Latex phagocytosis by polymorphonuclear leukocytes: Role of sialic acid groups

Chem.-Biol. Interactions, 33 (1980) 91--100

91

© Elsevier/North-Holland Scientific Publishers Ltd.

LATEX PHAGOCYTOSIS BY P O L Y M O R P H O N U C L E A R LEUKOCYTES: R O L E O F SIALIC ACID GROUPS

H. BEUKERS, F.A. DEIERKAUF, C.P. BLOM, MARTHA DEIERKAUF, C.C. SCHEFFERS and JELLE C. RIEMERSMA Laboratory for Medical Chemistry, 8ylvius Laboratorla, State University of Leiden, Wassenaarseweg 72, 2333 AL Leiden (The Netherlands) (Received April 15th, 1980) (Revision received August 1st, 1980) (Accepted August 24th, 1980)

SUMMARY

Polystyrene latex particles are rapidly phagocytized by rabbit polymorphonuclear (PMN) leukocytes. The uptake is influenced by macromolecules which have the effect of altering the surface charge of the latex particle. The influence of polylysines of varying chain length on the surface charge of latex particles and of P M N cells was studied by micro-electrophoresis. Charge reversal at the latex surface was found to occur at concentrations considerably below that at which the surface charge of the P M N cells is reversed. Phagocytosis of latex by P M N cells is enhanced in the presence of low concentrations of long-chain polylysines. The enhancement of phagocytosis is strongly reduced if P M N cells are treated with neuraminidase. This suggests participation of siliac acid groups in a stage of particle-cellinteraction which precedes engulfment.

INTRODUCTION Polystyrene latex sphemles with well-defined surface characteristics are availsble in mono-disperse suspensions. Such particles have been used extensively in studies of phagocytosis. This investigation deals with the uptake of latex particles by PMN leukocytes. Generally speaking particle recognition and attachment requires an interaction between the particle and cell-surface receptors; moreover, in many instances certain serum components are involved [1--4]. With latex or carbon particles the interaction between particle and cell surface is non-specific, and here the term 'foreign surface receptor' is sometimes used [5]. Opsonization by serum factors is n o t required for the uptake of such particles. It has been shown, however, that certain macromolecular substances enhance or inhibit their phagocytosis; for instance polylysine causes increased latex phagocytosis by

92 PMN cells [6,7]. A further study of this effect m a y provide additional insights regarding cell surface components involved in particle uptake. In some types o f mammalian cells sialic acid groups at the cell surface, as part of membrane glycoproteins, stabilize an external hydrophilic barrier [ 8 - 1 0 ] . Particles with a net positive surface charge, such as polylysin~ coated latex particles, may have a destabilizing effect by interacting with these and other negative groups at the cell surface. There is some evidence that sialic groups are involved in particle attachment, whereas h y d r o p h o b i c interactions may play a role in particle ingestion [11]. By studying the phagocytic behavior of PMN cells before and after neuraminidase treat~ ment information m a y be obtained regarding the function of sialic acid groups in phagocytosis. MATERIALS AND METHODS Polymorphonuclear leukocytes were obtained from peritoneal exudates of CH-rabbits as described by Hirsch [12]; in this procedure a few changes were made [7]. A stock suspension resulted containing 0.40 × 10 ~ cells/ml in protein-free Hanks' solution [13]. In our phagocytosis experiments 2.5 ml cell suspension (107 cells) was mixed at 37°C with a latex suspension, to which in some cases other substances had been added, to a final volume of 5 ml. Phagocytosis was stopped after 12 min by adding 4 ml 40 mM EDTA and cooling in ice [14]. Non-phagocytized material was removed by centrifuging at 1000 rev./min (4°C). Latex was determined in the sedim e n t e d cells after washing the cells by centrifugation. The cells were extracted with dioxane (purest quality) overnight, at r o o m temperature. Polystyrene latex was determined in the extract by s p e c t r o p h o t o m e t r y [ 15]. Polystyrene latex with a particle diameter of 0.48 p m was obtained from D o w Chemical Company. In some experiments we used latex spherules (0.50 p m ) and latex spherules containing covalently b o u n d NHrgroups, so-called aminolatex, {0.52 /~m, 0.125 mEq. NH2/g) obtained from Polysciences Inc. As a control, we assessed by electron microscopy whether the increase of latex uptake after certain additions was indeed due to internalized latex. For this purpose, a cell sediment containing latex was fixed overnight in 0.1 M cacodylate-glutaraldehyde (pH 7.4, 310 mOsm), post-fixed in 2% veronal-acetate-bufferend osmium tetroxide, and resuspended in 0.4% bovine serum albumin [16,17]. The albumin suspension was coagulated with 0.1 ml 12.5% glutaraldehyde and subsequently centrifuged. The pellet was dehydrated in ethanol and e m b e d d e d in Epon. Ultrathin sections of this material were stained either with 2% uranyl acetate and 0.4% leadcitrate, or with 1% lanthanum nitrate followed by the lead uranyl stain [18]. The sections are studied with a Zeiss EM9 electron microscope. Neuraminidase (EC 3.2.1.18) was obtained from Boehringer. For neuraminidase treatment PMN cells were incubated 30 min at 37°C with 1.3 mg enzyme/ml in Hanks' solution. The cells were washed by centrifugation (300 g) and

