Printed in Sweden Copyright ~ 1977 by Academic Press, Inc. All rights of reproduction in any form reserved ISSN 0014~t827
Experimental Cell Research 108 (1977) 175-184
I N T E R A C T I O N OF LIPOSOMES W I T H POLYMORPHONUCLEAR LEUKOCYTES II. Studies on the Consequences of Interaction C. DAHLGREN, E. KIHLSTR~)M, K.-E. MAGNUSSON, O. STENDAHL and C. TAGESSON 1
Department of Medical Microbiology, University of Link6ping, S-58185 Link6ping, Sweden
SUMMARY Polymorphonuclear leukocytes were incubated at 37°C with liposomes composed of phosphatidylcholine, cholesterol and dicetylphosphate and the cells obtained characterized with respect to properties that would disclose a modification of their surface properties. The cells excluded Trypan blue, did not leak ~lCr-labelled cytoplasmic proteins and responded as control cells to concanavalin A (ConA) with an increased HMS activity. Their volume was increased as revealed by the pulse-height distribution at electronic counting. The partition of the cells in aqueous biphasic systems containing polymers with covalently bound ligands showed that their negative surface charge density and their liability to hydrophobic interaction were decreased. The cells showed a reduced capacity to phagocytose S. typhimurium 395 MR 10 and S. typhimurium 395 MS opsonized with hyperimmune anti-MS IgG and a decreased random mobility. This suggests that the presence of exogenous lipid affects the molecular organization providing attachment of a particle to be phagocytosed and alters such surface properties of the cell that are linked to its motility. The results support the hypothesis that liposomes may be used to modify the physico-chemical surface properties of polymorphonuclear leukocytes and so their essential biological functions.
When liposomes encounter the surface of animal cells, their behaviour is not the same in all experimental systems--rather, the fate of the liposome seems to depend on the particular entities of each individual system [1-4]. Possible mechanisms of interaction between liposomes and cells thus include phagocytosis, liposome-cell fusion, lipid exchange and lipid adsorption. Apparently, the possible applications of the interaction depends heavily on which mechanisms that are actually operative. However, if fusion occurs, the interaction might be used as an 1 To whom reprint requests should be addressed. 12-771801
experimental tool for delivering molecules into the plasma membrane of an intact cell [5, 6]. Studies on the mode of interaction between liposomes and polymorphonuclear leukocytes (PMN cells) have lent support to the hypothesis that liposomes, rather than being phagocytosed, may fuse with the plasma membrane of the cell [7]. By implication, liposomal lipid is associated to the cells in a process that by conveying new material to the plasma membrane may modify the cell surface structure. We have therefore entertained the hypothesis that liposomes may be used to modify such Exp Cell Res 108 (1977)
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Dahlgren et al.
properties
of the PMN
cell that are perti-
n e n t to its b i o l o g i c a l f u n c t i o n s . A c c o r d ingly, experiments have been designed to investigate
whether
the
interaction
with
liposomes brings about such changes of the P M N c e l l t h a t d i s c l o s e a m o d i f i c a t i o n o f its surface properties.
In particular, we have
considered the possibility that such a modification might
substantiate
a relationship
between the physico-chemical
surface prop-
erties of PMN cells and essential biological function
s u c h as m o t i l i t y a n d p h a g o c y t i c
capacity. MATERIALS
AND
METHODS
Preparation o f liposomes Source and grades of chemicals have been described elsewhere [7]. Phosphatidylcholine (PC), cholesterol (Chol) and dicetylphosphate (DCP) in molar proportions as described below, were dissolved in chloroform in a roundbottom flask. After rotary evaporation of the solvent in vacuo at 40°C, the lipid film was dried under streaming nitrogen and then mechanically dispersed in water. The lipid dispersion was then subjected to ultrasonic vibration for 5 min in an MSE 100 W Ultrasonic Disintegrator and centrifuged at 20000 g for 30 rain. Only minute amounts (less than 5 %) of the lipids were then pelleted. Following centrifugation, the supernatant fluid was withdrawn and brought to isotonicity either with 3.4 % phosphate-buffered NaC1, pH 7.2, or with a salt solution containing the components of Gey's solution (Gey, 8) in double their usual concentration (2× Gey) but no albumin.
Polymorphonuclear leukocytes ( P M N cells) PMN cells were collected from the peritoneal cavity of guinea pigs 12 h after intraperitoneal injection of 25 ml 0.1% glycogen solution (Nutritional Biochemical Corp., Cleveland, Ohio). The exsudate was centrifuged (200 g, 5 min) and the cells washed once in Krebs-Ringer's phosphate buffer with 10 mM glucose, pH 7.2 (KRG); Ca 2+ and Mg 2÷ were omitted from the medium. To remove contaminating red cells the pellet was resuspended in 6 ml cold distilled water. After 15 sec, 2 ml 3.4% phosphate-buffered NaC1 was added. The cells were then centrifuged (200 g, 5 min) and washed once in KRG. The exsudate cells were finally suspended in KRG or Gey. More than 90% of the cells were polymorphonuclear leukocytes.
L i p o s o m e - P M N cell interaction Liposomes and PMN cells were incubated at 37°C so as to allow the association of liposomal lipid to the PMN cells. The cells were then characterized as de-
Exp CellRes 108 (1977)
scribed below. Details concerning the incubation conditions preceding the different analyses are given under respective heading.
Viability testing To evaluate the viability of the PMN cells after incubation with the liposomes, three methods were used. Vital staining. After incubation with different liposomes (PC-Chol-DCP in molar proportions 70 : 20 : 10 or 79 : 20 : 1, 3 ~zmol lipid/ml) the number of cells excluding Trypan blue (2 mg/ml) were counted in a Bfirker chamber. Measurement of 51Cr-release. The PMN cells were labelled with ~lCr in the following way: One ml of PMN cells (108 PMN/ml) in KRG without Ca ~+ was incubated at 20°C for 20 min with 1 ml of isotonic Na2 51CrO~ (50-100/~Ci/ml phosphate-buffered saline, pH 7.2 (PBS), NEN Chemicals GmbH, D6072 Dreieichenhein). After washing the cells four times in KRG (without Ca 2+) containing 1.0% albumin, they were suspended in KRG (107 cells/ml KRG) and allowed to adhere to the bottom of 5 cm plastic Petri dishes (1.5×107 PMN/dish). The liposomes (PC-Chol-DCP in molar proportions 79 : 20 : 1 or 70 : 20 : 10, 3 /~mol lipid/ml) or, in the control, PBS were then added. After 30 rain incubation, non-associated liposomes were decanted. More than 95 % of the cells remained adhering to the dishes in all cases. Thereafter, 4.5 ml of KRG was added and the cells incubated at 37°C. At indicated intervals, duplicate samples (0.25 ml) were removed, centrifuged (1 500 g, 10 min), and the supernatant analysed for 5~Cr radioactivity. The release was expressed as percentage of the activity released from the cells by 0.2 % (v/v) Triton X-100.
Metabolic response to Concanavalin A (ConA). PMN cells were suspended in KRG (with 0.2 mM glucose) and allowed to adhere to the bottom of 25 ml Erlenmayer flasks (10 T cells/flask). Liposomes (PCChol-DCP in molar proportions 70 : 20 : 10) were added and the cells incubated at 37°C for 30 min and washed four times. Then, ConA (Sigma grade III), KRG with 0.2 mM glucose and 0.5 p.Ci [l-14C]glucose (NEN Chemicals GmbH) were added to give a final volume of 3.0 ml (100 t~g ConA/ml). The release of 14CO2 was assayed as described elsewhere [9].
Sizing o f the P M N cells Twenty-five ml of a dispersion of liposomes (PCChol-DCP in molar proportions 70:20: 1, 0.26-3.20 /~mol lipid/ml PBS) was mixed with an equal volume of a PMN cell suspension (2×107 PMN/ml KRG) and incubated at 37°C. After 30 min, 1 ml samples were withdrawn and put in glass beakers with 39 ml PBS. The size of the PMN cells were then determined by counting in the Coulter Counter ZF with a 100channel pulse-height analyser, the Channelyzer C-1000 (Coulter Electronics Ltd., High Street, South Dunstable, Beds., UK). The counter was calibrated with polystyrene latex spherules (9.79/~m in diameter). The variables on the ZF were set at A = I , I=16 and T=8. On the Channelyzer, the Base Channel Threshold was --4 and the integration range 5-99. With this set of variables more than 95% of the cells were counted.
