43%) (61%) (65%) (35%)

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Fig. 5. Radioactivity accumulated by cells at different stages of maturation, after incubation for 24 h in the presence of 125I-labelled vinylamine-vinylpyrrolidone copolymer (10/~g/ml). Each graph represents the uptake by cells derived from a single donor.

obvious correlation between clearance and stage of cell maturation.

Discussion

In this paper we report methods for the quantitative measurement of pinocytosis by human monocytes. We are unaware of any previous work on this topic, other than the experiments reported briefly by Knight and Soutar (1982). ]zSI-labelled polyvinylpyrrolidone has been used in many cell types to measure fluid-phase pinocytosis. Like [14C]sucrose and [3H]dextran, two other widely used markers, it does not bind to cell-surface receptors, so that its rate of uptake is a direct measure of basal non-adsorptive pinocytotic accumulation. We found it impossible to measure any progressive accumulation of 125Ilabelled polyvinylpyrrolidone by monocytes in culture when reasonable cell numbers and acceptable amounts of isotope were used. Using the experimental conditions described by Knight and Soutar (1982), we obtained uptake values that did not differ appreciably from background values, and we are unable to explain their results. We have, however, been able to obtain reliable measurements of pinocytotic uptake in'monocytes, by increasing the duration of exposure of the cells to radiotracer, and by measuring non-specific adsorptive pinocytosis, rather than fluid-phase pinocytosis. Our experiments included three further features that we consider to be of great im-

portance. First, we have not simply assumed that the monocytes maintained in culture under appropriate conditions would mature into macrophages, but have confirmed this positively by monitoring a range of differentiation markers, as delineated by Davies and Lloyd (1989). Secondly, by demonstrating that colchicine (100/~g/ml) inhibited the accumulation of radiotracer, it can be confidently asserted that the accumulation seen represents uptake by pinocytosis and is not merely non-specific binding to cells. Thirdly, we have demonstrated that uptake is approximately linear with time: without this information it is impossible to interpret the results of single-time-point measurements in terms of the rates of pinocytotic capture. The values we have obtained for the pinocytotic uptake of 125I-labeUed polyvinylpyrrolidone in the presence of suramin (500 /~g/ml) can be compared with those reported by Pratten and Lloyd (1983), using rat peritoneal macrophages. The latter values were expressed per 106 cells but, when normalized to cell protein, are of the same order of magnitude as our values for human monocytes. Likewise our values for the uptake of vinylpyrrolidone-vinylamine copolymer are quantitatively very similar to those reported by Pratten et al. (1982) for uptake by rat peritoneal macrophages. Knight and Soutar (1982) measured the pinocytotic uptake of azsI-labelled polyvinylpyrrolidone at 2, 5, 7, 9, 12 and 15 days of monocyte maturation. Cells at all stages except day 2 showed

117

the same level of uptake. Day 2 cells, however, showed a value approximately one-fifth that of more mature cells. Our results do not substantiate this finding, and we conclude that the pinocytotic activity of monocytes does not change during their differentiation in vitro into macrophages.

Acknowledgement Dr. D.E. Davies thanks the Science and Engineering Research Council for a Research Studentship.

References Davies, D.E. and Lloyd, J.B. (1989) Monocyte-to-macrophage transition in vitro, a systematic study using human cells isolated by fractionation on Percoll. J. Immunol. Methods 118, 9. Duncan, R., Pratten, M.K. and Lloyd, J.B. (1979) Mechanism of polycation stimulation of pinocytosis. Biochim. Biophys. Acta 587, 463. Knight, B.L. and Soutar, A.K. (1982) Changes in the metabolism of modified and unmodified low-density lipoproteins during the maturation of cultured blood monocyte-macrophages from normal and homozygous familial hypercholesterolaemic subjects. Biochem. J. 125, 407. Lloyd, J.B. (1980) Insights into mechanisms of intraceUular protein turnover from studies on pinocytosis. In: Protein Degradation in Health and Disease. Ciba Foundation Symposium 75, p. 151.

Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265. Pratten, M.K. and Lloyd, J.B. (1979) Effects of temperature, metabolic inhibitors and some other factors on fluid-phase and adsorptive pinocytosis by rat peritoneal macrophages. Biochem. J. 180, 567. Pratten, M.K. and Lloyd, J.B. (1983) Effect of suramin on pinocytosis by resident rat peritoneal macrophages: an analysis using four different substrates. Chem.-Biol. Interactions 47, 79. Pratten, M.K., Duncan, R. and Lloyd, J.B. (1978) A comparative study of the effects of polyamino acids and dextran derivatives on pinocytosis in the rat yolk sac and the rat peritoneal macrophage. Biochim. Biophys. Acta 540, 455. Pratten, M.K., Duncan, R. and Lloyd, J.B. (1980) Adsorptive and passive pinocytic uptake. In: C.D. Ockleford and A. Whyte (Eds.), Coated Vesicles. Cambridge University Press, Cambridge, p. 179. Pratten, M.K., Cable, H.C., Ringsdorf, H. and Lloyd, J.B. (1982) Adsorptive pinocytosis of polycationic copolymers of vinylpyrrolidone with vinylamine by rat yolk sac and rat peritoneal macrophage. Biochim. Biophys. Acta 719, 424. Roberts, G., Williams, K.E. and Lloyd, J.B. (1980) Mechanism of stimulation of pinocytosis by trypan blue. Chem.-Biol. Interactions 32, 305. Steinman, R.M., Mellman, I.S., Muller, W.A. and Cohn, Z.A. (1983) Endocytosis and the recycling of plasma membrane. J. Cell Biol. 96, 1. Williams, K.E., Kidston, E.M., Beck, F. and Lloyd, J.B. (1975) Quantitative studies on pinocytosis. I. Kinetics of uptake of [125I]polyvinylpyrrolidone by rat yolk sac cultured in vitro. J. Cell Biol. 64, 113.