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Experimental Cell Research 108 (1977) 167-174

I N T E R A C T I O N OF L I P O S O M E S W I T H POLYMORPHONUCLEAR

LEUKOCYTES

I. Studies on the Mode o f Interaction O. STENDAHL and CHR. TAGESSON 1 D e p a r t m e n t o f M e d i c a l Microbiology, The University o f Link6ping, S-581 85 Linkb'ping, S w e d e n

SUMMARY Amphipathic long chain anions (dicetylphosphate) or cations (stearylamine) were inserted into the lipid lamellae of multilayered lecithin-cholesterol liposomes so as to obtain liposomes with varying surface charge. These liposomes were then analysed with respect to their interaction with polymorphonuclear leukocytes (PMN cells) at 37°C. The different liposomes showed ditlerent tendencies to associate to the cells: the more negatively charged, the greater the tendency to associate. The association was insensitive to metabolic inhibitors and proceeded without activation of the hexose monophosphate shunt. It was not influenced by omitting divalent cations from the medium. However, lowering the temperature below 30°C and further down inhibited the association. The kinetics of the association process suggests that the presence of dicetylphosphate on the liposome surface increased the maximal rate of association, whereas lowering the temperature decreased the accessibility to appropriate receptor sites in the PMN cell membrane. The results lend support to the hypothesis that controlled manipulations of the liposome composition may be used to engineer the introduction of lipids into PMN cells by different rates and mechanisms. This should have relevance to the possible modification of PMN cell behavior by introduction of components derived from liposomes.

Little is known about the properties which govern the outcome of a phagocyte-prey confrontation. Due to the structural complexity of biological membranes and cell surfaces, a precise description in molecular terms--of the relevant surface attributes is still remote. However, studies of the recognition and activation mechanisms involved in the phagocytic process have shown that such overall physico-chemical properties as charge and hydrophobicity of the particle to be phagocytosed are important [1-5]. The present communication aims at 1 To whom reprint requests should be addressed.

exploring the interaction between polymorphonuclear leukocytes (PMN cells) and liposomes. We first report data concerning the mode of interaction. The original purpose of the investigation was based on the anticipation that, if the mode of a phagocyte-prey interaction was determined by the surface structure of the prey, the use of well-defined model particles would prove useful in revealing the relative importance of different physico-chemical parameters involved. However, certain phospholipid vesicles that constitute but a weak endocytic stimulus may fuse with the plasma membrane of mammalian cells rather than being endocytosed [6-11]. In this process, Exp Cell Res 108 (1977)

168 the

Stendahl and Tagesson mammalian

cell m e m b r a n e

acquires

n e w p h o s p h o l i p i d m a t e r i a l . S u c h an i n c o r p o r a t i o n o f lipids into p h a g o c y t i c cells m a y create a valuable system for modifying their b e h a v i o u r an d f o r d i s c l o s i n g f u n c t i o n a l req u i r e m e n t s a n d m o l e c u l a r m e c h a n i s m s inv o l v e d in t h e a t t a c h m e n t a n d i n t e r n a l i z a t i o n p h a s e s o f t h e p h a g o c y t i c p r o c e s s . T h i s is th e s u b j e c t o f an a c c o m p a n y i n g p a p e r [12].