93 then resuspended. Released neuraminic acid was determined according to Warren [19]. Reference values for total neuraminic acid were corrected for deoxysugars by measuring the extinction at two wave lengths (549 and 532 nm). In experiments with added macromolecular substances we used Sigma products, namely poly-L-lysines (HBr) of molecular weight 3, 13, 32 and 60 × 103 Daltons (representing polymerization numbers o f 15, 60, 150 and 270). In addition poly-D-lysine (HBr) of M = 70 × 10 ~ Daltons (polymerization number 323), and poly-L-glutamic acid (Na) of M = 98 × 103 Daltons (polymerization number 565) were used, as well as L-lysyl-L-lysine (HC1). For the purpose of assessing the surface charge of latex particles and of PMN cells a micro-electrophoresis apparatus was used (Mark II, Rank Brothers, Cambridge, U.K.) with Pt-electrodes. For latex we employed a cylindrical cell. Polylysine was added in Hanks' buffer (pH 7.4) and the particle velocity was measured at 5.5 V/cm. For PMN cells in Hanks' we used a flat electrophoresis cell at 3.8 V/cm (37°C). Measurements were carried o u t on freshly prepared cell suspensions, with current reversal. Mobility was expressed in ~m/s/V/cm. For procedure see Vassar et al. [20]. To measure the extent of cell lysis due to polylysine, K ÷ was determined by flame p h o t o m e t r y (Eppendorf). For the same purpose lactate dehydrogenase leakage from the cells was determined by the pyruvatelactate method, in the supernatant of a cell suspension to which polylysine had been added (total volume 5 ml, 2.5 × 107 cells). RESULTS Quantitative determinations of latex phagocytosis, described under Materials and Methods, show an increased latex uptake in the presence of polylysines. The chain length of these substances is a factor of considerable importance. Lysyl-lysine has no effect. The stimulating effect in the series of polylysines of Fig. 1 starts in some cases at a very low concentration (e.g. 270-polylysine). With 15-polylysine, stimulation requires a polycation concentration of approx. 3 X 103 mM. The polylysines PL-60, PL-150, and PL-270 have an optimal concentration b e y o n d which the quantity of latex in the cell sediment starts to decrease. If we used latex particles with covalently bound amino groups, we obtained similar results, i.e. an approximate doubling of the uptake of the same type of latex without aminogroups. To determine the extent to which macromolecules influencing latex uptake are bound, either by the particle or b y the phagocyte surface, microelectrophoresis was used. We measured the electrophoretic mobility of latex beads as a function of polylysine concentration with various polylysines. As Fig. 2 indicates charge reversal of the particle surface occurs at a certain concentration of polylysine; this concentration is lowest in the case of PI~270. The polylysine concentration at which the mobilitycurves begin to rise is also the concentration where latex uptake under influence of polylysine starts to increase (Fig. 1).

