incorporation of clodronate-liposomes

Apoptotic cell death in activated monocytes following

230 Journal of Leukocyte Biology Volume 60, August 1996

incorporation of clodronate-liposomesCarsten B. Schmidt�Weber,* Michael Rittig,t Eberhard Buchner,* Ingeborg Hauser,tIrma Schmidt,� Ernesta PaIombo�Kinne,* Frank Emmrich�, Raimund W. Kinne�*Immunology Unit, Department ofMedicine III, tlrutittae ofAnatomy, and Department ofMedicine IV, University

ofErlangen-Nuremberg, and �Institute ofClinical immunology and Transfzuion Medicine, University of Leipzig,

Leipzig, Germany

Abstract: The present study was performed to elu-

cidate whether sterically stabilized liposomes laden

with clodronate, which lead to depletion of macro-

phages (M4s) and amelioration of experimental

autoimmune arthritis in vivo, selectively affect cells

of the m� lineage in vitro. The rates of incorporation

of drug-free, fluorescent liposomes and the rates of

cell death following exposure to clodronate-

liposomes were assessed in human peripheral blood

monocytes, as well as in polymorphonuclear leuko-

cytes (PMNs), T cells, endothelial cells, and fi-

broblasts, both at rest and following activation. Gel

electrophoresis of nuclear extracts and ultrastructu-

r&d analyses were performed to identify the modality

of cell death. Monocytes, particularly upon activa-

tion, were more efficient in incorporating sterically

stabilized liposomes than all other cells except PMNs.

Twenty percent of resting monocytes and up to 65%

of activated monocytes died within 24 h of exposure

to clodronate-liposomes, whereas the other celltypes, including PMNs, remained unaffected. Acti-

vated monocytes exposed to clodronate-liposomes,

but not resting or activated monocytes exposed to

drug-free liposomes, showed clear signs of apoptotic

cell death. In most of the assays, sterically stabilized

liposomes were more efficient than conventional

phosphatidylcholine-liposomes. Sterically stabilized

clodronate-liposomes preferentially affect cells of the

m4� lineage, particularly if activated. Selective elimina-

tion of activated M�s by apoptosis may explain both

therapeutic efficacy and safety of clodronate-

liposomes in experimental models of autoimmunity.

J. Leukoc. Rio!. 60: 230-244; 1996.

Key Words: phagocytosLc . PMNs . T cells . endothelial cells�

fibroblasts . sterically stabilized liposomes

INTRODUCTION

Lipid vesicles containing an aqueous phase can be used to

encapsulate water-soluble drugs to be conveyed to phago-

cytic cells [1]. A liposome preparation containing the

bisphosphonate clodronate (dichloromethylene-bisphos-

phonate [2] has been utilized to achieve selective elimina-

tion of macrophages (M�s) in liver and spleen [3] and

exploited as an anti-m4 principle in the treatment of

autoimmune disorders, such as experimental allergic en-

cephalomyelitis [4], autoimmune neuritis [5], and experi-

mental arthritides [6-10]. In all these inflammatory

conditions remarkable therapeutic efficacy has been attrib-

uted to selective elimination of m�s, which play a relevant

pathogenetic role both as immunoregulatory cells [11] and

as effectors of tissue destruction [12]. Clodnonate-

liposomes have also proved effective in preventing

lipopolysaccharide (LPS)-induced septic shock in healthy

mice, probably by inhibiting the production of tumor ne-

crosis factor-a (TNF-a) by liver M�s [13].

The present study was performed to determine the ca-

pacity of cells of the m4 lineage (human peripheral blood

monocytes) to incorporate clodronate-liposomes in culture,

either at rest or following stimulation with 1,25-dihydroxy-

vitamin D3 [1,25(OH)2D3] or LPS, that is, standard matu-

rational and/or activation stimuli capable of enhancing

phagocytosis in monocytes [14-17]. The incorporating ca-

pacity of monocytes was compared not only with that of

strongly phagocytic polymorphonuclear leukocytes (PMNs)

but also with that of other cell types involved in inflamma-

tory processes, such as T cells, endothelial cells, and fi-

broblasts, both at rest and following stimulation. In

addition, the viability of cells following incorporation of

liposomes containing two different concentrations of do-

dronate, dose dependently effective in the treatment of rat

adjuvant arthritis [10], was assessed by means of the try-

pan blue exclusion test. The appropriate controls were

used, that is, free clodronate and phosphate-buffered sa-

line (PBS)-laden (empty) liposomes; in selected expeni-

Abbreviations: AET, aminoethylisothiouronium bromide; DMEM, Dulbecco’s

modified Eagle’s medium; FCS, fetal calf serum; IL-1�, interleukin-1f�; LPS,

lipopolysaccharicle; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; M�, macrophage;

PBMC, peripheral blood mononuclear cell; PBS, phosphate-buffered saline; PC,

phosphatidylcholine; PEG-S, polyethyleneglycol-400-stearate; PMA, phorbol

myristate acetate; PMNs, polymorphonuclear leukocytes; TNF-a, tumor necrosis

factor-a.