Interaction o f liposomes with leukocytes. H
177
Phagocytosis system
10 000
5000
I
II
III
Fig. 1. Abscissa: PMN cell population; ordinate: 14CO2 release (cpm/30 min/10 TPMN). ConA-induced stimulation of the HMS-activity in different PMN cell populations (I, untreated, control cells, II, cells preincubated with 1.5 /xmol liposomal lipid/ml, III, cells preincubated with 3 ~mol liposomal lipid/ml).
Two-phase partitioning Twenty-five ml of a dispersion of liposomes (PCChol-DCP in molar proportions 79 : 20 : 1, 3.20 ~mol lipid/ml PBS) was mixed with an equal volume of a PMN cell suspension (2 × 107 PMN/ml KRG) and incubated at 37°C. After 30 min, 0.1 ml samples were withdrawn and pipetted into aqueous biphasic polymer systems [10]. These systems were prepared from 20% (w/w) stock solutions of poly(ethylene-glycol) (PEG) 6000 (Carbowax 6000, Union Carbide, New York), dextran T500 (Pharmacia Fine Chemicals, S-75104 Uppsala), 0.1 M tris (hydroxymetylaminomethane) (Tris) buffer, pH 7.0 and distilled water. The final system used contained 4.4% (w/w) PEG and 6.2% (w/w) dextran in 0.03 M Tris. After temperature equilibration at 4°C overnight in a separation funnel the two phases were separated from each other: the bottom phase rich in dextran, the top phase in PEG. Two ml of each phase were then pipetted into graded centrifuge tubes and the system made isotonic with sucrose. Thereafter 0.2 ml PEG was added, substituted with either bis-trimethyl-amino-PEG (TMA+-PEG) or palmitoyl-PEG (P-PEG, 0.13 mmol palmitic acid per g polymer). The PEG derivatives were dissolved in deionized water and added in amounts to substitute 6.25% (w/w) of the total PEG with TMA+-PEG or 0.2% (w/w)With P-PEG, respectively. The P-PEG was warmed to 45°C before the addition to the phase system. To each tube, 0.1 ml of the cell suspension (1 x 106 PMN cells) was pipetted. The tubes were then inverted 20 times for mixing and kept at 4°C for 45 min. Then, 1 ml samples were withdrawn from each phase and, after mixing, from the combined systems, which also contained the material adhering to the interface between the phases. The number of cells in each sample was then counted in the Channelyzer C-1000 and the percentage of cells in top and bottom phase and of those adhering to the interface calculated.
Monolayers of PMN cells were prepared by pipetting 3 ml of the leukocyte suspension (5×106 PMN/ml KRG) into 50×13 mm plastic tissue culture dishes (Flow Lab., Inglewood Calif.) on the bottom of which were fastened cellulose acetate filters (Millipore Corp.) [11]. The leukocytes were allowed to adhere to the filters for 60 min, and non-adhering leukocytes were washed off with KRG. Approx. 1×106 PMN cells adhered to each filter. To each dish, 3 ml liposome dispersion (3.5 /xmol lipid/ml PBS) or, in the control, 3 ml PBS was added. The dishes were then incubated at 37°C and after 30 min washed twice with KRG to remove unassociated liposomes. More than 95 % of the cells remained adhering to the filters. Their viability was checked with Trypan blue exclusion. The smooth Salmonella typhimurium 395 MS and its mutant rough strain MR 10 were grown for 16 h in nutrient broth (Difco) at 37°C, harvested by centrifugation (6000 g, 10 min) and washed once in PBS. The bacteria were then heat killed at 56°C for 1 h, washed twice in PBS and labelled with ~lCr [11]. To opsonize MS bacteria, rabbit hyperimmune anti-MS IgG was prepared and used as described elsewhere [12]. To the dishes with untreated PMN cells and to those with cells exposed to liposomes were added 5× l0 s unopsonized (R10) or opsonized (MS) bacteria suspended in 4.5 ml KRG. The dishes were then incubated at 37°C. At indicated intervals filters were removed, washed three times in PBS and measured for radioactivity in an Auto-Gamma Scintillation Counter (Intertechnique, Plaisir, France). The phagocytosis was expressed as percent of added radioactivity found per filter.
Motility m e a s u r e m e n t s 108 cells were divided in two equal portions. One portion (5× 107 cells) was suspended in 8 ml of the liposome dispertion (2.8/~mol lipid/ml Gey), the other in 8 ml Gey. The mixtures were incubated at 37°C for 30 min, centrifuged (200 g, 10 rain), and the cells
lxlO3
J j
0
i
2
3
0
o.~3
~
5x102
Figs 2, 3. Abscissa: vol (/~m;~); ordinate: no. of pulses. Fig. 2. Pulse-height (volume) distribution of PMN cells after 30 rain incubation with different concentrations of liposomes; • •., 0; - - -, 0.13; and - - , 1.60/~mol lipid/ml.
Exp CellRes 108 (1977)
178
Dahlgren et al. RESULTS
lx10 3
7:..
~,.IS
~." /'J '..L
{3.5
~ .-!/ //
o
;
i 2
30
~" I
I
I
3
4
5x10 2
Fig. 3. Pulse-height (volume) distribution of PMN cells after incubation with liposomes (1.6/zmol lipid/ml) for different times; -.-, 0; ---, 15; - - - , 30; and - - , 60 min.
washed in Gey. They were then suspended in Gey to 4 x 10r cells/ml and their viability checked with Trypan blue exclusion. The motility of the cells was then measured, using a modification of the method described by Cutler [13]. Agarose (1.5 g) was dissolved in 100 ml water by heating to 100°C. After cooling to 50°C, the agarose solution was mixed with an equal volume of prewarmed 2x Gey and 6 ml of the mixture poured into 60x 1.5 mm plastic tissue culture dishes. Six wells, 2.4 mm in diameter, were cut out in each dish. The wells were then filled with samples (8/zl) of the liposome-treated and untreated (incubated in Gey) PMN cells and the tissue culture dishes incubated at 37°C for 2 h. They were then fixed in methanol, the agarose removed and the cells stained with Giemsa. The migration distance, i.e. the linear distance between the margin of the well and the cell front, was measured with an ocular micrometer in a microscope with a 10x objective and 8x ocular lenses.
Viability More than 90% of cells treated witla the liposomes used throughout this investigation excluded Trypan blue at vital staining. Data on the release of 51Cr activity from 51Cr-labelled cells preincubated with different liposomes showed that during incubation at 37°C for 60 min, 6--8 % of the activity in treated cells and 5 % of the activity in untreated was released. Corresponding values after 120 min were 812 % and 6 %, respectively. The metabolic response to ConA in PMN cells preincubated with different concentrations of liposomes is shown in fig. 1. Thus, ConA stimulated the HMS activity to approximately the same extent in cells treated with 3/~mol liposomal lipid/ml and in untreated cells. It also appeared that in cells preincubated with 1.5 /~mol lipid/ml, ConA induced a greater increase in HMSactivity that it did in control cells. golumf
Fig. 2 shows the pulse-height distribution of PMN cells after incubation with liposomes. After 30 min incubation, the distribution was transposed towards higher thresholds. The magnitude of the transposi-
Table 1. Partition of P M N cells in aqueous biphasic polymer systems containing covalently bound ligands Per cent cells Cells
Two-phase system
Top phase
Interface
Bottom phase
Untreated PMN
Dextran-PEG Dextran-PEG/TMA+-PEG D extran-PEG/palmitoyl-PEG
3+ 1 42_+5 77 + 2
85 + 5 55 + 5 17 + 2
12 + 6 3+ 1 6 _+1
Liposome-treated PMN
Dextran-PEG Dextran-PEG/TMA +-PEG Dextran-PEG/palmitoyl-PEG
2+1 18+2 51 + 7
89+6 80+2 44 + 6
9+6 2+ 1 5+ 2
Exp Cell Res 108 (1977)
Interaction of liposomes with leukocytes. H 2.0
2.0
R10
MS* [gG
1.0
1.0
179
Lx/
J 30
60
90
120
30
60
90
120
Fig. 4. Abscissa: time (min); ordinate: % bacteria phagocytosed/filter. Phagocytosis of (left) S. typhimurium 395 MR 10; (right) S. typhimurium 395 MS opsonized with anti-MS IgG by untreated and liposome-treated PMN cells. A, Untreated cells; A, untreated cells+NaF (5x 10-2 M); O, liposome-treated cells; 0, liposometreated cells+NaF (5x 10-~ M).
tion increased gradually with increasing concentrations of liposomal lipid; at 1.6 tzmol/ml, the increase over untreated cells was 29_+4% (mean of four experiments). As illustrated in fig. 3, the magnitude of the transposition at this lipid concentration increased with increasing time of incubation.