94

o i50

o /

o

..~ 0

[]

5 x 100

°"""~ o . ~ d -

°'"-- °

.~.

0

-~

-6

-~

-Z

-~

-~

-i

10g finol p~ly-L-lysine cone {raM] Fig. I. Increase of the quantity of polystyrene latex in the cellsediment of po]ymorphonuclear leukocy~s in the presence of poly-L-lysine. The amount of latex ~ expressed as % of control (po]ylysine absent). Curves 1--5 represent the addition of 270-polylysine, 150-polylysine, 60-po]ylysine, 15-polylysine, and lysyl-lysineresp. All values -+ i0. Each p o i n t is t h e m e a n o f 4 m e a s u r e m e n t s .

• 4.0

o~-O

o/O/°-I

~ ~ ~ o -

÷2D

E o

Ig

..D O

-2D

-'.0'

-o

-7

-6

-5

-4

log finol poly-L-lysinecone.(mM)

-2

-2

Fig. 2. E l e c t r o p h o r e t i e m o b i l i t y o f p o l y s t y r e n e l a t e x b e a d s a n d P M N l e u k o e y t e s as a f u n c t i o n o f t h e c o n c e n t r a t i o n o f a d d e d poly-L-lysines. Curves 1--3 r e p r e s e n t t h e a d d i t i o n o f resp. 2 7 0 - p o l y l y s i n e , 1 5 0 - p o l y l y s i n e , a n d 60-polylysine. All vaules ± 0.4. E a c h p o i n t is t h e m e a n o f 4 d e t e r m i n a t i o n s . Curve 4 r e p r e s e n t s t h e m o b i l i t y o f P M N cells in t h e p r e s e n c e o f increasing q u a n t i t i e s o f 2 7 0 - p o l y l y s i n e ; values ± 0.2.

95 Apparently polylysines bind at the latex particle surface. Since polylysine in a certain concenlration range may be bound by the phagocyte as well as by the particle, this needs however not be the only factor responsible for increased latex uptake. PMN cells are normally negatively charged in Hanks' solution. They acquire a net positive charge only when the polylysines added are present in a concentration much higher than is required for reversing the charge on latex. Thus for instance 10 -3 mM PL-270 was required for the charge reversal of PMN cells; for charge reversal o f latex 10-6--10 -s mM PL-270 was already sufficient (Fig. 2). A decreasing latex uptake beyond the optimal polylysine-concentrations (Fig. 1) is due to cytolysis and not due to the fact that beyond this point

(control)

2rrNn12rn=n

20 .'-

7

.-."

15

10 tO U

iii 5

.-"

iii IJ

~6

B (polylysine added)

J~

E c

20

15

iiiii iii

10

..

R

0-4

5-8

9-12

13L16'

. .

>16 "

latex uptake, porticleslcellsection

Fig. 3. Effect of 270-poly-L-lysine on latex phagocytosis after respectively 2 (left columns) and 12 rain (right, dotted columns). Latex particles were counted in electron micrographs of 50 different sections of polymorphonuelear leukoeytes per preparation.

96 the PMN cells and the latex particles are both positive. As the data assembled in Table I shows cytolysis, measured as an abrupt rise of the K ÷- and LDH-concentrations in the extracellular medium, starts at a critical concentration of polylysine. From this concentration onwards cytolysis reduces the n u m b e r of cells participating in phagocytosis. Micro-electrophoresis experiments give meaningful results only at low concentrations where cell damage is negligible. The range of used polysine-concentrations ends somewhat below the concentration required for maximal latex uptake. In the presence of lysed cells or cell fragments mobility measurements are unreliable, as indicated by the d o t t e d line segments in Fig. 2. An alternative m e t h o d to determine latex uptake by PMN cells instead of s p e c t r o p h o t o m e t r y is to c o u n t on electron micrographs the number of particles per cell section. In separate whole cells observed in sections from the same fixed and e m b e d d e d pellet the internalized latex spherules were counted. Different sections separated by a distance of 20 pm were studied until 50 cells per pellet had been examined. On the basis of the electron micrographs we arrived at an estimate of the average number of latex particles per cell section. In the presence of polylysine the average number of latex spherules per cell is increased b y a factor two (see Fig. 3), confirming the spectrophotometric findings. With available staining methods it was possible to differentiate clearly between internalized and adsorbed latex particles. The EM technique permitted an estimate of the internalized latex. After counting several sections