MATERIALS

AND METHODS

Preparation of liposomes Source and grades of chemicals were as follows: Chromatographically pure phosphatidylcholine was prepared from egg yolks essentially as described by Rhodes & Lea [13] but modified according to Pangborn [14]. The egg yolks were vigorously shaken in methanol before extraction. Silicic acid column chromatography was used for the separation of phospholipids [ 15]. Cholesterol and dicetylphosphate were purchased from Sigma Chemical Co., whereas stearylamine was obtained from Koch-Light Laboratory Ltd. All the chemicals were analytical grade. Phosphatidylcholine (PC), cholesterol (Chol) and dicetylphosphate (DCP) or stearylamine (SA), in molar proportions as described below, were dissolved in chloroform in a round bottom flask. To label the liposomes radioactively, a small amount (10 /xCi) of [1,2-3H]cholesterol (103 mCi/mg) or [14C]phosphatidylcholine (1.5 Ci/mmole of phosphorus) (NEN Chemicals GmbH, D6072 Dreieichenhain, Germany) was added. A thin film of lipids was formed on the surface of the flasks after rotary evaporation of the solvent in vacuo at 40°C. The lipid film was dried under streaming nitrogen and then dispersed mechanically in phosphate-buffered saline solution, pH 7.2 (PBS), by putting the flasks on a rotary shaker for 90 rain at room temperature. The liposome dispersion was then allowed to stand at room temperature for another 90 rain. Finally, the liposomes were centrifuged at 20000 g for 15 min. The supernatant was discarded and the pellet resuspended in PBS or Krebs-Ringer phosphate buffer containing 10 mM glucose (KRG), pH 7.2.

Liposome-PMN cell interaction Monolayers of PMN cells were prepared as follows: Three ml of the PMN cell suspension were pipetted into 5 cm Petri dishes, on the bottom of which were fastened cellulose acetate filters (Millipore Corp.). The leukocytes were allowed to adhere to the filters for 60 min, and non-adhering leukocytes and contaminating erythrocytes were washed off with KRG. Approx. 1× 106 PMN cells adhered to each filter. To each dish, 4.5 ml of the appropriate liposome dispersion (2/xmol lipid) was added, and the dishes were incubated at desired temperature. At indicated times the filters were removed and washed thoroughly three times in cold saline solution. To analyze how inhibitors of glycolysis, iodoacetic acid (IAA) and sodiumfluoride (NaF) influenced the interaction, the inhibitors were added to the monolayers in concentrations known to inhibit phagocytosis of particles such as bacteria. The cells were then preincubated at 37°C for 30 min before the addition of liposomes or bacteria (S. typhimurium 395 MR10, heat-killed and 125I-labelled as described elsewhere

[2]).

Radioactivity measurements The washed filters were put into scintillation vials and 1 ml of Soluene (Packard Instr. Co., Downers Grove, Ill.) was added. After solubilization, 10 ml of Insta-Gel (Packard Instr. Co.) was added and the radioactivity measured in an Auto Liquid Scintillation Counter (Packard). '~SI-radioactivity was measured in an AutoGamma Scintillation Counter (Intertechnique, Plaisir, France). The interaction between liposomes (or bacteria) and PMN cells was expressed either as percent of added radioactivity found per filter, or as the total amount of Jail]cholesterol or [14C]phosphatidylcholine found per filter. Hexose monophosphate shunt (HMS) activity was assayed as described elsewhere [17].

RESULTS

Influence of liposome surface charge Liposomes

with different surface

charge

s h o w e d c l e a r d i f f e r e n c e s in t h e i r i n t e r a c t i o n with PMN amounts

cells (fig. 1). W i t h i n c r e a s i n g

of DCP

(1-10 m o l % )

a greater

a m o u n t o f l i p o s o m a l lipid a s s o c i a t e d w i t h

Polymorphonuclear leukocytes (PMN cells) PMN cells were collected from the peritoneal cavity of rabbits 12 h after the injection of 100 ml of 0,1% glycogen solution (Nutritional Biochemical Corporation, Cleveland, Ohio). The exsudate was centrifuged (200 g, 10 min), washed twice in KRG and then suspended in KRG to 6× 106 cells/ml. More than 90% of the cells were polymorphonuclear leukocytes. Their viability was checked by Trypan blue exclusion.

