Phagocytosis by Rabbit Polymorphonuclear Leukocytes: The Effect of Albumin and Polyamino Acids on Latex Uptake FRANS A. DEIERKAUF, HARM BEUKERS, MARTHA DEIERKAUF AND JELLE C. RIEMERSMA Laboratoriurn voor Medische Chemie, Sylvius Laboratoria, Uniuersity of Leiden, Wassenaarseweg 72, Leiden, The Netherlands
ABSTRACT
Albumin in low concentrations (0.001-0.01 weight percent) was found to be a n effective inhibitor of phagocytosis of polystyrene latex beads by rabbit polymorphonuclear leukocytes. Polyglutamic acid proved to be a n inhibitor of latex uptake at even lower concentrations. Polylysine stimulates phagocytosis, maximal stimulation occurring a t 0.002%polylysine. These findings are discussed with reference to the surface properties of latex particles and leukocytes, and particularly with reference to electrostatic interactions in phagocytosis. Polymorphonuclear leukocytes take up var- Stossel, '74; Griffin et al., '75; Jacques, '75). ious kinds of particles by phagocytosis. Quan- This study considers the effects of some mactitative studies have shown that uptake can romolecular inducers and suppressors of be stimulated or inhibited by changing the phagocytosis which lend themselves to quanparticle or the leukocyte cell surface. We used titative study. The quantity of polystyrene polymorphonuclear cells obtained from rabbit latex taken up by the cells was measured exudates to study the effects of various macro- spectrophotometrically after extraction with molecular substances added to the medium. dioxane (Roberts and Quastel, '63; Kvarstein, Polystyrene latex spherules were chosen as '69). phagocytosible particles because they can be The importance of electric surface charge in obtained in well-defined sizes and with repro- phagocytosis was emphasized earlier by ducible surface characteristics. Nagura et al. ('731, who found a stimulatory Phagocytosis can be viewed as a continuous effect of certain polycations, such a s protprocess following contact and recognition of a amine sulfate. A negatively charged macroparticle by the cell. A number of steps can be molecule such as chondroitin sulfate had no conceptualized within this process. A parti- effect. Using polymorphonuclear leukocytes cle's engulfment begins after i t is attached to we have extended these observations to a few the cell's surface, and results in its being sur- other polyanions and polycations. In contrast rounded by cell membrane in a so-called t o Nagura's observations we found a strongly phagosome. A complicated enzymatic mecha- inhibitory effect of negatively charged molenism subsequently brings about the particle's cules such a s albumin and polyglutamic acid. ingestion and disposal. While older studies of Polylysine stimulated phagocytosis. The data phagocytosis tended to view the process in indicate that cell surface charge is a n imporphysico-chemical terms (Mudd et al., '34; Wil- tant parameter in phagocytic activity. kins, '67), energy requirements have recently MATERIALS AND METHODS come under close scrutiny, and attention has Polystyrene latex with a particle diameter been given to various biochemical aspects (Cohn and Morse, '60; Karnovsky, '62). Even of 0.481 p was obtained from Dow Chemical in this newer perspective, however, attention Company. To remove emulsifier and other must be given to the surface interactions oc- soluble substances, portions of 10 ml 1%latex curring between particles and cell membranes were dialyzed over 48 hours at 0°C against 4 in the initial steps of attachment and engulf- x 2 liters of water (Hul and Vanderhoff, '68). ment (Rabinovitch, '67, '70; Stossel et al., '72; Received Nov. 1, '76. Accepted Feb. 10, '77. J. CELL. PHYSIOL., 92: 169-176.
169
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F. DEIERKAUF, H. BEUKERS, M. DEIERKAUF AND J. RIEMERSMA
Crystalline human serum albumin (Fluka), bovine serum albumin (Sigma), polylysine hydrobromide, M = 70,000 (L and D, Sigma) and polyglutamic acid, M = 98,000 and 74,000 (respectively L and D, Sigma) were obtained in the highest purity commercially available. Peritoneal exudates were obtained from CH-rabbits weighing 3 kg (Centraal Proefdieren Bedrijf, Zeist, The Netherlands). Individual animals were used a t two-week intervals, and each time 200 ml sterile isotonic saline (38°C) was injected intraperitoneally; this solution contained 1.5 mg glycogen per ml. The cells were collected after four hours by washing with a solution containing 4 g sodium citrate dihydrate and 9 g NaCl per liter (Hirsch, '56). The solution was introduced in two portions of 150 ml. To remove contaminating debris the exudate was passed through surgical gauze and collected in a 500 ml polypropylene container. The yield was 300-350 ml cell suspension. The suspension was centrifuged for ten minutes a t room temperature, a t 165 g, and the sediment resuspended in 50 ml proteinfree Hanks' solution (Martin and Green, '58). Centrifugation was repeated after a cell count and a suspension was prepared containing 0.4 x lo7 cells per ml. The cells were used immediately. Phagocytosis experiments were carried out in Hanks' medium in such a way that 2.5 mi stock suspension (lo7cells) were mixed with latex suspension and other additions to a final standard volume of 5 ml; the temperature was 37°C. Phagocytosis was stopped a t the required time by adding 4 ml 40 mM EDTA (Kvarstein, '69) and cooling on ice. After centrifugation a t low speed (1,000 rpm for 10 minutes) excess latex was removed with the supernatant. Adherent latex particles were washed from the sedimented cells by resuspension in 40 ml Hanks' solution containing 4 mM EDTA and centrifugation a t 4°C and 1,000 rpm. This washing procedure was repeated, with a final centrifugation a t 1,250 rpm. The sedimented cells were extracted with 5 ml dioxane overnight a t 23-23"C, A spectroscopic grade of dioxane has to be used, for instance Merk Uvasol stabilized with 2.5 ppm BHT. The tube contents were mixed and then centrifuged at 5,000 rpm for 15 minutes. Polystyrene was determined in the supernatant by measuring the extinction a t 255 nm (Roberts and Quastel, '63).