250'

-~ 200. 0

150-

•..-q 100Q.

°~-~°-~-°~

x

"6

-- . . . .

50-

log final polyomino acid conc. lmM)

Fig. 4. Latex phagocytosis by polymorphonuclear leukocytes (PMNL) as influenced by polyaminoacids and neuraminidase (1) Untreated PMNL, effect of poly-D-lysine (PL 323), (2) neuraminidase-treated effect o f poly-D-lysine, (3) untreated PMNL, effect of poly-L-glutamic acid and (4) neuraminidase-treated PMNL, effect o f poly-L-glutamic acid. Each point averaged from 6 determinations. Error range for the polylysine treated cells ± 10, for the polyglutamic acid treated cells ± 6. For experimental details see text.

97 TABLE I EFFECTIVE CONCENTRATION OF VARIOUS POLYLYSINES WITH REGARD TO CERTAIN PHENOMENA OBSERVED DURING LATEX PHAGOCYTOSIS BY POLYMORPHONUCLEAR LEUKOCYTES Log added polylysine (retool/l)

Start of increased latex uptake Charge reversal of latex spherules Maximum of latexuptake Beginning of cytolysis

270-polylysine

150-polylysine

60-polylysine

--5.8

--5.0

--5.0

--5.6

--4.9

--4.8

--3.8

--3.3

--2.4

--4.6

--3.8

--2.9

per sample (50) relative estimates of latex uptake are obtained. These can be used for comparative purposes in assessing the effect of polylysine. Dehydration and embedding cause a departure from the in vivo conditions, observable with light microscopy. Using the light microscope we found no evidence for uptake of latex in specific regions of the cell. One way to influence cell surface charge is to remove neuraminic acid groups from the cell surface. The upper curves of Fig. 4 show the phagocytosis-stimulating effect of PL-323 before neurarninidase treatment (curve 1), and after neumminidase treatment (curve 2). Apparently the stimulating effect of polylysine is lessened in cells from which neuraminic groups are removed. The lower curve indicate the effects of poly-L-glutamic acid before neumminidase (curve 3), and after neuraminidase (curve 4). The shift of the curve here is in the opposite direction of that in the case of polylysine. In an earlier study we showed that polyglutamic acid reduces phagocytic uptake of latex by P M N cells [7]. Removal of neuraminic acid groups counteracts this effect. DISCUSSION

The foregoing results suggest that added polycations or polyanions, by influencing the charges on either the particle or the cell surface, have an effect on phagocytosis. It has been established that PMN cells contain at their external surface negatively charged neuraminic groups as well as carboxyl groups belonging to amino acids of membrane proteins [20,21]. The latex particles are also negatively charged, sulfate groups are present at their surface deriving from the m e t h o d of preparation. These sulfate groups are present even after prolonged dialysis [22]. Our micro-electrophoresis experiments indicate that cationic polymers such as polylysine