Erp CellRes 108(1977)

t h e cells. O n t h e o t h e r h a n d , i n t r o d u c t i o n o f S A (1-10

mol%)

into P C - C h o l liposomes

did n o t i n f l u e n c e t h e i r a p p a r e n t i n t e r a c t i o n w i t h t h e cells as c o m p a r e d w i t h u n c h a r g e d l i p o s o m e s ( P C - C h o l ; m o l a r r at i o 8 : 2). L o w c o n c e n t r a t i o n s o f D C P ( 0 . 1 - 1 . 0 m o l % ) did not increase the association, whereas 5 and

Interaction of liposomes with leukocytes. I

169

Table 1. The influence of temperature and inhibitors of glycolysis on the association of bacteria or liposomes to polymorphonuclear leukocytes

Salmonella typhimurium 395 MR10 to approx. 20 % of values attained in the absence of inhibitor (table 1). In contrast, lowering the temperature from 37 to 30°C and further down inhibited the association Association (% of controls) a (fig. 2). After incubation for 120 min at 4°C, Temp. °C Inhibitor the amount of radioactivity associated with 37° 4° IAA b N a F c Particles cells corresponded to only 30-45% of values obtained after incubation for 120 min Bacteria (S. typhimuat 37°C (table 1). rium MR 10) 100 15 24 16 The question then arises whether the Liposomes (70 : 20 : 10) ~ 100 30 100 92 Liposomes (79 : 21 : 1)d 100 45 100 95 interaction with liposomes evokes such a metabolic burst in the leukocytes that is a Controls, radioactivity associated with the cells after incubation at 37°C, 120 min. known to occur during phagocytosis. Table b 5× 10-3 M. c 5×10 2M. 2 shows that there was no significant ind Molar ratio phosphatidylcholine--cholesterol--dicecrease in 14CO2-production from [1-14C]tylphosphate. glucose and thus no significant activation of the hexose monophosphate shunt. To investigate the possibility that the dif10 tool% increased the association after 120 ferent liposomal lipids became associated min incubation at 37°C from 0.15% (un- to the cells by different rates, PC-Cholcharged liposomes) to 0.25 and 0.45%, DCP liposomes were co-labelled with both respectively. In most experiments, the [3H]cholesterol and [14C]phosphatidylchoamount of radioactivity associated cor- line and incubated with PMN cells at responded to 3-10 nmoles of liposomal lipid 37°C. It then seemed, both with weakly per 106 PMN cells. The cells remained in- charged liposomes (containing 1 mol% tact upon association as judged by their DCP) and strongly charged liposomes (conability to exclude Trypan blue. In a series of experiments, the requirements for Ca 2+ and Mg 2+ were tested. No O.A j o changes in reaction rates were observed o when Ca 2+ and/or Mg ~+ were omitted from the medium. 0,5

o/o

Characteristics of the interaction If the association of liposomal lipid to the PMN cells were due to endocytic activity in the cells, the process would be inhibited in the presence of metabolic inhibitors and at low temperature. H o w e v e r , the effect of 5×10 -3 M iodoacetic acid (IAA) or 5× 10-~ M NaF on the process was inconspicious although these concentrations of inhibitor reduced the phagocytic uptake of

i

30

i

60

°

I

90

i

120

Figs 1, 2. Abscissa: incubation time (rain); ordinate: association (% of added radioactivity per 106 cells).

Fig. I. Association of different liposomes to polymorphonuclear leukocytcs. II, PC-Chol, molar ratio 80 : 20; O, PC-Ghol-SA, molar ratio 70 : 20 : 10; n , PC-ChoI-DCP, molar ratio 79:20: 1; G, PC-CholDCP, molar ratio 75 : 20 : 5; ©, PC-Chol-DCP, molar ratio 70:20: 10. I-~H]Cholestero] was used as liposome marker. Exp Cell Res 108 (1977)

Stendahl and Tagesson

170 0.5

Q

0.4

0.3 20 °

0,2

tations in fig. 4) suggests that lowering the temperature reduced the l/K,, of the process, i.e. the association affinity. In contrast, the maximal rate of association was not reduced by lowering the temperature from 37 ° to 4°C.