Reprint requests: R.W. Kinne, Institute ofClinical Immunology and Transfusion

Medicine, University ofLeipzig, DelitzscherStr. 141, D-04129 Leipzig, Germany.

Carsten B. Schmidt-Weber’s present address: Immunology Unit, Institute of

Pathology, Brigham and Woman’s Hospital, Harvard Medical School, Boston, MA

02115

Received December 4, 1995; revised April 17, 1996; accepted April 19, 1996.

Schmidt-Weber et al. Cellular effects of clodronate-liposomes 231

ments, the effect of the combination of free clodronate and

empty liposomes on the viability of monocytes was also

tested. Ultrastructural analysis of monocytes exposed to

clodronate-liposomes or controls and gel electrophoretic

analysis of their nuclear extracts were also performed to

identify the modality of cell death. These analyses were

also performed with PMNs, whereas in the case of endothe-

hal cells the in situ terminal deoxynucleotidyl transferase

and nick translation assay (TUNEL [18]) was performed.

Sterically stabilized liposomes formed by polyethylene-

conjugated lipids have been described that have prolonged

circulation times and improved biological activities

I19-21], because of less rapid clearance by the mononu-

clear phagocyte system and thus prolonged availability for

peripheral organs [211. In our own investigations,

liposomes composed of polyethyleneglycol-400-stearate

(PEG-S, which confers steric stability), sodium dodecyl

sulfate (which confers negative charge), and cholesterol

have displayed remarkable antiarthnitic properties in ex-

perimental models of arthritis following intravenous ad-

ministration [8, 10]. An additional goal of the present

study was therefore to evaluate the efficacy of this formu-

lation in comparison with conventional liposome prepara-

tions composed of phosphatidyicholine (PC) and

cholesterol [22].

MATERIALS AND METHODS

Preparation of multilamellar liposomes

Multilamellar PEG-S or PC liposomes were prepared by evaporating 10

mL of a chloroform-methanol mixture containing either 1 19 mg of PEG-

S (0.18 mmol; Caesar and Loretz, Hilden, Germany), 13 mg of sodium

dodecyl sulfate (0.045 mmol), and 68 mg of cholesterol (0.18 mmol;

both Sigma, Deisenhofen, Germany; molar ratio 4:1:4, respectively) or

174.4 rug of PC (Sigma) and 25.6 mg of cholesterol (Sigma; molar ratio

6:1 [22]) under vacuum at 60#{176}Cin a rotation evaporator. Twenty milli-

liters of PBS (0.15 M NaCI, 6.5 mM Na2HPO4, 1.5 mM KH2PO4, pH

7.4) or PBS containing disodium clodronate tetrahydrate (molecular

weight 360.2; kind gift of Boehringer Mannheim, Mannheim, Germany)

was added to the lipid film; this led to spontaneous formation of empty

or clodronate-liposome suspensions with 10 g lipid/L.

Intraliposomal encapsulation of clodronate was obtained by adding

PBS containing either 50 g/L (high dose) or 10 gIL (low dose) clodronate

to the lipid film. The suspensions were rotated without vacuum for 1 h

at 60#{176}C.Free clodronate was removed by dialysis (up to 300 mL of

liposome dispension, 3 x 8 h dialysis against 5 L of PBS). The in-

traliposomal concentration of clodronate was determined as previously

described [23]. As stock solutions, the high-dose clodronate-liposome

suspension contained 317 jig clodronate/mL (0.88 mM) and the low-

dose preparation 198 �tg clodronate/mL (0.55 mM). The free clodronate

stock solution contained doses fivefold higher (1.6 mg/mL; 4.4 mM)

than those contained in the high-dose liposomes, for congruity with

previous treatment studies [8, 10], that is, to arbitrarily compensate for

enhanced drug delivery through encapsulation.

Labeling of liposomes with fluorescent carbocyanine

Empty and clodronate-laden PEG-S or PC liposomes were labeled with

lipid-soluble [1,1’-dioctadecyl-3,3,3’,3’-tetramethyl]-indocarbocyanine

perchlorate (carbocyanine; Molecular Probes, Eugene, OR) according to

Claassen [24]. Briefly, 100 RL of liposomes (1 mg lipid/100 �tL) was

mixed with 40 �tL of the dye (1 �.tg/mL in ethanol) and incubated for 10

mm at room temperature. The preparation was then centrifuged

( 100,000g; 45 mm, room temperature) and washed once with fluores-

cent dye-free PBS.