RESULTS AND DISCUSSION
On the basis of differential counts and of electron microscopy, it was found that the suspensions obtained by the exudate method contained practically no other cell types except polymorphonuclear leukocytes. The yield usually obtained was 4-6 X 10*cells per animal. Under the standard conditions described in the MATERIALS AND METHODS section, the quantity of latex taken up after 12 minutes was found to be proportional to the latex quantity added (fig. 1). Electron microscopic examination reveals that the latex particles are engulfed not in clusters, but one by one. Particles are found embedded in a quantity of fluid, but we also observed a number which had membranes closely adhering to the particle surface (fig. 2). The fluid may correspond to entrapped extracellular medium or to the contents of emptied intracellular granules. Under our experimental conditions latex adhering externally after incubation was evidently washed away completely. The measurements of latex uptake t o be discussed refer to particles completely engulfed. It should be mentioned here that latex phagocytosis by amoebae occurs without uptake of extracellular medium, while latex particles in this case are taken up in clusters (Korn and Weisman, '67; Weisman and Korn, '67). The curve representing latex uptake with time shows a steep rise during the first 30 seconds after latex addition. Then the uptake increases a t a slower rate (fig. 4: control). Earlier authors have given time curves for latex uptake in which the rapid phase lasts as long as 20 minutes (Kvarstein, '69). The speed of uptake probably depends on whether glycogen-stimulated cells are used, as in the present study, or unstimulated cells derived from whole blood (Kvarstein, '69; Boyum, '68). With exudate cells obtained after stimulation with caseinate-saline a plateau of uptake was reached after ten minutes (Roberts and Quastel, '63). The usual buffer for phagocytosis experiments was Hanks' solution; a Krebs-Ringer solution gave similar results, indicating that the ionic composition of the buffer solution may be varied within certain limits. However, phagocytosis was affected by the presence of various organic macromolecules in the medium. Human or bovine serum albumin added in varying concentrations t o the Hanks' me-
PHAGOCYTOSIS BY POLYMORPHONUCLEAR LEUKOCYTES
171
latex uptake ,ug / l o 7 leukocytes
1000
I
800 600
100 200
100 200
300
LOO 500
600 700 800 p L 1 YOlatex
Fig. 1 Total uptake of polystyrene latex spherules (0.481 diameter) by rabbit polymorphonuclear leukocytes. Various quantities of PSL were added to lo7 PMN cells. Medium: Hanks' solution, final volume 5 ml, temperature 37OC, incubation time 12 minutes. Each point represents the average of a t least two determinations.
Fig. 2 Detail of a polymorphonuclear leukocyte after phagocytosis of latex particles for three minutes. Besides latex particles (arrowheads) azurophil granules (arrows) and specific granules (barred arrows) are present in the cytoplasm. N, nucleus. Glutaraldehyde fixation, osmium tetroxide postfixation. Uranyl-lead staining. X 17,000.
dium gave a clear-cut concentration-dependent reduction of latex uptake (fig. 3). Purification of the commercial albumin preparations by charcoal adsorption treatment (Chen, '67) or by dialysis gave the same
results. In a concentration of 0.1% albumin (w/v) uptake was reduced to zero. Electron microscopic examination showed a complete absence of latex spherules inside the leukocytes. The effect on the time curves for latex
172
F. DEIERKAUF. H. BEUKERS, M. DEIERKAUF AND J . RIEMERSMA
YO of latex uptake in control
100
i
0 = human albumin A = bovine albumin
a0 60 LO
20
log % albumin conc Fig. 3 Influence of albumin (HSA and BSA) on latex uptake by PMN leukocytes after 12 minutes; conditions as described in legend to figure 1.
latex uptoke ,ug / l o 7 Leukocytes
200
8o 40
1
I Y
1 I
10
I
30
I
50
I
70
I
I
90
1
120
180 seconds o A
control 0,001% albumin 0.003% albumin
Fig. 4 Time curve of latex uptake (PSL particles, 0.481 X diameter) by PMN leukocytes and the influence of two albumin (BSA) concentrations. Conditions as described in legend to figure 1. All values t 8 fig.
uptake of 0.001% and 0.003% bovine serum albumin respectively is shown in figure 4. It should be noted that after initial rapid phagocytosis a plateau is reached in the presence of albumin. The initial speed of latex uptake is the same, but the total capacity is much lower. Added albumin may thus affect membrane ingestion rather than particle attachment. Little is known about the interaction of albumin with leukocyte membranes and the membranes of other mammalian cells. Transport of albumin into mammalian cells has been demonstrated (Ryser, '701, but so far no evidence has been presented that this process interferes with phagocytosis. In the case of
phagocytosis of starch granules by polymorphonuclear leukocytes, the data suggest that fluid uptake is occurring which indirectly causes measurable albumin uptake (Chang, '69). I t appears unlikely that albumin molecules and latex particles would compete for uptake sites in a way resembling the competition that has been found between denatured acetylated albumin aggregates on one side and colloidal carbon particles on the other (Normann, '74). Albumin is bound by latex particles, and the characteristics of binding have been thoroughly investigated (Oss and Singer, '66). A remarkable difference was found between
PHAGOCYTOSIS BY POLYMORPHONUCLEAR LEUKOCYTES
173
A poly-D- lysine
YO of latex uptake in control
A poly-L- lysine o albumin. o poly-D-glutamic acid rn poly-L - glutarnic acid
300
20 0
100
\EJ
-L
I
-3
T------.