0,1 i

30

,

60

90

i

120

t

i

i

30

60

90

Fig. 2. (A) Influence of temperature; (B) inhibitors of energy metabolism on liposome association (PCChol-DCP, molar ratio 70:20: 10) to PMN cells. ©, Control; A, IAA, 5x 10-3 M iodoacetic acid; [3, NaF, 5 x 10-2 M sodiumfluoride.

taining 10 mol% DCP) that the 3H/z4Cratios in the applied liposomes and in the treated cells were close to unity (table 3).

Kinetics of the interaction The differences between weakly and strongly charged liposomes in their tendency to interact with PMN cells may depend on differences in the accessibility to appropriate receptors on the cell surface or on differences that otherwise affect the rate of association. Considering the interaction with a given concentration of liposomes (S) an association process with velocity V, a double reciprocal plot (1/V as a function of 1/S) may be used to assess the maximal rate of association (Vmax) and the affinity (or association strength (l[Km) in the liposomePMN cell interaction. As illustrated in fig. 3, the presence of DCP on the liposomal surface increased the maximal rate of association to PMN cells but did not alter the PMN cell-liposome affinity. Analogously, the reduced tendency to associate at 4°C as compared to 37°C may reflect altered association affinity or altered association rate. Comparing the kinetics of the interaction at the different temperatures (illustrated as Lineweaver-Burk represenExp Cell Res 108 (1977)

DISCUSSION

i

120

Possible mechanisms of interaction between liposomes and PMN cells include (1) phagocytosis, i.e. the phagocyte regards the liposome a prey; (2) liposome-PMN cell fusion, i.e. the outer lipid lamellae of the liposomes fuse with the plasma membrane of the PMN cell; (3) lipid exchange, i.e. lipid molecules from the liposomes and from the plasma membrane of the PMN cell are interchanged; (4) adsorption of lipids from the liposomes to the PMN cell surface. Endocytosis of liposomes and (phospho)lipid vesicles may take place in a number of experimental sysvems. According to Batzri & Korn [8], unilamellar eggPC or dimyristoyl-PC vesicles are taken up by endocytosis into Acanthamoeba castellani, and according to Papahadjopoulos et al. [ 18], dipalmitoyl PC-Chol-SA vesicles may

Table 2. HMS activity a in polymorphonuclear leukocytes during interaction with bacteria or liposomes Particles

14CO2-release (cpm)

Bacteria (S. typhimurium MR 10) Liposomes (70 : 20 : 10)b Liposomes (79 : 20 : 1)b None

1 850 220 270 225

a Measured as 14CO2 released during 60 min from 5×10 ~ PMN cells incubated with 0.5 /~Ci [ I J 4 C ] glucose and liposomes (2/zmol lipid) or bacteria (2.5 x 108 S. typhimurium 395 MR 10) in a total volume of 3 ml. b Molar ratio phosphatidylcholine-cholesterol--dicetylphosphate.

Interaction of liposomes with leukocytes. I

171

Table 3. Association of 3H and 14Cactivity to polymorphonuclear leukocytes after incubation with [14C]phosphatidylcholine-[3H]cholesterol-labelled liposomes *H/a4C ratio Liposome composition (molar ratio)

In applied liposome dispersion

Associated with cells after interaction a

PC (70) : Chol (20) : DCP (10) PC (79) : Chol (20) : DCP (1)

6.9 6.4

5.5 5.6

Identical values were obtained after 15, 30, 60 and 120 rain incubation.

be incorporated into mouse 3T3 and L929 cells primarily by way of endocytosis. Poste & Papahadjopoulos [11] have also reported that egg PC vesicles and dipalmitoyl-PC--distearyl PC-phosphatidyl-serine (PS) vesicles are incorporated into 3T3 cells largely by endocytosis. Gregoriadis et al. have used multilamellar PC-Chol-DCP (or SA) liposomes as vectors to introduce biologically active molecules into the lysosomal apparatus of rat liver cells [19, 20], mouse peritoneal macrophages [21] and HeLa cells [22]. Weissmann et al., arguing that the phospholipid surfaces of liposomes constitute but a weak endocytic stimulus to the lysosomal system, have coated PC-CholDCP liposomes with aggregated immunoglobulin and so obtained an endocytic uptake of liposomes into phagocytes of the smooth dogfish [23]. To determine the mode of entry and association of different liposomes with the different cells, techniques involving tissue fractionation [ 19, 20, 22], electron microscopy [24, 25] and ultrastructural cytochemistry [23] have been employed. Consistent results have been obtained, indicating that liposomes may be interiorized into cells through classical endocytosis. The association of liposomes with the cells is then inhibited at 4°C or in the presence of inhibitors of glycolysis [11]. However, the systems at issue in the pres-