Isolation of cells

Monocytes

These cells were exposed to clodronate-liposomes either in cell culture

following cell purification or in whole blood before subsequent purifica-

tion.

In the first case, heparinized peripheral blood (or buffy coats for the

analysis of DNA fragmentation, kindly provided by Dr. Zingsen, Blood

Bank, University of Erlangen-Nuremberg, Germany) was diluted 1:1 in

PBS and centrifuged on Lymphoprep according to the supplier’s recom-

mendations (Nycomed, Oslo, Norway). Peripheral blood mononuclear

cells (PBMCs) accumulated in the interphase were harvested and resus-

pended in RPMI 1640 medium containing 10% fetal calf-serum (FCS),

15 mM HEPES, 10 �tg/mL penicillin, and 10 U/mL streptomycin (all

Gibco, Eggenstein, Germany; henceforth RPMI/10% FCS). The cells

were allowed to adhere on plastic cell culture Petri dishes (size 100 X

20 mm) or 96-well plates for 1 h (Nunc, Wiesbaden, Germany). Nonad-

herent cells were removed by washing five times with warm RPMI/2%

FCS. In order to prevent cell activation via repeated detachment and

adherence, monocytes were not removed from the cell culture dish after

the initial adherence step.

In the case of exposure to liposomes in whole blood, PBMCs were

subsequently depleted of T cells by rosetting with sheep erythrocytes

previously treated with 2-aminoethylisothiouronium bromide (AET, 140

mM; Sigma). This procedure was chosen to exclude purification steps

relying on active adherence of monocytes to plastic, which may he

altered following incorporation of clodronate-liposomes. Briefly,

heparinized peripheral blood was diluted 1: 1 with 3% dextran (Pharma-

cia, Freiburg, Germany) in 0.9% NaCI and sedimented for 20 mm in an

upright position at room temperature. The leukocyte-rich supernatant

was aspirated and centrifuged at 5#{176}Cfor 10 mm at 250g. The pellet was

resuspended in saline and subjected to Lymphoprep purification as

described above in order to obtain PBMCs. The PBMCs (1 x 10�) were

then resuspended in 3 mL of RPMI/30% FCS, and 3 mL of a 3% (v/v)

solution of AET-treated sheep erythrocytes was added. The cells were

centrifuged (750g, 7 mm, room temperature) and the pellet incubated

for 30 mm on ice. Following gentle resuspension of the pellet, the

volume of the suspension was adjusted to 15 mL with RPMI/10% FCS.

Lymphoprep purification was performed, the cells were harvested from

the interface and adjusted to 106 cells/mL.

PMNs

To circumvent the problem of massive degranulation of PMNs usually

observed upon in vitro culture, which may interfere with phagocytosis,

PMNs were incubated with liposomes in whole blood before isolation;

purification was performed at the end of the incubation period. Follow-

ing dextran sedimentation and Lymphoprep centrifugation (see above),

the pellet was incubated for 30 s with 20 mL of 0.2% NaCl at 4#{176}Cto

lyse the erythrocytes; isotonicity was restored by adding 20 mL of b.6%

NaCl and the PMNs were washed once with 0.9% NaCl by centrifuga-

tion (5#{176}Cfor 10 mm at 250g).

T-cells. . . 8

PBMCs were isolated as described above and adjusted to 1.5 X 10cells/mL. One milliliter of this cell suspension was added to a nylon

wool column, containing 1.2 g of nylon wool (Polysciences, St. Goar,

Germany) in a 10-mL syringe, which had been preincubated with

RPMI/bO% FCS. The cells were allowed to enter the column and then

kept for 45 mm in an incubator (37#{176}C,5% C02). Fifteen milliliters of

warm RPMI/10% FCS were used to elute T cells from the column (purity

>90%).

Endothelial cells PMNs


The cells were harvested from the human iliac vein and artery of two

brain-dead patients using collagenase digestion [25]. Cells were cul-

tured in medium Mb99 enriched with 20% FCS, bovine hypothalamic

growth factor (10 �ig/mL [26]), and heparin (20 U/mL; all Gibco).

Fibro blasts

Cells were harvested from one human rheumatoid arthritis synovectomy

sample kindly provided by Prof. G. Weseloh, University of Erlangen,

Nuremberg. The tissues were transferred into sterile PBS containing

0.1% trypsin (Boehringer Mannheim) and finely minced with scissors.