-2
log
YO conc
Fig. 5 Influence of polyamino acids (L and D) on uptake of PSL particles, by PMN leukocytes, as compared poly-L-lysine (M = 70,000),poly-D-glutamic to influence of BSA. Added substances poly-D-lysine (M = 70,000), acid (M = 74,000) and poly-L-glutamic acid (M = 98,000).Experimental conditions as described in legend to figure 1. All values for polylysine f 5 and for albumin and polyglutamic acid & 8.
bovine and human albumin: per cm2 of latex concentrations both surfaces will be positively surface much less bovine albumin was bound charged and repulsion would increase. The hythan of human albumin. Van der Waals-Lon- pothesis t h a t electrostatic interactions bedon and electrostatic interactions both are tween cell and particle play a n important role in phagocytosis is compatible with the typicalimportant in the binding reaction. To assess the effect of charge interactions, ly biphasic curve for the polylysine effect. phagocytosis was studied in the presence of Cationic polymers have been shown to be polyglutamic acid and polylysine respectively. bound by latex particles, thereby altering These substances affected phagocytosis in an their surface charge (Gregory, '76). The inopposite sense. Polylysine causes increased creased latex uptake occurring in the presence latex uptake, while polyglutamic acid inhib- of small concentrations of polylysine is probaited uptake, even more strongly than albumin bly due to a facilitated interaction between (fig. 5 ) . Both the D- and the L-forms of these cell membrane and particle. High concentrapolymers were employed; no explanation can tions of polylysine would result in positively as yet be given as to why there is a difference charged surfaces of both cell and particle, and electrostatic repulsion could make phagocybetween the effects of these optical isomers. Polymorphonuclear leukocytes have a t tosis more difficult. This may explain the detheir external surface negative charges be- crease of uptake beyond 0.002%polylysine (fig. longing t o membrane-bound neuramic-acid 5 ) . In addition, higher polylysine concentraand to the carboxyl groups of membrane pro- tions could result in the formation of polymer teins (Vassar and Kendall, '68; Seaman et al., bridges between cells, cell clumping and cell '69). Polystyrene latex spherules contain sur- lysis (Katchalsky, '59). The extent to which the macromolecules face charges due t o sulfate groups originating from the method of preparation and persisting influencing latex uptake are bound by the in part even after prolonged dialysis (Hul and latex particle's surface and by the cell surface requires further study. Polystyrene latex parVanderhoff, '68). Polylysine reduces the negative charge den- ticles were treated with gelatine derivatives, sity a t either surface and thus diminishes both anionic and cationic, in such a way that electrostatic repulsion. At high polylysine their surface properties were altered; clear-
174
F. DEIERKAUF, H. BEUKERS, M. DEIERKAUF AND J. RIEMERSMA
cut effects on phagocytosis were observed in in vivo experiments (Wilkins, '67). Under certain conditions polylysine has been shown to induce in vitro phagocytosis (De Vries e t al., '55; Buchanan-Davidson e t al., '60). A naturally occurring basic tetrapeptide, tuftsin, has been shown to stimulate phagocytosis and to require membrane sialic acid for its action (Constantopoulos and Najjar, '73). Certain polyanionic macromolecules, on the other hand, suppress phagocytosis. Bacillus anthrax interferes with the host defense mechanism by means of its capsular poly-D-glutamic acid which inhibits uptake by host phagocytes (Zwartouw and Smith, '56). A modulation of the phagocytosis potential by various molecules appears, therefore, to be physiologically significant. Apart from its negatively charged groups the latex particle surface contains relatively large hydrophobic regions (Oss and Singer, '66; Oss and Gilman, '72). The particle-medium interface is likely to accumulate various substances which reduce interfacial tension. The particle surface would thereby in effect become more hydrophilic. A lowered interfacial tension between particle and medium may suppress phagocytosis (Mudd et al., '34). The interfacial tension between phagocyte and medium is low to begin with and is probably less sensitive to macromolecular additions. If we assume t h a t albumin is bound to the latex surface by hydrophobic interactions, this surface would become more hydrophilic. However, a t the same time, albumin binding increases the negative surface charge. Given the observed similar effects of polyglutamic acid and albumin, and the opposite effects of polycations, the most likely interpretation of the albumin effect is t h a t electrostatic repulsion between cell and particle is increased, thereby inhibiting one of the early steps of cell-particle interaction. Treatment of r a t macrophages with cationic polyelectrolytes has been shown to result in enhanced phagocytosis (Nagura e t al., '73). The effect of various proteins on granulocyte surface charges was studied by Gallin et al. ('75), who suggested t h a t granulocyte chemotaxis is modulated by substances which decrease or increase surface charge. Our data show clearly opposite effects of negatively and positively charged polyelectrolytes on phagocytosis by granulocytes. Further work is needed to assess the importance of cell surface