ent investigation, i.e. liposomes composed of egg phosphatidylcholine, cholesterol and dicetylphosphate interacting with rabbit PMN cells at 37°C, were insensitive to glycolytic inhibitors: iodoacetic acid and sodium fluoride in concentrations sufficient to inhibit phagocytosis of bacteria were without effect on the rate by which lipid molecules associated with the cells. Furthermore, the association proceeded without such an activation of the hexose monophosphate shunt that is elicited by a phago-

3000

z~

o 2000

I000

¢'.

,;6

.;0e

Figs 3, 4. Abscissa: (A) liposome conc. (S), cpm [3H]cholesterol/ml; (B) I[S; ordinate: (A) association rate (V), cpm 3H/106 cells/30 min; (B) 1IV. Fig. 3. Influence of liposome surface charge on liposome-PMN cell interaction. (A) Relation between liposome concs; ©, PC-Chol-DCP, molar ration 70 : 20 : 10; A, 79 : 20 : 1 ; and rate of association to PMN cells; (B) data expressed as double reciprocal plots. Exp Cell Res 108 (1977~

Stendahl and Tagesson

172

B

•

ZOO0

1000

/ ,,5;7. 106

2x10 G

Fig. 4. Influence of temperature on l i p o s o m e - P M N cell interaction. (A) Relation between liposome concs: P C - C h o l - D P C , molar ratio 70 : 21 : 10, and rate of association to PMN cells at O, 37°C; O, 4°C; (B) data expressed as double reciprocal plots.

cytic stimulus [26] or accompanies phagocytic ingestion [27]. These results are interpreted to indicate that in the present systems, the liposomes were not endocytosed. However, liposome components may be incorporated into cells by other, nonexclusive mechanisms--indeed, fusion and endocytosis have been proposed as alternate mechanisms for the uptake of lipidsoluble and water-soluble molecules [8]. Papahadjopoulos et al. have argued that fusion of unilamellar vesicles with the plasma membrane of cultured mammalian cells is favoured when the vesicle lipids are above their gel-liquid transition temperature [7]. The present results are also consistent with the hypothesis that fusion has occurred. In accordance with the findings of Poste & Papahadjopoulos [11] the decreased association at lower temperature may reflect a temperature-dependent alteration in the functional state of the PMN cell membrane. The kinetics of the association suggests that the affinity between the interacting structures is reduced at lower temperature. This may be expected if the Exp Cell Res 108 (1977)

fluidity of the membrane lipids is decreased and the accessibility to appropriate receptor sites in the cell membrane is reduced. Papahadjopoulos et al. [28] have studied fusion between vesicles prepared from individual phospholipid species and found that vesicles containing lipids that were in a liquidcrystalline state were more susceptible to fusion than vesicles composed of lipids that were in the solid phase. It was also demonstrated that whereas uncharged PC vesicles showed only a limited capacity to fuse, extensive fusion occurred between negatively charged phosphatidylserine (PS) vesicles incubated in the presence of CaCI~ and between vesicles prepared from greater than 50 % PS in PC in the presence of CaC12 and albumin. These findings may have bearing upon the mechanism by which the presence of negatively charged DCP on the liposome surface increased the maximal rate of lipidPMN cell association. In the chemically and physically well-defined systems investigated by Papahadjopoulos et al. [28], a Ca2+-induced segregation or clustering of individual phospholipid species into separate domains was demonstrated and postulated to allow charged regions in apposed vesicles to preferentially fuse with each other. However, the precise relationship between Ca2+-induced segregation and fusion is not clear. In the present investigation, no effect was obtained by omitting Ca 2+ from the medium. At the moment, we can offer no conclusive explanation for this discrepancy between the interaction in simple, well-defined model systems and the interaction in complex systems, such as the liposome-PMN cell system. As to the predominant mechanism of liposome-PMN cell interaction, we thus consider fusion of the lipid lamellae with the cell membrane the explanation most consistent with the described observations. If