Two-phase partition Table 1 shows the partition of liposometreated PMN cells in aqueous polymer systems. In systems of dextran and PEG, only 2 (_+1) % of the cells were found in the PEGrich top phase; the bulk, 89 (_+6)%, was found at the interface between the phases and the remainder, 9 (_+6) %, in the dextranrich bottom phase. Corresponding values for untreated, control cells were 3 (-+ 1)%, 85 (-+5)% and 12 (-+6)%, respectively. However, in systems containing TMA +PEG only 18 (_+2)% of the liposome-treated as compared to 42 (-+5)% of control cells accumulated in the top phase. Corresponding values in systems containing P-PEG were 51 (-+7) % and 77 (-+2) %, respectively.
Phagocytic capacity As illustrated in fig. 4, the liposome-treated PMN cells showed a reduced capacity to
Fig. 5. Migration of (top) untreated; (bottom) liposome-treated PMN cells from a well cut out in tissue culture dishes with agarose.
phagocytose S. typhimurium 395 MR 10 and S. typhimurium 395MS opsonized with hyperimmune anti-MS IgG. The uptake of R 10 was thus 23 % and that of opsonized MS 32 % of the uptake attained by untreated cells in 120 min. In the presence of 5x 10-2 M NaF, the uptake was inhibited to essentially the same level in the treated and in the untreated cells. Exp Cell Res 108 (1977)
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20 []
-ai- -~ - i - - } 10
-G" 0000 123~56
123~56
Fig. 6. Abscissa: expt no.; ordinate: migration distance (arb. units). Random mobility of HI, untreated; E], liposometreated PMN cells. Each square represents the mean of six different values obtained with the particular PMN cell population used in the experiment. Dashed line, mean of the six expts; vertical bar, S.E.M.
Motility The typical migration of untreated and liposome-treated PMN cells is shown in fig. 5. It thus appeared that the treated cells showed a clear reduction in random mobility. In a series of six experiments the migration of the treated cells was reduced as compared with the controls in five (fig. 6). The migration distance then ranged from 6 % to 29 % of the distance migrated by the untreated cells.
DISCUSSION The use of artificial phospholipid vesicles, i.e. liposomes, has been reported to produce a variety of modifications concerning the physiology of mammalian cells. These include the use of vesicles as carriers to cells of entrapped materials [14, 15] and as agents promoting cell fusion [16] but also as tools for modifying the lipid composition of the membrane of intact cells. It was thus reported that a decrease in the membrane fluidity of malignant lymphoma cells leading to decreased virulence, was obtained Exp Cell Res 108 (1977)
after treating the cells with cholesterol-containing liposomes [17]. Recently, Martin & MacDonald showed that the hapten 2,4dinitrophenyl- aminocaproyl- phosphatidylethanolamine (DNP-Cap-PE) may be inserted into the membrane of red cells through fusion of DNP-Cap-PE liposomes with the cells [6]. We have obtained some evidence to support the idea of using liposomes to insert lipid molecules into the plasma membrane of intact polymorphonuclear leukocytes [7]. Thus, as to the predominant mechanism of interaction between liposomes and PMN cells, we have considered fusion of the lipid lamellae with the cell membrane the explanation most consistent with the previously described observations. The experiments described in this paper were designed to study the eventual consequences of the interaction, i.e. to investigate whether liposome-treated PMN cells show characteristics that disclose a modification of their surface properties. The liposomes used in this investigation was prepared by brief ultrasonication whereas the liposomes used to study the mode of interaction were not sonicated at all. In view of the differences that may be obtained with different liposomes, this point merits comment. The association of liposomal lipid to the PMN cells was independent of the sonication as far as characteristics of the interaction are concerned: the association was insensitive to metabolic inhibitors, proceeded without activation of the HMS, was not influenced by omitting divalent cations but was inhibited at low temperature. These findings are in agreement with those by Poste & Papahadjopoulos [18], who have shown that similar results are obtained with both unilamellar and multilamellar vesicles and argued that vesicle composition rather than size is important in determining the path-
Interaction of liposomes with leukocytes. H way of uptake. However, with the use of sonicated liposomes, Significantly more lipid associated to the PMN cells. A necessary prerequisite for the evaluation of any experiments performed was therefore that the treated cells stayed alive. To evaluate the viability of the cells in the current study several methods were used. More than 90 % of the cells excluded Trypan blue at vital staining. During incubation at 37°C, the leakage of ~lCrlabelled intrace!lular proteins (mol. wt 80 000-250 000) [19] from treated cells were close to that from untreated: minor amounts (less than 10%) of the ~lCr activity was released during 60 min. Since this test system is a sensitive indicator of cytotoxicity [20], the results were taken to indicate that liposome-treated PMN cells did not loose their viability. Furthermore, ConA perturbed the cell membrane and stimulated the HMS activity in the treated cells. Assuming that the metabolic effects are mediated by lectin bound to the cell surface and not by ConA which has been pinocytosed [21], this finding suggests that the liposome-treated PMN cells maintained the function of (a) the ConA receptor; (b) the transduction mechanism conveying the signal; and (c) the biochemical target affected. Indeed, PMN cells preincubated with 1.5/zmol liposomal lipid/ml showed an enhanced metabolic response to ConA. This finding may indicate a change in the number of ConA-binding sites or in the molecular organization providing transduction of the signal. If the liposome bilayers fuse with the cell membrane, one would expect the interaction to cause an increase in cell surface area and, possibly, an increase in cell volume [2]. Experiments were therefore designed to determine the size of liposometreated PMN cells, using an electronic par-
181
ticle counter provided with a pulse-height analyzer. After incubation with liposomes, the pulse-height (volume) distribution of the cells was transposed towards higher thresholds. The findings that the magnitude of the transposition increased with concentrations of liposomal lipid and, for a given concentration, with the time of incubation suggest that the liposome-mediated introduction of lipids is a process by which the volume of the PMN cells is increased. Assuming that this is accomplished by an increase in cell surface area, the observations are consistent with the formation of a cell membrane comprising exogenous, liposomederived lipid. Attempts were then made to describe, in physico-chemical terms, the surface changes inflicted by the exogenous lipid through determining the partition of liposome-treated cells in aqueous biphasic polymer systems. In systems of dextran and PEG such cells partitioned as control, untreated cells and in systems containing PEG-bound ligands, bis-trimethylaminoand palmitoyl-residues covalently bound to the PEG molecule, both untreated and liposome-treated cells collected to the PEGrich top phase. This redistribution demonstrated the tendency to interact with positively charged TMA+-PEG and hydrophobic palmitoyl-PEG, respectively. However, the magnitude of the redistribution of liposome-treated cells was less than that of controls in both systems investigated. This indicates that on treated cells, the number of binding sites per surface area for TMA + and palmitoyl residues were reduced. The increase in cell surface area as discussed above may result in a reduction in negative surface charge density and subsequently a reduction in the affinity for TMA +. Since this affinity is due mainly to the negatively charged sialic acid-rich glycoproteins at the periphery of the cell, such an increase in Exp Cell Res 108 (1977)
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Dahlgren et al.