charge on phagocytic activity under in vivo conditions. ACKNOWLEDGMENTS
We wish to thank Mr. S.Broers, Laboratory of Pharmacology, University of Leiden, for his help in obtaining rabbit exudates, and Doctor P. Brederoo, Laboratory for Electron Microscopy, University of Leiden, for t h e electron micrograph reproduced a s figure 2. LITERATURE CITED Boyum, A. 1968 Separation of leucocytes from blood and bone marrow. Scand. J. Clin. Lab. Invest., (Suppl.), 97. Bucbanan-Davidson, D. J., C. V. Seastone and M. A. Stahman 1960 Action of synthetic polylysine on the growth and phagocytosis of bacteria in vitro. J. Bacteriol., 80: 590-594. Chang, Y.-H. 1969 Studies of phagocytosis, I Uptake of human serum albumin as a measure of the degree of phagocytosis in vivo. Exp. Cell Res., 54: 42-48. Chen, R. F. 1967 Removal of fatty acids from serum albumin by charcoal treatment. J. Biol. Chem., 242: 173181. Cohn, 2. A.: and S.I. Morse 1960 Functional and metabolic properties of polymorphonuclear leucocytes. J. Exp. Med., 111; 667-687. Constantopoulos, A,, and V. Najjar 1973 The requirement for membrane sialic acid in the stimulation of phagocytosis by the natural tetrapeptide, tuftsin. J. Biol. Chem., 248: 3819-3822. De Vries, A,, J. Salgo, Y. Matotb, A. Nevo and E. Katchalski 1955 Effect of basic polyamino acids on phagocytosis in vitro. Arch. Int. Pharmacodyn., 104: 1-10, Gallin, J. I., J. R. Durocher and A. P. Kaplan 1975 Interaction of leukocyte chemotactic factors with the cell surface, I. Chemotactic factor induced changes in human granulocyte surface charge. J. Clin. Invest., 55: 967-974. Gregory, J. 1967 The effect of cationic polymers on the colloidal stability of latex particles. J. Coll. Interf. Sci., 25: 35-44. Griffin, F. M., Jr., J. A. Griffin, J. E. Leider and S.C. Silverstein 1975 Studies on the mechanism of phagocytosis. J. Exp. Med., 142: 1263-1282. Hirsch, J . G. 1956 Phagocytin: A bactericidical substance from PMN leucocytes. J. Exp. Med., 103: 589611. Hul, H. J. van den, and J. W. Vanderhoff 1968 Well characterized monodisperse latexes. J. Coll. Interf. Sci., 28: 336337. Jacques, P. L. 1975 The endocytic uptake of macromolecules. In: Pathobiology of cell membranes. B. F. Trump and A. U. Arstila, eds. Academic Press, pp. 255-279. Karnovsky, M. L. 1962 Metabolic basis of phagocytic activity. Physiol. Rev., 42: 143-168. Katchalsky, A. 1959 Interaction of basic polyelectrolytes with the red blood cell. Biochim. Biophys. Acta, 33: 120.128. Korn, E. D., and R. A. Weisman 1967 Phagocytosis of latex beads by Acanthamoeba. J. Cell. Biol., 34: 219-222. Kvarstein, B. 1969 The effect of temperature, metabolic inhibitors and EDTA on phagocytosis of PSL by human leucocytes. Scand. J. Clin. Lab. Invest., 24; 271-277. Martin, S. P., and R. Green 1958 Methods for the study of surviving leucocytes. Meth. Med. Res., 7: 136-141. Mudd, S., M. McCutcheon and B. Lucke 1934 Phagocytosis. Physiol. Rev., 14: 210-275.
PHAGOCYTOSIS BY POLYMORPHONUCLEAR LEUKOCYTES Nagura, H., J. Asai, Y. Katsumata and K. Kojima 1973 Role of electric surface charge of cell membrane in phagocytosis. Acta. Path. Jap., 23: 279-290. Normann, S. J. 1974 Kinetics of phagocytosis. Lab. Invest., 31: 161-169. Oss, C. J., and C. G. Gillman 1972 Phagocytosis as a surface phenomenon. J. Reticuloendothel. SOC.,12: 283-292. Oss, C. J. van, and J. M. Singer 1966 The binding of immune globulins and other proteins by polystyrene latex particles. J. Reticuloendothel. SOC.,3: 29-40. Rabinovitch, M. 1967 The dissociation of the attachment and ingestion phases of phagocytosis by macrophages. Exp. Cell. Res., 46: 19-28. 1970 Phagocytic recognition. In: Mononuclear phagocytes. R. van Furth, ed. Blackwell, Oxford, pp. 299315. Roberts, J., and J. H. Quastel 1963 Particle uptake by PMN leucocytes and Ehrlich Ascites-carcinoma cells. Biochem. J., 89: 150-156. Ryser, H. J. P. 1970 Transport of macromolecules, esp. pro-
175
teins into mammalian cells. Proc. Fourth Inter. Cong. Pharmacol. Basel 1969, 3: 96.132. Seaman, G. V. F., P. S.Vassar and M. J . Kendall 1969 Electrophoretic studies on human PMN leucocytes and erythrocytes. Arch. Biochem. Biophys., 135: 356-362. Stossel, T. P. 1974 Phagocytosis 1-111.New Eng. J . Med., 290: 717-723, 774-780, 833.839. Stossel, T. P., R. J. Mason, J. Hartwig and M. Vaughan 1972 Quantitative studies of phagocytosis by polymorphonuclear leucocytes. J. Clin. Invest., 51: 615-624. Vassar, P. S., and M. J. Kendall 1969 Electrophoresis of human leucocytes. Arch. Biochem. Biophys., 135: 350355. Weisman, R. A,, and E. D. Korn 1967 Phagocytosis of latex beads by Acanthamoeba. Biochem., 6: 485-497. Wilkins, D. J. 1967 The biological recognition of foreign from native particles as a problem in surface chemistry. J. Coll. Interf. Sci., 25: 84-89. Zwartouw, H. T., and H. Smith 1956 Polyglutamic acid from Bacillus anthracis. Biochem. J., 63: 437-442.