98 at low concentrations are b o u n d preferably by the latex particles and n o t b y the cells [23]. Surface-modified latex particles with adsorbed polycations behave like amino-latex particles containing covalently b o u n d amino groups. Polylysines of the largest chain length are effective at the lowest concentrations (10 -s mM). Analogous results have been obtained with regard to the phagocytic uptake o f Streptococcus faecalis by PMN leukocytes; PL-375 stimulated uptake at a m u c h lower concentration than PL-6 [24]. Polylysine causes cross-bridging of cells, i.c. clumping, and at high concentrations cytolysis [25--27]. Polylysine concentrations below those o f maximal phagocytic uptake did n o t produce considerable clumping and lysis. In this range reliable uptake determinations could be performed, as well as mobility measurements by micro-electrophoresis. Ingested latex particles could be distinguished from adherent latex particles by means of electron microscopy, if appropriate stains were used. In cell preparations to which latex was added together with polylysine, there was a roughly two-fold enhancement of phagocytosis (measured as the average number o f latex particles per cell). With polylysines of adequate chain length, the surface charge of latex particles is altered at low polylysine concentrations as shown by microelectrophoresis. The concentration where charge reversal begins coincides with the concentration at which enhanced phagocytic uptake is observed (Fig. 1 and 2). The cellular surface is still negative at this polylysine concentration. Polylysine thus stimulates latex uptake when b o u n d to the latex particle surface rather than to the cell surface. Covalently b o u n d amino groups, as are present in amino-latex, have the same effect, and here certainly there is only secondarily an interaction between amino groups and the cell surface. The way in which modified latex particles interact with the PMN cell surface probably involves membrane-bound neuraminic groups. We found that incubation of intact PMN cells with neuraminidase removed approximately 18% of the available neuraminic acid. Similar data are given by Tsan et al. [28]. Removal of cell surface neuraminic acid by neuraminidase greatly reduced the polylysine-caused enhancement of phagocytoais. Moreover the inhibition of latex uptake b y poly-L-glutamic acid is diminished after neuraminidase treatment of PMN cells. It appears that electrostatic forces are responsible for the first interaction between the latex particle and the cell surface. The observations suggest that sialic groups are important in the uptake of latex particles by PMN cells. In the stage preceding engulfment particles m a y be attached to the cell-surface by means of 'foreignsurface receptors'. They are mainly b o u n d by ionic interactions. Subsequent engulfment m a y occur via a zipper-mechanism as postulated by Griffin [29,30]. Such a mechanism probably requires contact between hydrophobic areas of the latex surface and the cell surface. After the establishm e n t o f cell-particle contact, differences in interfacial tensiop will determine whether particle engulfment will occur [11,31]. Although the effects of certain macromolecular substances have been studied mainly under in