Interaction of liposomes with leukocytes. I lipid exchange processes were important, one would expect the different liposome components to exhibit different rates of exchange with the cell membrane lipidscholesterol would exchange at a faster rate than phosphatidylcholine [29] and the liposome components would not become cellassociated in the same proportions as they were in the applied dispersion. Thus, the finding that the association of cholesterol was slower than that of phosphatidylcholine makes lipid exchange as a major mechanism of interaction unlikely. Finally, the introduction of positively charged SA on the surface of the liposomes did not influence their interaction with the negatively charged cells. This finding together with the lack of requirements for cations in the interaction between negatively charged liposomes and the cells indicate that the association was not primarily due to electrostatic attraction or brought about by ionic bridging. However, for the lack of electron microscopy or electron microscopic autoradiography, they do not rule out the possibility that liposomes were absorbed to the PMN cell surface without being fused with the cell membrane. Tissue fractionation data, however, showed that the association of liposomes and PMN cells leads to the formation of a subcellular element in which liposome derived lipid is truly integrated and which co-sediments with 5'-nucleotidase activity [30]. Furthermore, the demonstration that biologically active materials trapped within liposomes can be recovered from within cells [7, 18, 23, 31] and produce highly specific changes in cell metabolism and behaviour [7, 18, 23, 31] argues strongly against the possibility that liposomes merely bind to the cell surface. It is true that to rigorously demonstrate the existence of a fusion mechanism it must be shown that a number of criteria, not dealt

173

with in the present study, are met. Following cellular uptake of proportional amounts of lipid and trapped marker substance (i.e. molecules truly soluble and internalized within the aqueous spaces of the liposomes) the marker should be free in the cytoplasm of the cell and neither compartmentalized in the lysosomal apparatus nor in some other membrane-bound vesicular body. Although a fusion mechanism was not unequivocally demonstrated, the present observations lend support to the hypothesis that controlled manipulations of the liposome composition may be used to engineer the introduction of lipids into intact PMN cells. This should have bearing on the possible use of liposomes for the transplantation of foreign molecules into an intact phagocytic cell. The possibility that this is an experimental tool for modifying the surface structure and properties of phagocytic cells awaits further experimentation. The technical assistance of Ellinor Granstr6m and Britinger Thor6n is gratefully acknowledged. This study was supported by grant 16X-2183-10 from the Swedish Medical Research Council.

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25. Rahman, Y & Wright, B J, J cell bio165 (1965) 112. 26. Romeo, D, Jug, M, Zabuchi, G & Rossi, F, FEBS lett 42 (1974) 90. 27. Sbarra, A J, Paul, B B, Jacobs, A A, Strauss, R R & Mitchell, G W, Jr, J reticuloendothel soc 12 (1972) 109. 28. Papahadjopoulos, D, Poste, G, Schaeffer, B E & Vail, W J, Biochim biophys acta 352 (1974) 10. 29. Ehnholm, C & Zilversmit, D B, J biol chem 248 (1973) 1719. 30. Tagesson, C, Stendahl, O & Dahlgren, C, Proc 1st Eur conf on phagocytic leukocytes (Movement, metabolism and bacterial mechanisms of phagocytes) Trieste 1976. In press. 31. Gregoriadis, G, Enzyme therapy in lysosomal storage diseases (ed I M Taber, G I M Hoogwinkel & W Th Daems) p. 131. North-Holland, Amsterdam (1974). Received January 31, 1977 Accepted March 9, 1977