surface area is likely to overshadow any effect of introducing minute amounts of charged lipid in the membrane bilayer. The reduced liability to hydrophobic interaction indicates either that components governing such interaction were lost from the treated cells or that the presence of exogenous lipid in these cells increased the total hydrophobic interaction within their membranes such that interaction with the hydrophobic probe was impeded. We have found no evidence for the first hypothesis and favour the second. The data thus demonstrate that the interaction with liposomes modified the physicochemical surface properties of the PMN cells. It seemed reasonable to hypothesize that such modifications would influence the behaviour of the PMN cells in biological systems. The present experiments show that the interaction with liposomes alters the phagocytic activity and the random mobility of the cells. They do not, however, disclose the precise characteristics of the functional modification, i.e. the mode by which the exogenous lipid exerts its effects. The effects on phagocytosis may thus be derived from alterations in either or both of two phagocytic steps; the attachment and the internalization. The phagocytosis of Salmonella typhimurium 395 MR 10 and of Salmonella typhimurium 395 MS opsonized with anti-MS IgG is governed by the physico-chemical surface properties of the respective particle [12, 22]. Both particles are, in contrast to the phagocytosis-resistant smooth strain Salmonella typhimurium 395 MS, negatively charged [23] and hydrophobic [2224]. On the basis of the physico-chemical surface properties of particles in relation to their liability to phagocytosis, we [23, 24] and others [22] have put forward the hypothesis that negative surface charge and Exp Cell Res 108 (1977)
hydrophobicity of a particle to be phagocytosed promote its attachment to the phagocytic cell surface. In the current investigation, liposome-treated PMN cells showed a reduced capacity to phagocytose. S. typhimurium 395 MR 10 and S. typhimurium 395 MS opsonized with anti-MS IgG. Since the treated cells as compared with untreated, control cells are less negatively charged and less liable to hydrophobic interaction, our finding is consistent with the hypothesis that attachment is brought about by hydrophogic-hydrophobic bonding and charged-linked associations such as ionic bridges. We thus suggest that the presence of exogenous lipid in the plasma membrane of the liposometreated PMN cells affects the molecular organisation providing attachment of the particle to be phagocytosed. Dianzani et al. have shown that an increase in plasmamembrane cholesterol is accompanied by a significant reduction in the phagocytic uptake of latex particles or lipid droplets by macrophages [25]. These findings are consistent with ours, since cholesterol may induce condensation of phospholipid molecules, restrict motion of acyl chains and increase their perpendicular orientation [26, 27], all of which would increase the total hydrophobic interactions within the phagocytic cell membrane. On the other hand, Heiniger et al. have shown that L cells (mouse fibroblasts) which are deficient in sterol synthesis are unable to endocytose horseradish peroxidase at normal rates [28]. Provided phagocytosis in macrophages and endocytosis in cultured L cells are accomplished by the same basic mechanism these findings indicate that the cholesterol/phospholipid ratio in phagocytic cell membranes must be maintained within a fairly narrow range to ensure their proper biological functioning. This point may be clarified by the
Interaction of liposomes with leukocytes. H
183
possible use of liposomes in introducing surface charge density and liability to phospholipids together with or without hydrophobic interaction may alter the adhecholesterol in the phagocytic cell membrane. siveness or the deformability of the cells, The possibility that the exogenous lipid two properties closely linked to their motilimpaired the triggering of the internalization ity [34-36]. phase cannot be precluded. A decrease in It is evident that the experiments persurface charge has been shown to reduce formed do not permit any explanation as to the membrane-mediated triggering of oxida- the specific structural alterations that may tive metabolism during phagocytosis [29, be responsible for the registered effects. 30]. Analogously, this triggering may be Yet, we interpret the present findings to inreduced by manipulations that change the dicate that the system used may create a membrane permeability for certain ions tool for modifying the behaviour of PMN (Ca2 +, MgZ+), known to be pertinent for the cells and to disclose functional requireactivation of PMN cells [31, 32]. In the ments and molecular mechanisms for the present system, however, ConA evokes attachment and internalization phases of normal or even augmented increase in HMS the phagocytic process. activity. Although internalization and oxiThe technical assistance of Ellinor Granstr6m and dative metabolism may not be closely co- Britinger Thor6n is gratefully acknowledged. This ordinated during all experimental condi- study was supported by grant 16X-2183-10 from The Swedish Medical Research Council. tions [29], this finding suggests that the metabolic excitability of the liposomeREFERENCES treated cells was unimpaired. In addition, Gregoriadis, G, New Englj reed 295 (1976) 704. the demonstration that the metabolic in- 2.1. Batzri, S & Korn, E D, J cell biol 66 (1975) 621. hibitor sodium fluoride inhibited phago- 3. Ehnholm, C & Zilversmit, D B, J biol chem 248 (1973) 1719. cytosis also in the liposome-treated cells 4. Papahadjopoulos, D, Poste, G & Mayhew, E, shows that internalization does proceed in B iochim biophys acta 363 (1974) 404. Pagano, R E & Huang, L, J cell bio167 (1975) 49. these cells. However, the rate of this pro- 5. 6. Martin, F C & MacDonald, R C, J cell biol 70 cess remains to be determined. (1976) 515. To explain the decreased random mobil- 7. Stendahl, O & Tagesson, C, Exp cell res 108 (1977) 167. ity in the liposome-treated cells, some hy- 8. Keller, H U & Sorkin, E, Int arch allergy 31 (1967) 575. pothesis may be put forward. The impair9. Br6te, L & Stendahl, O, Acta chir Scand 141 ment can not be accounted for by extensive (1975) 565. phagocytosis of liposomes; the criteria for 10. Albertsson, P-/~, Partition of cell particles and macromolecules. Almqvist & Wiksell, Uppsala, such a mode of interaction are not met [7]. Academic Press, New York (1971). O & Edebo, L, Acta pathol microbiol It might be accounted for by the lysis of 11. Stendahl, Scand sect B 80 (1972) 481. contaminating red cells and a subsequent 12. Stendahl, O, Tagesson, C & Edebo, L, Infect immun 10 (1974) 316. release of chemotactic material [33], form13. Cutler, J, Proc soc exp biol med 147 (1974) 471. ing a negative gradient keeping the cells 14. Gregoriadis, G, New Englj reed 295 (1976) 704. D, Poste, G & Mayhew, E, back in the well. Preliminary experiments 15. Papahadjopoulos, Biochim biophys acta 363 (1974) 404. show, however, that although the random 16. Papahadjopoulos, D, Poste, G & Schaeffer, B E, Biochim biophys acta 323 (1973) 23. mobility of the cells are impaired, their 17. Inbar, M & Shinitzky, M, Proc natl acad sci US 71 ability to respond to chemotactic stimuli (1974) 2128. 18. Poste, G & Papahadjopoulos, D, Proc natl acad sci during these experimental conditions are US 73 (1976) 1603. unaffected. More likely, the alterations in 19. Henney, C S, J immunol 110 (1973) 73. Exp Cell Res 108 (1977)
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20. Scharmann, W, Infect immun 13 (1976) 836. 21. Romeo, D, Jug, M, Zabucchi, G & Rossi, F, FEBS lett 42 (1974) 90. 22. Cunningham, R K, S6derstr6m, T O, Gillman, C F & van Oss, C J, Immunol commun 4 (1974) 429. 23. Stendahl, O, Magnusson, K-E & Tagesson, C, Proc 1st Eur conf on phagocytic leukocytes (Movement, metabolism and bactericidal mechanisms of phagocytes) Trieste (1976). in press. 24. Edebo, L, Lindstr6m, F, SkOldstam, L, Stendahl, O & Tagesson, C, Immunol commun 6 (1975) 87. 25. Dianzani, M U, Torrielli, M V, Canuto, R A, Carcea, R & Feo, F, J pathol 118 (1976) 193. 26. Steim, G M, Mitochondria biogenesis and bioenergetics. Biomembranes: molecular arrangements and transport mechanisms. FEBS, 8th meeting (ed S G Van Denbergh, P Borst, L L M van Deenen, J C Riemersma, E C Slater & J M Targer) p. 185. North-Holland, Amsterdam (1972). 27. Chapman, D, Biological membranes (ed D Chapman & D F t t Wallach) vol. 2, p. 91. Academic Press, New York (1973).
E.W Cell Res 108 (1977)
28. Heiniger, H J, Kandutsch, A A & Chen, H W, Nature 263 (1976) 515. 29. Tsan, M F & McIntyre, P A, J exp med 143 (1976) 1308. 30. Tsan, M F, Newman, B & McIntyre, P A, Brit j haematol 33 (1976) 189. 31. Romeo, D, Zabucchi, G, Miami, N & Rossi, F, Nature 253 (1975) 542. 32. Schell-Frederick, E, FEBS lett 48 (1975) 37. 33. Bessis, M, Antibiotics and chemotherapy, vol. 19, p. 369. Karper, Basel (1974). 34. Lichman, M A & Weed, R I, Blood 39 (1972) 301. 35. Carter, S B, Nature 213 (1967) 256. 36. Miller, M E, The phagocytic cell in host resistance (ed J A Bellanti & D H Dayton) p. 295. Raven Press, New York (1975).