ABSTRACT
Albumin in low concentrations (0.001-0.01 weight percent) was found to be a n effective inhibitor of phagocytosis of polystyrene latex beads by rabbit polymorphonuclear leukocytes. Polyglutamic acid proved to be a n inhibitor of latex uptake at even lower concentrations. Polylysine stimulates phagocytosis, maximal stimulation occurring a t 0.002%polylysine. These findings are discussed with reference to the surface properties of latex particles and leukocytes, and particularly with reference to electrostatic interactions in phagocytosis. Polymorphonuclear leukocytes take up var- Stossel, '74; Griffin et al., '75; Jacques, '75). ious kinds of particles by phagocytosis. Quan- This study considers the effects of some mactitative studies have shown that uptake can romolecular inducers and suppressors of be stimulated or inhibited by changing the phagocytosis which lend themselves to quanparticle or the leukocyte cell surface. We used titative study. The quantity of polystyrene polymorphonuclear cells obtained from rabbit latex taken up by the cells was measured exudates to study the effects of various macro- spectrophotometrically after extraction with molecular substances added to the medium. dioxane (Roberts and Quastel, '63; Kvarstein, Polystyrene latex spherules were chosen as '69). phagocytosible particles because they can be The importance of electric surface charge in obtained in well-defined sizes and with repro- phagocytosis was emphasized earlier by ducible surface characteristics. Nagura et al. ('731, who found a stimulatory Phagocytosis can be viewed as a continuous effect of certain polycations, such a s protprocess following contact and recognition of a amine sulfate. A negatively charged macroparticle by the cell. A number of steps can be molecule such as chondroitin sulfate had no conceptualized within this process. A parti- effect. Using polymorphonuclear leukocytes cle's engulfment begins after i t is attached to we have extended these observations to a few the cell's surface, and results in its being sur- other polyanions and polycations. In contrast rounded by cell membrane in a so-called t o Nagura's observations we found a strongly phagosome. A complicated enzymatic mecha- inhibitory effect of negatively charged molenism subsequently brings about the particle's cules such a s albumin and polyglutamic acid. ingestion and disposal. While older studies of Polylysine stimulated phagocytosis. The data phagocytosis tended to view the process in indicate that cell surface charge is a n imporphysico-chemical terms (Mudd et al., '34; Wil- tant parameter in phagocytic activity. kins, '67), energy requirements have recently MATERIALS AND METHODS come under close scrutiny, and attention has Polystyrene latex with a particle diameter been given to various biochemical aspects (Cohn and Morse, '60; Karnovsky, '62). Even of 0.481 p was obtained from Dow Chemical in this newer perspective, however, attention Company. To remove emulsifier and other must be given to the surface interactions oc- soluble substances, portions of 10 ml 1%latex curring between particles and cell membranes were dialyzed over 48 hours at 0°C against 4 in the initial steps of attachment and engulf- x 2 liters of water (Hul and Vanderhoff, '68). ment (Rabinovitch, '67, '70; Stossel et al., '72; Received Nov. 1, '76. Accepted Feb. 10, '77. J. CELL. PHYSIOL., 92: 169-176.
169
170
F. DEIERKAUF, H. BEUKERS, M. DEIERKAUF AND J. RIEMERSMA
Crystalline human serum albumin (Fluka), bovine serum albumin (Sigma), polylysine hydrobromide, M = 70,000 (L and D, Sigma) and polyglutamic acid, M = 98,000 and 74,000 (respectively L and D, Sigma) were obtained in the highest purity commercially available. Peritoneal exudates were obtained from CH-rabbits weighing 3 kg (Centraal Proefdieren Bedrijf, Zeist, The Netherlands). Individual animals were used a t two-week intervals, and each time 200 ml sterile isotonic saline (38°C) was injected intraperitoneally; this solution contained 1.5 mg glycogen per ml. The cells were collected after four hours by washing with a solution containing 4 g sodium citrate dihydrate and 9 g NaCl per liter (Hirsch, '56). The solution was introduced in two portions of 150 ml. To remove contaminating debris the exudate was passed through surgical gauze and collected in a 500 ml polypropylene container. The yield was 300-350 ml cell suspension. The suspension was centrifuged for ten minutes a t room temperature, a t 165 g, and the sediment resuspended in 50 ml proteinfree Hanks' solution (Martin and Green, '58). Centrifugation was repeated after a cell count and a suspension was prepared containing 0.4 x lo7 cells per ml. The cells were used immediately. Phagocytosis experiments were carried out in Hanks' medium in such a way that 2.5 mi stock suspension (lo7cells) were mixed with latex suspension and other additions to a final standard volume of 5 ml; the temperature was 37°C. Phagocytosis was stopped a t the required time by adding 4 ml 40 mM EDTA (Kvarstein, '69) and cooling on ice. After centrifugation a t low speed (1,000 rpm for 10 minutes) excess latex was removed with the supernatant. Adherent latex particles were washed from the sedimented cells by resuspension in 40 ml Hanks' solution containing 4 mM EDTA and centrifugation a t 4°C and 1,000 rpm. This washing procedure was repeated, with a final centrifugation a t 1,250 rpm. The sedimented cells were extracted with 5 ml dioxane overnight a t 23-23"C, A spectroscopic grade of dioxane has to be used, for instance Merk Uvasol stabilized with 2.5 ppm BHT. The tube contents were mixed and then centrifuged at 5,000 rpm for 15 minutes. Polystyrene was determined in the supernatant by measuring the extinction a t 255 nm (Roberts and Quastel, '63).