99 vitro conditions, it appears not unlikely that phagocytosis in vivo is regulated by similar processes. REFERENCES 1 S.V. Boyden, J.R. North and S.H. Faulkner, Complement and the activity of phagocytes, in: G.E. Wolstenholme and J. Knights (Eds.), Complement, CIBA Foundation Symposium, Churchill, London, 1965, p. 190. 2 J. Newsome, Phagocytosis by human neutrophils, Nature, 214 (1967) 1092. 3 M. Rabinovich, Phagocytic recognition, in: R. van Furth (Ed.), Mononuclear Phagocytes, Blackwell, Oxford, 1970, p. 299. 4 T.P. Stossel, Phagocytosis I--HI, New Eng. J. IVied., 290 (1974) 717, 774, 833. 5 M.N.J. Waiters and J.M. Papadimitriou, Phagocytosis, a review, C.R.I. Crit. Rev. Toxicol., (1978) 377. 6 H. Nagura, J. Asai, J. Katsumata and K. Kojima, Role of electric surface charge of cell membranes in phagocytosis, Acta Pathol. Jap., 23 (1973) 279. 7 F.A. Deierkauf, H. Beukers, M. Deierkanf and J.C. Riemersma, Phagocytosis by rabbit PMN leukocytes, the effect of albumin and polyamino acids on latex uptake, J. Cell. Physiol., 92 (1977) 169. 8 J. Noseworthy, H. Korehak, M.L. Karnovsky, Phagocytosis and the sialic acid of the surface of PMN leukocytes, J. Cell. Physiol., 79 (1972) 91. 9 P.P.H. de Bruyn, The role of siaiated glycoproteins in endocytosis, permeability and transmural passage in the myeloid endothelium, J. Histochem. Cytochem., 27 (1979) 1174. 10 P.M. Quinton and C.W. Philpott, A role for anionic sites in epithelial architecture, J. Cell Biol., 56 (1973) 787. 11 H. Beukers, F.A. Deierkauf, C.P. Blom, M. Deierkauf and J.C. Riemersma, Effects of albumin on the phagocytosis of polystyrene spherules by rabbit PMN leukocytes, J. Cell. Physiol., 97 (1978) 29. 12 J.G. Hirsch, Phagocytin, a bactericidal substance from PMN leukocytes, J. Exp. Med., 103 (1956) 589. 13 S.P. Martin and R. Green, Methods for the study of surviving leukocytes, Methods Med. Res., 7 (1958) 136. 14 B. Kvarstein, The effect of temperature, metabolic inhibitors and EDTA on phagocytosis of PSL by human leukocytes, Scand. J. Clin. Lab. Invest., 24 (1969) 271. 15 J. Roberts and J.H. Quastel, Particle uptake by PMN leukocytes and Ehrlich aseitescarcinoma cells, Biochem. J., 89 (1963) 150. 16 A.M. Glauert, Fixation, dehydration and embedding of biological specimens, NorthHolland Publ. Company, Amsterdam, 1975. 17 J. Jansen, C.J.L.M. Meyer, P. van der Valk, W.C. de Bruyn, P.C.J. Leyh, G.J. den Ottolander and R. van Furth, Phagocytic potential of hairy cells, Scand. J. Haematol., 23 (1979) 69. 18 P.R. Lewis and D.P. Knight, Staining methods for sectioned material, North-Holland Publ. Company, Amsterdam, 1977. 19 L. Warren, The thiobarbituric acid A ~ y of sialic acids, J. Biol. Chem., 234 (1959) 1971. 20 P.S. Vassar, M.J. Kendall and G.V.F. Seaman, Electrophoresis of human leukocytes, Arch. Biochem. Biophys., 135 (1969) 350. 21 G.V.F. Seaman, P.S. Vassar and M.J. Kendall, Electrophoretic studies on human PMN leukocytes and erythrocytes, Arch. Biochem. Biophys., 135 (1969) 356. 22 H.J. van den Hull and J.W. Vanderhoff, Well characterised monodisperse latexes, J. Coll. Interface Sci., 28 (1968) 336. 23 J. Gregory, The effect of cationic polymers on the colloid stability of latex particles, J. Coll. Interface Sci., 25 (1967) 35.

100 24 W. Pruzanski and S. Saito, The influence of natural and synthetic cationic substances on phagocytic activity of human PMN cells, Exp. Cell Res., 117 (1978) 1. 25 A. Katchalsky, D. Danon and A. Nevo, Interactions of basic polyelectrolytes with the red blood cell, Biochim. Biophys. Acta, 33 (1959) 120. 26 D. Danon, C. Howe and L.T. Lee, Interaction of polylysine with soluble components of human erythrocytes membranes, Biochim. Biophys. Acta, 101 (1965) 201. 27 M. Mamelak, S.K. Wissig~ R. Bogoroch and J.S. Edelman, Physiological and morphological effects of poly-L-lysine on the toad bladder, J. Membrane Biol., 1 (1969) 144. 28 M.F. Tsan and P.A. McIntyre, The requirement for membrane sialic acid in the stimulation of superoxide production during phagocytosis by human PMN leukocytes, J. Exp. Med., 143 (1976) 1308. 29 F.M. Griffin, J.A. Griffin, J.C. Leider and S.C. Silverstein, Studies on the mechanism of phagocytosis, J. Exp. Med., 142 (1975) 1263. 30 F.M. Griffin and S.C. Silverstein, Segmental response of the macrophage plasma membrane to a phagocytic stimulus, J. Exp. Med., 139 (1974) 323. 31 C.J. van Oss, Phagocytosis as a surface phenomenon, Ann. Rev. Microbiol., 32 (1978) 19.