Received January 31, 1977 Accepted March 10, 1977
Experimental Cell Research 108 (1977) 175-184
I N T E R A C T I O N OF LIPOSOMES W I T H POLYMORPHONUCLEAR LEUKOCYTES II. Studies on the Consequences of Interaction C. DAHLGREN, E. KIHLSTR~)M, K.-E. MAGNUSSON, O. STENDAHL and C. TAGESSON 1
Department of Medical Microbiology, University of Link6ping, S-58185 Link6ping, Sweden
SUMMARY Polymorphonuclear leukocytes were incubated at 37°C with liposomes composed of phosphatidylcholine, cholesterol and dicetylphosphate and the cells obtained characterized with respect to properties that would disclose a modification of their surface properties. The cells excluded Trypan blue, did not leak ~lCr-labelled cytoplasmic proteins and responded as control cells to concanavalin A (ConA) with an increased HMS activity. Their volume was increased as revealed by the pulse-height distribution at electronic counting. The partition of the cells in aqueous biphasic systems containing polymers with covalently bound ligands showed that their negative surface charge density and their liability to hydrophobic interaction were decreased. The cells showed a reduced capacity to phagocytose S. typhimurium 395 MR 10 and S. typhimurium 395 MS opsonized with hyperimmune anti-MS IgG and a decreased random mobility. This suggests that the presence of exogenous lipid affects the molecular organization providing attachment of a particle to be phagocytosed and alters such surface properties of the cell that are linked to its motility. The results support the hypothesis that liposomes may be used to modify the physico-chemical surface properties of polymorphonuclear leukocytes and so their essential biological functions.
When liposomes encounter the surface of animal cells, their behaviour is not the same in all experimental systems--rather, the fate of the liposome seems to depend on the particular entities of each individual system [1-4]. Possible mechanisms of interaction between liposomes and cells thus include phagocytosis, liposome-cell fusion, lipid exchange and lipid adsorption. Apparently, the possible applications of the interaction depends heavily on which mechanisms that are actually operative. However, if fusion occurs, the interaction might be used as an 1 To whom reprint requests should be addressed. 12-771801
experimental tool for delivering molecules into the plasma membrane of an intact cell [5, 6]. Studies on the mode of interaction between liposomes and polymorphonuclear leukocytes (PMN cells) have lent support to the hypothesis that liposomes, rather than being phagocytosed, may fuse with the plasma membrane of the cell [7]. By implication, liposomal lipid is associated to the cells in a process that by conveying new material to the plasma membrane may modify the cell surface structure. We have therefore entertained the hypothesis that liposomes may be used to modify such Exp Cell Res 108 (1977)
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properties
of the PMN
cell that are perti-
n e n t to its b i o l o g i c a l f u n c t i o n s . A c c o r d ingly, experiments have been designed to investigate
whether
the
interaction
with
liposomes brings about such changes of the P M N c e l l t h a t d i s c l o s e a m o d i f i c a t i o n o f its surface properties.
In particular, we have
considered the possibility that such a modification might
substantiate
a relationship
between the physico-chemical
surface prop-
erties of PMN cells and essential biological function
s u c h as m o t i l i t y a n d p h a g o c y t i c
capacity. MATERIALS
AND
METHODS
Preparation o f liposomes Source and grades of chemicals have been described elsewhere [7]. Phosphatidylcholine (PC), cholesterol (Chol) and dicetylphosphate (DCP) in molar proportions as described below, were dissolved in chloroform in a roundbottom flask. After rotary evaporation of the solvent in vacuo at 40°C, the lipid film was dried under streaming nitrogen and then mechanically dispersed in water. The lipid dispersion was then subjected to ultrasonic vibration for 5 min in an MSE 100 W Ultrasonic Disintegrator and centrifuged at 20000 g for 30 rain. Only minute amounts (less than 5 %) of the lipids were then pelleted. Following centrifugation, the supernatant fluid was withdrawn and brought to isotonicity either with 3.4 % phosphate-buffered NaC1, pH 7.2, or with a salt solution containing the components of Gey's solution (Gey, 8) in double their usual concentration (2× Gey) but no albumin.
Polymorphonuclear leukocytes ( P M N cells) PMN cells were collected from the peritoneal cavity of guinea pigs 12 h after intraperitoneal injection of 25 ml 0.1% glycogen solution (Nutritional Biochemical Corp., Cleveland, Ohio). The exsudate was centrifuged (200 g, 5 min) and the cells washed once in Krebs-Ringer's phosphate buffer with 10 mM glucose, pH 7.2 (KRG); Ca 2+ and Mg 2÷ were omitted from the medium. To remove contaminating red cells the pellet was resuspended in 6 ml cold distilled water. After 15 sec, 2 ml 3.4% phosphate-buffered NaC1 was added. The cells were then centrifuged (200 g, 5 min) and washed once in KRG. The exsudate cells were finally suspended in KRG or Gey. More than 90% of the cells were polymorphonuclear leukocytes.
L i p o s o m e - P M N cell interaction Liposomes and PMN cells were incubated at 37°C so as to allow the association of liposomal lipid to the PMN cells. The cells were then characterized as de-
Exp CellRes 108 (1977)
scribed below. Details concerning the incubation conditions preceding the different analyses are given under respective heading.
Viability testing To evaluate the viability of the PMN cells after incubation with the liposomes, three methods were used. Vital staining. After incubation with different liposomes (PC-Chol-DCP in molar proportions 70 : 20 : 10 or 79 : 20 : 1, 3 ~zmol lipid/ml) the number of cells excluding Trypan blue (2 mg/ml) were counted in a Bfirker chamber. Measurement of 51Cr-release. The PMN cells were labelled with ~lCr in the following way: One ml of PMN cells (108 PMN/ml) in KRG without Ca ~+ was incubated at 20°C for 20 min with 1 ml of isotonic Na2 51CrO~ (50-100/~Ci/ml phosphate-buffered saline, pH 7.2 (PBS), NEN Chemicals GmbH, D6072 Dreieichenhein). After washing the cells four times in KRG (without Ca 2+) containing 1.0% albumin, they were suspended in KRG (107 cells/ml KRG) and allowed to adhere to the bottom of 5 cm plastic Petri dishes (1.5×107 PMN/dish). The liposomes (PC-Chol-DCP in molar proportions 79 : 20 : 1 or 70 : 20 : 10, 3 /~mol lipid/ml) or, in the control, PBS were then added. After 30 rain incubation, non-associated liposomes were decanted. More than 95 % of the cells remained adhering to the dishes in all cases. Thereafter, 4.5 ml of KRG was added and the cells incubated at 37°C. At indicated intervals, duplicate samples (0.25 ml) were removed, centrifuged (1 500 g, 10 min), and the supernatant analysed for 5~Cr radioactivity. The release was expressed as percentage of the activity released from the cells by 0.2 % (v/v) Triton X-100.
Metabolic response to Concanavalin A (ConA). PMN cells were suspended in KRG (with 0.2 mM glucose) and allowed to adhere to the bottom of 25 ml Erlenmayer flasks (10 T cells/flask). Liposomes (PCChol-DCP in molar proportions 70 : 20 : 10) were added and the cells incubated at 37°C for 30 min and washed four times. Then, ConA (Sigma grade III), KRG with 0.2 mM glucose and 0.5 p.Ci [l-14C]glucose (NEN Chemicals GmbH) were added to give a final volume of 3.0 ml (100 t~g ConA/ml). The release of 14CO2 was assayed as described elsewhere [9].
Sizing o f the P M N cells Twenty-five ml of a dispersion of liposomes (PCChol-DCP in molar proportions 70:20: 1, 0.26-3.20 /~mol lipid/ml PBS) was mixed with an equal volume of a PMN cell suspension (2×107 PMN/ml KRG) and incubated at 37°C. After 30 min, 1 ml samples were withdrawn and put in glass beakers with 39 ml PBS. The size of the PMN cells were then determined by counting in the Coulter Counter ZF with a 100channel pulse-height analyser, the Channelyzer C-1000 (Coulter Electronics Ltd., High Street, South Dunstable, Beds., UK). The counter was calibrated with polystyrene latex spherules (9.79/~m in diameter). The variables on the ZF were set at A = I , I=16 and T=8. On the Channelyzer, the Base Channel Threshold was --4 and the integration range 5-99. With this set of variables more than 95% of the cells were counted.