RESULTS AND DISCUSSION
On the basis of differential counts and of electron microscopy, it was found that the suspensions obtained by the exudate method contained practically no other cell types except polymorphonuclear leukocytes. The yield usually obtained was 4-6 X 10*cells per animal. Under the standard conditions described in the MATERIALS AND METHODS section, the quantity of latex taken up after 12 minutes was found to be proportional to the latex quantity added (fig. 1). Electron microscopic examination reveals that the latex particles are engulfed not in clusters, but one by one. Particles are found embedded in a quantity of fluid, but we also observed a number which had membranes closely adhering to the particle surface (fig. 2). The fluid may correspond to entrapped extracellular medium or to the contents of emptied intracellular granules. Under our experimental conditions latex adhering externally after incubation was evidently washed away completely. The measurements of latex uptake t o be discussed refer to particles completely engulfed. It should be mentioned here that latex phagocytosis by amoebae occurs without uptake of extracellular medium, while latex particles in this case are taken up in clusters (Korn and Weisman, '67; Weisman and Korn, '67). The curve representing latex uptake with time shows a steep rise during the first 30 seconds after latex addition. Then the uptake increases a t a slower rate (fig. 4: control). Earlier authors have given time curves for latex uptake in which the rapid phase lasts as long as 20 minutes (Kvarstein, '69). The speed of uptake probably depends on whether glycogen-stimulated cells are used, as in the present study, or unstimulated cells derived from whole blood (Kvarstein, '69; Boyum, '68). With exudate cells obtained after stimulation with caseinate-saline a plateau of uptake was reached after ten minutes (Roberts and Quastel, '63). The usual buffer for phagocytosis experiments was Hanks' solution; a Krebs-Ringer solution gave similar results, indicating that the ionic composition of the buffer solution may be varied within certain limits. However, phagocytosis was affected by the presence of various organic macromolecules in the medium. Human or bovine serum albumin added in varying concentrations t o the Hanks' me-
PHAGOCYTOSIS BY POLYMORPHONUCLEAR LEUKOCYTES
171
latex uptake ,ug / l o 7 leukocytes
1000
I
800 600
100 200
100 200
300
LOO 500
600 700 800 p L 1 YOlatex
Fig. 1 Total uptake of polystyrene latex spherules (0.481 diameter) by rabbit polymorphonuclear leukocytes. Various quantities of PSL were added to lo7 PMN cells. Medium: Hanks' solution, final volume 5 ml, temperature 37OC, incubation time 12 minutes. Each point represents the average of a t least two determinations.
Fig. 2 Detail of a polymorphonuclear leukocyte after phagocytosis of latex particles for three minutes. Besides latex particles (arrowheads) azurophil granules (arrows) and specific granules (barred arrows) are present in the cytoplasm. N, nucleus. Glutaraldehyde fixation, osmium tetroxide postfixation. Uranyl-lead staining. X 17,000.
dium gave a clear-cut concentration-dependent reduction of latex uptake (fig. 3). Purification of the commercial albumin preparations by charcoal adsorption treatment (Chen, '67) or by dialysis gave the same
results. In a concentration of 0.1% albumin (w/v) uptake was reduced to zero. Electron microscopic examination showed a complete absence of latex spherules inside the leukocytes. The effect on the time curves for latex
172
F. DEIERKAUF. H. BEUKERS, M. DEIERKAUF AND J . RIEMERSMA
YO of latex uptake in control
100
i
0 = human albumin A = bovine albumin
a0 60 LO
20
log % albumin conc Fig. 3 Influence of albumin (HSA and BSA) on latex uptake by PMN leukocytes after 12 minutes; conditions as described in legend to figure 1.
latex uptoke ,ug / l o 7 Leukocytes
200
8o 40
1
I Y
1 I
10
I
30
I
50
I
70
I
I
90
1
120
180 seconds o A
control 0,001% albumin 0.003% albumin
Fig. 4 Time curve of latex uptake (PSL particles, 0.481 X diameter) by PMN leukocytes and the influence of two albumin (BSA) concentrations. Conditions as described in legend to figure 1. All values t 8 fig.
uptake of 0.001% and 0.003% bovine serum albumin respectively is shown in figure 4. It should be noted that after initial rapid phagocytosis a plateau is reached in the presence of albumin. The initial speed of latex uptake is the same, but the total capacity is much lower. Added albumin may thus affect membrane ingestion rather than particle attachment. Little is known about the interaction of albumin with leukocyte membranes and the membranes of other mammalian cells. Transport of albumin into mammalian cells has been demonstrated (Ryser, '701, but so far no evidence has been presented that this process interferes with phagocytosis. In the case of
phagocytosis of starch granules by polymorphonuclear leukocytes, the data suggest that fluid uptake is occurring which indirectly causes measurable albumin uptake (Chang, '69). I t appears unlikely that albumin molecules and latex particles would compete for uptake sites in a way resembling the competition that has been found between denatured acetylated albumin aggregates on one side and colloidal carbon particles on the other (Normann, '74). Albumin is bound by latex particles, and the characteristics of binding have been thoroughly investigated (Oss and Singer, '66). A remarkable difference was found between
PHAGOCYTOSIS BY POLYMORPHONUCLEAR LEUKOCYTES
173
A poly-D- lysine
YO of latex uptake in control
A poly-L- lysine o albumin. o poly-D-glutamic acid rn poly-L - glutarnic acid
300
20 0
100
\EJ
-L
I
-3
T------.
-2
log
YO conc
Fig. 5 Influence of polyamino acids (L and D) on uptake of PSL particles, by PMN leukocytes, as compared poly-L-lysine (M = 70,000),poly-D-glutamic to influence of BSA. Added substances poly-D-lysine (M = 70,000), acid (M = 74,000) and poly-L-glutamic acid (M = 98,000).Experimental conditions as described in legend to figure 1. All values for polylysine f 5 and for albumin and polyglutamic acid & 8.
bovine and human albumin: per cm2 of latex concentrations both surfaces will be positively surface much less bovine albumin was bound charged and repulsion would increase. The hythan of human albumin. Van der Waals-Lon- pothesis t h a t electrostatic interactions bedon and electrostatic interactions both are tween cell and particle play a n important role in phagocytosis is compatible with the typicalimportant in the binding reaction. To assess the effect of charge interactions, ly biphasic curve for the polylysine effect. phagocytosis was studied in the presence of Cationic polymers have been shown to be polyglutamic acid and polylysine respectively. bound by latex particles, thereby altering These substances affected phagocytosis in an their surface charge (Gregory, '76). The inopposite sense. Polylysine causes increased creased latex uptake occurring in the presence latex uptake, while polyglutamic acid inhib- of small concentrations of polylysine is probaited uptake, even more strongly than albumin bly due to a facilitated interaction between (fig. 5 ) . Both the D- and the L-forms of these cell membrane and particle. High concentrapolymers were employed; no explanation can tions of polylysine would result in positively as yet be given as to why there is a difference charged surfaces of both cell and particle, and electrostatic repulsion could make phagocybetween the effects of these optical isomers. Polymorphonuclear leukocytes have a t tosis more difficult. This may explain the detheir external surface negative charges be- crease of uptake beyond 0.002%polylysine (fig. longing t o membrane-bound neuramic-acid 5 ) . In addition, higher polylysine concentraand to the carboxyl groups of membrane pro- tions could result in the formation of polymer teins (Vassar and Kendall, '68; Seaman et al., bridges between cells, cell clumping and cell '69). Polystyrene latex spherules contain sur- lysis (Katchalsky, '59). The extent to which the macromolecules face charges due t o sulfate groups originating from the method of preparation and persisting influencing latex uptake are bound by the in part even after prolonged dialysis (Hul and latex particle's surface and by the cell surface requires further study. Polystyrene latex parVanderhoff, '68). Polylysine reduces the negative charge den- ticles were treated with gelatine derivatives, sity a t either surface and thus diminishes both anionic and cationic, in such a way that electrostatic repulsion. At high polylysine their surface properties were altered; clear-
174
F. DEIERKAUF, H. BEUKERS, M. DEIERKAUF AND J. RIEMERSMA
cut effects on phagocytosis were observed in in vivo experiments (Wilkins, '67). Under certain conditions polylysine has been shown to induce in vitro phagocytosis (De Vries e t al., '55; Buchanan-Davidson e t al., '60). A naturally occurring basic tetrapeptide, tuftsin, has been shown to stimulate phagocytosis and to require membrane sialic acid for its action (Constantopoulos and Najjar, '73). Certain polyanionic macromolecules, on the other hand, suppress phagocytosis. Bacillus anthrax interferes with the host defense mechanism by means of its capsular poly-D-glutamic acid which inhibits uptake by host phagocytes (Zwartouw and Smith, '56). A modulation of the phagocytosis potential by various molecules appears, therefore, to be physiologically significant. Apart from its negatively charged groups the latex particle surface contains relatively large hydrophobic regions (Oss and Singer, '66; Oss and Gilman, '72). The particle-medium interface is likely to accumulate various substances which reduce interfacial tension. The particle surface would thereby in effect become more hydrophilic. A lowered interfacial tension between particle and medium may suppress phagocytosis (Mudd et al., '34). The interfacial tension between phagocyte and medium is low to begin with and is probably less sensitive to macromolecular additions. If we assume t h a t albumin is bound to the latex surface by hydrophobic interactions, this surface would become more hydrophilic. However, a t the same time, albumin binding increases the negative surface charge. Given the observed similar effects of polyglutamic acid and albumin, and the opposite effects of polycations, the most likely interpretation of the albumin effect is t h a t electrostatic repulsion between cell and particle is increased, thereby inhibiting one of the early steps of cell-particle interaction. Treatment of r a t macrophages with cationic polyelectrolytes has been shown to result in enhanced phagocytosis (Nagura e t al., '73). The effect of various proteins on granulocyte surface charges was studied by Gallin et al. ('75), who suggested t h a t granulocyte chemotaxis is modulated by substances which decrease or increase surface charge. Our data show clearly opposite effects of negatively and positively charged polyelectrolytes on phagocytosis by granulocytes. Further work is needed to assess the importance of cell surface