Interaction o f liposomes with leukocytes. H
177
Phagocytosis system
10 000
5000
I
II
III
Fig. 1. Abscissa: PMN cell population; ordinate: 14CO2 release (cpm/30 min/10 TPMN). ConA-induced stimulation of the HMS-activity in different PMN cell populations (I, untreated, control cells, II, cells preincubated with 1.5 /xmol liposomal lipid/ml, III, cells preincubated with 3 ~mol liposomal lipid/ml).
Two-phase partitioning Twenty-five ml of a dispersion of liposomes (PCChol-DCP in molar proportions 79 : 20 : 1, 3.20 ~mol lipid/ml PBS) was mixed with an equal volume of a PMN cell suspension (2 × 107 PMN/ml KRG) and incubated at 37°C. After 30 min, 0.1 ml samples were withdrawn and pipetted into aqueous biphasic polymer systems [10]. These systems were prepared from 20% (w/w) stock solutions of poly(ethylene-glycol) (PEG) 6000 (Carbowax 6000, Union Carbide, New York), dextran T500 (Pharmacia Fine Chemicals, S-75104 Uppsala), 0.1 M tris (hydroxymetylaminomethane) (Tris) buffer, pH 7.0 and distilled water. The final system used contained 4.4% (w/w) PEG and 6.2% (w/w) dextran in 0.03 M Tris. After temperature equilibration at 4°C overnight in a separation funnel the two phases were separated from each other: the bottom phase rich in dextran, the top phase in PEG. Two ml of each phase were then pipetted into graded centrifuge tubes and the system made isotonic with sucrose. Thereafter 0.2 ml PEG was added, substituted with either bis-trimethyl-amino-PEG (TMA+-PEG) or palmitoyl-PEG (P-PEG, 0.13 mmol palmitic acid per g polymer). The PEG derivatives were dissolved in deionized water and added in amounts to substitute 6.25% (w/w) of the total PEG with TMA+-PEG or 0.2% (w/w)With P-PEG, respectively. The P-PEG was warmed to 45°C before the addition to the phase system. To each tube, 0.1 ml of the cell suspension (1 x 106 PMN cells) was pipetted. The tubes were then inverted 20 times for mixing and kept at 4°C for 45 min. Then, 1 ml samples were withdrawn from each phase and, after mixing, from the combined systems, which also contained the material adhering to the interface between the phases. The number of cells in each sample was then counted in the Channelyzer C-1000 and the percentage of cells in top and bottom phase and of those adhering to the interface calculated.
Monolayers of PMN cells were prepared by pipetting 3 ml of the leukocyte suspension (5×106 PMN/ml KRG) into 50×13 mm plastic tissue culture dishes (Flow Lab., Inglewood Calif.) on the bottom of which were fastened cellulose acetate filters (Millipore Corp.) [11]. The leukocytes were allowed to adhere to the filters for 60 min, and non-adhering leukocytes were washed off with KRG. Approx. 1×106 PMN cells adhered to each filter. To each dish, 3 ml liposome dispersion (3.5 /xmol lipid/ml PBS) or, in the control, 3 ml PBS was added. The dishes were then incubated at 37°C and after 30 min washed twice with KRG to remove unassociated liposomes. More than 95 % of the cells remained adhering to the filters. Their viability was checked with Trypan blue exclusion. The smooth Salmonella typhimurium 395 MS and its mutant rough strain MR 10 were grown for 16 h in nutrient broth (Difco) at 37°C, harvested by centrifugation (6000 g, 10 min) and washed once in PBS. The bacteria were then heat killed at 56°C for 1 h, washed twice in PBS and labelled with ~lCr [11]. To opsonize MS bacteria, rabbit hyperimmune anti-MS IgG was prepared and used as described elsewhere [12]. To the dishes with untreated PMN cells and to those with cells exposed to liposomes were added 5× l0 s unopsonized (R10) or opsonized (MS) bacteria suspended in 4.5 ml KRG. The dishes were then incubated at 37°C. At indicated intervals filters were removed, washed three times in PBS and measured for radioactivity in an Auto-Gamma Scintillation Counter (Intertechnique, Plaisir, France). The phagocytosis was expressed as percent of added radioactivity found per filter.
Motility m e a s u r e m e n t s 108 cells were divided in two equal portions. One portion (5× 107 cells) was suspended in 8 ml of the liposome dispertion (2.8/~mol lipid/ml Gey), the other in 8 ml Gey. The mixtures were incubated at 37°C for 30 min, centrifuged (200 g, 10 rain), and the cells
lxlO3
J j
0
i
2
3
0
o.~3
~
5x102
Figs 2, 3. Abscissa: vol (/~m;~); ordinate: no. of pulses. Fig. 2. Pulse-height (volume) distribution of PMN cells after 30 rain incubation with different concentrations of liposomes; • •., 0; - - -, 0.13; and - - , 1.60/~mol lipid/ml.
Exp CellRes 108 (1977)
178
Dahlgren et al. RESULTS
lx10 3
7:..
~,.IS
~." /'J '..L
{3.5
~ .-!/ //
o
;
i 2
30
~" I
I
I
3
4
5x10 2
Fig. 3. Pulse-height (volume) distribution of PMN cells after incubation with liposomes (1.6/zmol lipid/ml) for different times; -.-, 0; ---, 15; - - - , 30; and - - , 60 min.
washed in Gey. They were then suspended in Gey to 4 x 10r cells/ml and their viability checked with Trypan blue exclusion. The motility of the cells was then measured, using a modification of the method described by Cutler [13]. Agarose (1.5 g) was dissolved in 100 ml water by heating to 100°C. After cooling to 50°C, the agarose solution was mixed with an equal volume of prewarmed 2x Gey and 6 ml of the mixture poured into 60x 1.5 mm plastic tissue culture dishes. Six wells, 2.4 mm in diameter, were cut out in each dish. The wells were then filled with samples (8/zl) of the liposome-treated and untreated (incubated in Gey) PMN cells and the tissue culture dishes incubated at 37°C for 2 h. They were then fixed in methanol, the agarose removed and the cells stained with Giemsa. The migration distance, i.e. the linear distance between the margin of the well and the cell front, was measured with an ocular micrometer in a microscope with a 10x objective and 8x ocular lenses.
Viability More than 90% of cells treated witla the liposomes used throughout this investigation excluded Trypan blue at vital staining. Data on the release of 51Cr activity from 51Cr-labelled cells preincubated with different liposomes showed that during incubation at 37°C for 60 min, 6--8 % of the activity in treated cells and 5 % of the activity in untreated was released. Corresponding values after 120 min were 812 % and 6 %, respectively. The metabolic response to ConA in PMN cells preincubated with different concentrations of liposomes is shown in fig. 1. Thus, ConA stimulated the HMS activity to approximately the same extent in cells treated with 3/~mol liposomal lipid/ml and in untreated cells. It also appeared that in cells preincubated with 1.5 /~mol lipid/ml, ConA induced a greater increase in HMSactivity that it did in control cells. golumf
Fig. 2 shows the pulse-height distribution of PMN cells after incubation with liposomes. After 30 min incubation, the distribution was transposed towards higher thresholds. The magnitude of the transposi-
Table 1. Partition of P M N cells in aqueous biphasic polymer systems containing covalently bound ligands Per cent cells Cells
Two-phase system
Top phase
Interface
Bottom phase
Untreated PMN
Dextran-PEG Dextran-PEG/TMA+-PEG D extran-PEG/palmitoyl-PEG
3+ 1 42_+5 77 + 2
85 + 5 55 + 5 17 + 2
12 + 6 3+ 1 6 _+1
Liposome-treated PMN
Dextran-PEG Dextran-PEG/TMA +-PEG Dextran-PEG/palmitoyl-PEG
2+1 18+2 51 + 7
89+6 80+2 44 + 6
9+6 2+ 1 5+ 2
Exp Cell Res 108 (1977)
Interaction of liposomes with leukocytes. H 2.0
2.0
R10
MS* [gG
1.0
1.0
179
Lx/
J 30
60
90
120
30
60
90
120
Fig. 4. Abscissa: time (min); ordinate: % bacteria phagocytosed/filter. Phagocytosis of (left) S. typhimurium 395 MR 10; (right) S. typhimurium 395 MS opsonized with anti-MS IgG by untreated and liposome-treated PMN cells. A, Untreated cells; A, untreated cells+NaF (5x 10-2 M); O, liposome-treated cells; 0, liposometreated cells+NaF (5x 10-~ M).
tion increased gradually with increasing concentrations of liposomal lipid; at 1.6 tzmol/ml, the increase over untreated cells was 29_+4% (mean of four experiments). As illustrated in fig. 3, the magnitude of the transposition at this lipid concentration increased with increasing time of incubation.
Two-phase partition Table 1 shows the partition of liposometreated PMN cells in aqueous polymer systems. In systems of dextran and PEG, only 2 (_+1) % of the cells were found in the PEGrich top phase; the bulk, 89 (_+6)%, was found at the interface between the phases and the remainder, 9 (_+6) %, in the dextranrich bottom phase. Corresponding values for untreated, control cells were 3 (-+ 1)%, 85 (-+5)% and 12 (-+6)%, respectively. However, in systems containing TMA +PEG only 18 (_+2)% of the liposome-treated as compared to 42 (-+5)% of control cells accumulated in the top phase. Corresponding values in systems containing P-PEG were 51 (-+7) % and 77 (-+2) %, respectively.
Phagocytic capacity As illustrated in fig. 4, the liposome-treated PMN cells showed a reduced capacity to
Fig. 5. Migration of (top) untreated; (bottom) liposome-treated PMN cells from a well cut out in tissue culture dishes with agarose.
phagocytose S. typhimurium 395 MR 10 and S. typhimurium 395MS opsonized with hyperimmune anti-MS IgG. The uptake of R 10 was thus 23 % and that of opsonized MS 32 % of the uptake attained by untreated cells in 120 min. In the presence of 5x 10-2 M NaF, the uptake was inhibited to essentially the same level in the treated and in the untreated cells. Exp Cell Res 108 (1977)
180
Dahlgren et al.
20 []
-ai- -~ - i - - } 10
-G" 0000 123~56
123~56
Fig. 6. Abscissa: expt no.; ordinate: migration distance (arb. units). Random mobility of HI, untreated; E], liposometreated PMN cells. Each square represents the mean of six different values obtained with the particular PMN cell population used in the experiment. Dashed line, mean of the six expts; vertical bar, S.E.M.
Motility The typical migration of untreated and liposome-treated PMN cells is shown in fig. 5. It thus appeared that the treated cells showed a clear reduction in random mobility. In a series of six experiments the migration of the treated cells was reduced as compared with the controls in five (fig. 6). The migration distance then ranged from 6 % to 29 % of the distance migrated by the untreated cells.
DISCUSSION The use of artificial phospholipid vesicles, i.e. liposomes, has been reported to produce a variety of modifications concerning the physiology of mammalian cells. These include the use of vesicles as carriers to cells of entrapped materials [14, 15] and as agents promoting cell fusion [16] but also as tools for modifying the lipid composition of the membrane of intact cells. It was thus reported that a decrease in the membrane fluidity of malignant lymphoma cells leading to decreased virulence, was obtained Exp Cell Res 108 (1977)
after treating the cells with cholesterol-containing liposomes [17]. Recently, Martin & MacDonald showed that the hapten 2,4dinitrophenyl- aminocaproyl- phosphatidylethanolamine (DNP-Cap-PE) may be inserted into the membrane of red cells through fusion of DNP-Cap-PE liposomes with the cells [6]. We have obtained some evidence to support the idea of using liposomes to insert lipid molecules into the plasma membrane of intact polymorphonuclear leukocytes [7]. Thus, as to the predominant mechanism of interaction between liposomes and PMN cells, we have considered fusion of the lipid lamellae with the cell membrane the explanation most consistent with the previously described observations. The experiments described in this paper were designed to study the eventual consequences of the interaction, i.e. to investigate whether liposome-treated PMN cells show characteristics that disclose a modification of their surface properties. The liposomes used in this investigation was prepared by brief ultrasonication whereas the liposomes used to study the mode of interaction were not sonicated at all. In view of the differences that may be obtained with different liposomes, this point merits comment. The association of liposomal lipid to the PMN cells was independent of the sonication as far as characteristics of the interaction are concerned: the association was insensitive to metabolic inhibitors, proceeded without activation of the HMS, was not influenced by omitting divalent cations but was inhibited at low temperature. These findings are in agreement with those by Poste & Papahadjopoulos [18], who have shown that similar results are obtained with both unilamellar and multilamellar vesicles and argued that vesicle composition rather than size is important in determining the path-
Interaction of liposomes with leukocytes. H way of uptake. However, with the use of sonicated liposomes, Significantly more lipid associated to the PMN cells. A necessary prerequisite for the evaluation of any experiments performed was therefore that the treated cells stayed alive. To evaluate the viability of the cells in the current study several methods were used. More than 90 % of the cells excluded Trypan blue at vital staining. During incubation at 37°C, the leakage of ~lCrlabelled intrace!lular proteins (mol. wt 80 000-250 000) [19] from treated cells were close to that from untreated: minor amounts (less than 10%) of the ~lCr activity was released during 60 min. Since this test system is a sensitive indicator of cytotoxicity [20], the results were taken to indicate that liposome-treated PMN cells did not loose their viability. Furthermore, ConA perturbed the cell membrane and stimulated the HMS activity in the treated cells. Assuming that the metabolic effects are mediated by lectin bound to the cell surface and not by ConA which has been pinocytosed [21], this finding suggests that the liposome-treated PMN cells maintained the function of (a) the ConA receptor; (b) the transduction mechanism conveying the signal; and (c) the biochemical target affected. Indeed, PMN cells preincubated with 1.5/zmol liposomal lipid/ml showed an enhanced metabolic response to ConA. This finding may indicate a change in the number of ConA-binding sites or in the molecular organization providing transduction of the signal. If the liposome bilayers fuse with the cell membrane, one would expect the interaction to cause an increase in cell surface area and, possibly, an increase in cell volume [2]. Experiments were therefore designed to determine the size of liposometreated PMN cells, using an electronic par-
181
ticle counter provided with a pulse-height analyzer. After incubation with liposomes, the pulse-height (volume) distribution of the cells was transposed towards higher thresholds. The findings that the magnitude of the transposition increased with concentrations of liposomal lipid and, for a given concentration, with the time of incubation suggest that the liposome-mediated introduction of lipids is a process by which the volume of the PMN cells is increased. Assuming that this is accomplished by an increase in cell surface area, the observations are consistent with the formation of a cell membrane comprising exogenous, liposomederived lipid. Attempts were then made to describe, in physico-chemical terms, the surface changes inflicted by the exogenous lipid through determining the partition of liposome-treated cells in aqueous biphasic polymer systems. In systems of dextran and PEG such cells partitioned as control, untreated cells and in systems containing PEG-bound ligands, bis-trimethylaminoand palmitoyl-residues covalently bound to the PEG molecule, both untreated and liposome-treated cells collected to the PEGrich top phase. This redistribution demonstrated the tendency to interact with positively charged TMA+-PEG and hydrophobic palmitoyl-PEG, respectively. However, the magnitude of the redistribution of liposome-treated cells was less than that of controls in both systems investigated. This indicates that on treated cells, the number of binding sites per surface area for TMA + and palmitoyl residues were reduced. The increase in cell surface area as discussed above may result in a reduction in negative surface charge density and subsequently a reduction in the affinity for TMA +. Since this affinity is due mainly to the negatively charged sialic acid-rich glycoproteins at the periphery of the cell, such an increase in Exp Cell Res 108 (1977)
182
Dahlgren et al.