charge on phagocytic activity under in vivo conditions. ACKNOWLEDGMENTS
We wish to thank Mr. S.Broers, Laboratory of Pharmacology, University of Leiden, for his help in obtaining rabbit exudates, and Doctor P. Brederoo, Laboratory for Electron Microscopy, University of Leiden, for t h e electron micrograph reproduced a s figure 2. LITERATURE CITED Boyum, A. 1968 Separation of leucocytes from blood and bone marrow. Scand. J. Clin. Lab. Invest., (Suppl.), 97. Bucbanan-Davidson, D. J., C. V. Seastone and M. A. Stahman 1960 Action of synthetic polylysine on the growth and phagocytosis of bacteria in vitro. J. Bacteriol., 80: 590-594. Chang, Y.-H. 1969 Studies of phagocytosis, I Uptake of human serum albumin as a measure of the degree of phagocytosis in vivo. Exp. Cell Res., 54: 42-48. Chen, R. F. 1967 Removal of fatty acids from serum albumin by charcoal treatment. J. Biol. Chem., 242: 173181. Cohn, 2. A.: and S.I. Morse 1960 Functional and metabolic properties of polymorphonuclear leucocytes. J. Exp. Med., 111; 667-687. Constantopoulos, A,, and V. Najjar 1973 The requirement for membrane sialic acid in the stimulation of phagocytosis by the natural tetrapeptide, tuftsin. J. Biol. Chem., 248: 3819-3822. De Vries, A,, J. Salgo, Y. Matotb, A. Nevo and E. Katchalski 1955 Effect of basic polyamino acids on phagocytosis in vitro. Arch. Int. Pharmacodyn., 104: 1-10, Gallin, J. I., J. R. Durocher and A. P. Kaplan 1975 Interaction of leukocyte chemotactic factors with the cell surface, I. Chemotactic factor induced changes in human granulocyte surface charge. J. Clin. Invest., 55: 967-974. Gregory, J. 1967 The effect of cationic polymers on the colloidal stability of latex particles. J. Coll. Interf. Sci., 25: 35-44. Griffin, F. M., Jr., J. A. Griffin, J. E. Leider and S.C. Silverstein 1975 Studies on the mechanism of phagocytosis. J. Exp. Med., 142: 1263-1282. Hirsch, J . G. 1956 Phagocytin: A bactericidical substance from PMN leucocytes. J. Exp. Med., 103: 589611. Hul, H. J. van den, and J. W. Vanderhoff 1968 Well characterized monodisperse latexes. J. Coll. Interf. Sci., 28: 336337. Jacques, P. L. 1975 The endocytic uptake of macromolecules. In: Pathobiology of cell membranes. B. F. Trump and A. U. Arstila, eds. Academic Press, pp. 255-279. Karnovsky, M. L. 1962 Metabolic basis of phagocytic activity. Physiol. Rev., 42: 143-168. Katchalsky, A. 1959 Interaction of basic polyelectrolytes with the red blood cell. Biochim. Biophys. Acta, 33: 120.128. Korn, E. D., and R. A. Weisman 1967 Phagocytosis of latex beads by Acanthamoeba. J. Cell. Biol., 34: 219-222. Kvarstein, B. 1969 The effect of temperature, metabolic inhibitors and EDTA on phagocytosis of PSL by human leucocytes. Scand. J. Clin. Lab. Invest., 24; 271-277. Martin, S. P., and R. Green 1958 Methods for the study of surviving leucocytes. Meth. Med. Res., 7: 136-141. Mudd, S., M. McCutcheon and B. Lucke 1934 Phagocytosis. Physiol. Rev., 14: 210-275.
PHAGOCYTOSIS BY POLYMORPHONUCLEAR LEUKOCYTES Nagura, H., J. Asai, Y. Katsumata and K. Kojima 1973 Role of electric surface charge of cell membrane in phagocytosis. Acta. Path. Jap., 23: 279-290. Normann, S. J. 1974 Kinetics of phagocytosis. Lab. Invest., 31: 161-169. Oss, C. J., and C. G. Gillman 1972 Phagocytosis as a surface phenomenon. J. Reticuloendothel. SOC.,12: 283-292. Oss, C. J. van, and J. M. Singer 1966 The binding of immune globulins and other proteins by polystyrene latex particles. J. Reticuloendothel. SOC.,3: 29-40. Rabinovitch, M. 1967 The dissociation of the attachment and ingestion phases of phagocytosis by macrophages. Exp. Cell. Res., 46: 19-28. 1970 Phagocytic recognition. In: Mononuclear phagocytes. R. van Furth, ed. Blackwell, Oxford, pp. 299315. Roberts, J., and J. H. Quastel 1963 Particle uptake by PMN leucocytes and Ehrlich Ascites-carcinoma cells. Biochem. J., 89: 150-156. Ryser, H. J. P. 1970 Transport of macromolecules, esp. pro-
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teins into mammalian cells. Proc. Fourth Inter. Cong. Pharmacol. Basel 1969, 3: 96.132. Seaman, G. V. F., P. S.Vassar and M. J . Kendall 1969 Electrophoretic studies on human PMN leucocytes and erythrocytes. Arch. Biochem. Biophys., 135: 356-362. Stossel, T. P. 1974 Phagocytosis 1-111.New Eng. J . Med., 290: 717-723, 774-780, 833.839. Stossel, T. P., R. J. Mason, J. Hartwig and M. Vaughan 1972 Quantitative studies of phagocytosis by polymorphonuclear leucocytes. J. Clin. Invest., 51: 615-624. Vassar, P. S., and M. J. Kendall 1969 Electrophoresis of human leucocytes. Arch. Biochem. Biophys., 135: 350355. Weisman, R. A,, and E. D. Korn 1967 Phagocytosis of latex beads by Acanthamoeba. Biochem., 6: 485-497. Wilkins, D. J. 1967 The biological recognition of foreign from native particles as a problem in surface chemistry. J. Coll. Interf. Sci., 25: 84-89. Zwartouw, H. T., and H. Smith 1956 Polyglutamic acid from Bacillus anthracis. Biochem. J., 63: 437-442.