surface area is likely to overshadow any effect of introducing minute amounts of charged lipid in the membrane bilayer. The reduced liability to hydrophobic interaction indicates either that components governing such interaction were lost from the treated cells or that the presence of exogenous lipid in these cells increased the total hydrophobic interaction within their membranes such that interaction with the hydrophobic probe was impeded. We have found no evidence for the first hypothesis and favour the second. The data thus demonstrate that the interaction with liposomes modified the physicochemical surface properties of the PMN cells. It seemed reasonable to hypothesize that such modifications would influence the behaviour of the PMN cells in biological systems. The present experiments show that the interaction with liposomes alters the phagocytic activity and the random mobility of the cells. They do not, however, disclose the precise characteristics of the functional modification, i.e. the mode by which the exogenous lipid exerts its effects. The effects on phagocytosis may thus be derived from alterations in either or both of two phagocytic steps; the attachment and the internalization. The phagocytosis of Salmonella typhimurium 395 MR 10 and of Salmonella typhimurium 395 MS opsonized with anti-MS IgG is governed by the physico-chemical surface properties of the respective particle [12, 22]. Both particles are, in contrast to the phagocytosis-resistant smooth strain Salmonella typhimurium 395 MS, negatively charged [23] and hydrophobic [2224]. On the basis of the physico-chemical surface properties of particles in relation to their liability to phagocytosis, we [23, 24] and others [22] have put forward the hypothesis that negative surface charge and Exp Cell Res 108 (1977)
hydrophobicity of a particle to be phagocytosed promote its attachment to the phagocytic cell surface. In the current investigation, liposome-treated PMN cells showed a reduced capacity to phagocytose. S. typhimurium 395 MR 10 and S. typhimurium 395 MS opsonized with anti-MS IgG. Since the treated cells as compared with untreated, control cells are less negatively charged and less liable to hydrophobic interaction, our finding is consistent with the hypothesis that attachment is brought about by hydrophogic-hydrophobic bonding and charged-linked associations such as ionic bridges. We thus suggest that the presence of exogenous lipid in the plasma membrane of the liposometreated PMN cells affects the molecular organisation providing attachment of the particle to be phagocytosed. Dianzani et al. have shown that an increase in plasmamembrane cholesterol is accompanied by a significant reduction in the phagocytic uptake of latex particles or lipid droplets by macrophages [25]. These findings are consistent with ours, since cholesterol may induce condensation of phospholipid molecules, restrict motion of acyl chains and increase their perpendicular orientation [26, 27], all of which would increase the total hydrophobic interactions within the phagocytic cell membrane. On the other hand, Heiniger et al. have shown that L cells (mouse fibroblasts) which are deficient in sterol synthesis are unable to endocytose horseradish peroxidase at normal rates [28]. Provided phagocytosis in macrophages and endocytosis in cultured L cells are accomplished by the same basic mechanism these findings indicate that the cholesterol/phospholipid ratio in phagocytic cell membranes must be maintained within a fairly narrow range to ensure their proper biological functioning. This point may be clarified by the
Interaction of liposomes with leukocytes. H
183
possible use of liposomes in introducing surface charge density and liability to phospholipids together with or without hydrophobic interaction may alter the adhecholesterol in the phagocytic cell membrane. siveness or the deformability of the cells, The possibility that the exogenous lipid two properties closely linked to their motilimpaired the triggering of the internalization ity [34-36]. phase cannot be precluded. A decrease in It is evident that the experiments persurface charge has been shown to reduce formed do not permit any explanation as to the membrane-mediated triggering of oxida- the specific structural alterations that may tive metabolism during phagocytosis [29, be responsible for the registered effects. 30]. Analogously, this triggering may be Yet, we interpret the present findings to inreduced by manipulations that change the dicate that the system used may create a membrane permeability for certain ions tool for modifying the behaviour of PMN (Ca2 +, MgZ+), known to be pertinent for the cells and to disclose functional requireactivation of PMN cells [31, 32]. In the ments and molecular mechanisms for the present system, however, ConA evokes attachment and internalization phases of normal or even augmented increase in HMS the phagocytic process. activity. Although internalization and oxiThe technical assistance of Ellinor Granstr6m and dative metabolism may not be closely co- Britinger Thor6n is gratefully acknowledged. This ordinated during all experimental condi- study was supported by grant 16X-2183-10 from The Swedish Medical Research Council. tions [29], this finding suggests that the metabolic excitability of the liposomeREFERENCES treated cells was unimpaired. In addition, Gregoriadis, G, New Englj reed 295 (1976) 704. the demonstration that the metabolic in- 2.1. Batzri, S & Korn, E D, J cell biol 66 (1975) 621. hibitor sodium fluoride inhibited phago- 3. Ehnholm, C & Zilversmit, D B, J biol chem 248 (1973) 1719. cytosis also in the liposome-treated cells 4. Papahadjopoulos, D, Poste, G & Mayhew, E, shows that internalization does proceed in B iochim biophys acta 363 (1974) 404. Pagano, R E & Huang, L, J cell bio167 (1975) 49. these cells. However, the rate of this pro- 5. 6. Martin, F C & MacDonald, R C, J cell biol 70 cess remains to be determined. (1976) 515. To explain the decreased random mobil- 7. Stendahl, O & Tagesson, C, Exp cell res 108 (1977) 167. ity in the liposome-treated cells, some hy- 8. Keller, H U & Sorkin, E, Int arch allergy 31 (1967) 575. pothesis may be put forward. The impair9. Br6te, L & Stendahl, O, Acta chir Scand 141 ment can not be accounted for by extensive (1975) 565. phagocytosis of liposomes; the criteria for 10. Albertsson, P-/~, Partition of cell particles and macromolecules. Almqvist & Wiksell, Uppsala, such a mode of interaction are not met [7]. Academic Press, New York (1971). O & Edebo, L, Acta pathol microbiol It might be accounted for by the lysis of 11. Stendahl, Scand sect B 80 (1972) 481. contaminating red cells and a subsequent 12. Stendahl, O, Tagesson, C & Edebo, L, Infect immun 10 (1974) 316. release of chemotactic material [33], form13. Cutler, J, Proc soc exp biol med 147 (1974) 471. ing a negative gradient keeping the cells 14. Gregoriadis, G, New Englj reed 295 (1976) 704. D, Poste, G & Mayhew, E, back in the well. Preliminary experiments 15. Papahadjopoulos, Biochim biophys acta 363 (1974) 404. show, however, that although the random 16. Papahadjopoulos, D, Poste, G & Schaeffer, B E, Biochim biophys acta 323 (1973) 23. mobility of the cells are impaired, their 17. Inbar, M & Shinitzky, M, Proc natl acad sci US 71 ability to respond to chemotactic stimuli (1974) 2128. 18. Poste, G & Papahadjopoulos, D, Proc natl acad sci during these experimental conditions are US 73 (1976) 1603. unaffected. More likely, the alterations in 19. Henney, C S, J immunol 110 (1973) 73. Exp Cell Res 108 (1977)
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20. Scharmann, W, Infect immun 13 (1976) 836. 21. Romeo, D, Jug, M, Zabucchi, G & Rossi, F, FEBS lett 42 (1974) 90. 22. Cunningham, R K, S6derstr6m, T O, Gillman, C F & van Oss, C J, Immunol commun 4 (1974) 429. 23. Stendahl, O, Magnusson, K-E & Tagesson, C, Proc 1st Eur conf on phagocytic leukocytes (Movement, metabolism and bactericidal mechanisms of phagocytes) Trieste (1976). in press. 24. Edebo, L, Lindstr6m, F, SkOldstam, L, Stendahl, O & Tagesson, C, Immunol commun 6 (1975) 87. 25. Dianzani, M U, Torrielli, M V, Canuto, R A, Carcea, R & Feo, F, J pathol 118 (1976) 193. 26. Steim, G M, Mitochondria biogenesis and bioenergetics. Biomembranes: molecular arrangements and transport mechanisms. FEBS, 8th meeting (ed S G Van Denbergh, P Borst, L L M van Deenen, J C Riemersma, E C Slater & J M Targer) p. 185. North-Holland, Amsterdam (1972). 27. Chapman, D, Biological membranes (ed D Chapman & D F t t Wallach) vol. 2, p. 91. Academic Press, New York (1973).
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28. Heiniger, H J, Kandutsch, A A & Chen, H W, Nature 263 (1976) 515. 29. Tsan, M F & McIntyre, P A, J exp med 143 (1976) 1308. 30. Tsan, M F, Newman, B & McIntyre, P A, Brit j haematol 33 (1976) 189. 31. Romeo, D, Zabucchi, G, Miami, N & Rossi, F, Nature 253 (1975) 542. 32. Schell-Frederick, E, FEBS lett 48 (1975) 37. 33. Bessis, M, Antibiotics and chemotherapy, vol. 19, p. 369. Karper, Basel (1974). 34. Lichman, M A & Weed, R I, Blood 39 (1972) 301. 35. Carter, S B, Nature 213 (1967) 256. 36. Miller, M E, The phagocytic cell in host resistance (ed J A Bellanti & D H Dayton) p. 295. Raven Press, New York (1975).
Received January 31, 1977 Accepted March 10, 1977