The resulting suspension was mixed and digested for 30 mm at 37#{176}C

with 0.b% trypsin in PBS under constant stirring. After removal of the

trypsin-PBS, freshly prepared 0.1% collagenase P (Boehringer

Mannheim) in Dulbecco’s modified Eagle’s medium (DMEM), 10% FCS

(Gihco) was added for 2 h at 37#{176}Cunder shaking to further digest the

tissue. The cell suspension was then filtered through a sterile sieve and

the cells were collected by centrifugation (338g, 6 mm, room tempera-

ture). After washing twice with serum-free RPMI medium, the cells were

resuspended and cultured at 37#{176}C,5% C02 in DMEM supplemented

with penicillin (bOO U/mL), streptomycin (100 p.tg/mL), HEPES (0.05

mM), and 10% FCS (all Gibco) in small culture flasks. Seven to 10 days

later, the confluent, adherent cells were trypsinized (0.25% trypsin,

0.02% EDTA; Gibco) and further passaged every 5 to 6 days approxi-

mately at 1:4 dilution.

Assessment of liposome incorporation

The capacity of incorporating liposomes was first screened in all cell

types using empty PEG-S or PC liposomes, since clodronate-liposomes

induce cell death [2]. It would thus be difficult to distinguish the

genuine incorporation capacity of each cell type from the likely decrease

or arrest of vesicle internalization due to clodronate-liposome-induced

cell death. Subsequently, the incorporation of empty versus clodronate-

laden PEG-S liposomes was determined in monocytes, in order to verify

whether the intraliposomal presence of clodronate affected internaliza-

tion.

The evaluation of liposome incorporation was based on both the

percentage of cells carrying fluorescent signal (1, �bO%; 2, approxi-

mately 50%; 3, �90% of the cells) and the average intensity of the

signal within the cells (-, negative; +, weak; ++, moderate; +++,

strong).

Monocytes

The uptake of PEG-S or PC liposomes was determined by adding 5 �tL

of carbocyanine-laheled liposomes (50 �ig of lipid) to resting or stimu-

lated monocytes (1 x iO’1 cells/100 �tLiwell) in 16-well chamber slides

(Nunc). The chamber slides were washed three times with warm

RPMI/10% FCS 10 mm or 3, 13, or 24 h after adding the carbocyan-

inc-labeled liposomes, that is, until no extracellular signal was visible

at microscopy; they were then fixed, embedded with aqueous mounting

medium Permafluor (Immunotech, Marseille, France), and evaluated by

ultraviolet-light microscopy using a Zeiss-Axiophot microscope (Zeiss,

Oberkochen, Germany).

Stimulation of purified monocytes with b,25(OH)2D3 or LPS (both 10

ng/mL; Sigma, Deisenhofen, Germany) was tested in each case accord-

ing to two different schedules; that is, the stimuli were added either 24

h before or simultaneously with the addition of liposomes. Optimal

phagocytosis was observed after 24 h of stimulation in the case of

1,25(OH)2D3, but upon simultaneous stimulation in the case ofLPS. For

subsequent uptake and cell viability experiments, the optimized ached-

ules were applied.

The uptake of carbocyanine-labeled liposomes was assessed following

addition of 1 mL of empty PEG-S or PC liposomes to 4 mL of

heparinized blood for bO mm, 20 mm, 1 h, 3 h, and 24 h, both with and

without stimulation. Following isolation (see above), PMNs were placed

onto glass slides with flat wells (10k cells/100 jiL; Menzel,

Braunschweig, Germany), fixed, and embedded with aqueous mounting

medium Permafluor. In selected cases, degranulation was surveyed at 0,

15, and 20 mm following transfer of the PMNs to glass slides.

Stimulation of PMNs in whole blood was performed with 100 �tg/mL

LPS simultaneously with the addition of liposomes. The concentration

of LPS in this case was higher than that used for monocytes in cell

culture conditions (10 ng/mL), to compensate arbitrarily for possible

neutralizing effects of whole blood [27].

T cells

Five microliters of carbocyanine-labeled empty PEG-S or PC liposomes

(50 pg of lipid) were added to resting or activated T cells (10�/100�tIjwell) and the cells were incubated for 10 mm, 24 h, and 72 h in

16-well tissue culture chamber slides. At the end of the incubation

period, cells were removed from the chamber slides and washed twice

(338g, 10 mm, room temperature) with RPMI/10% FCS to remove free

liposomes. Subsequently, cytospins were prepared by centrifuging the

cell suspensions (5 x i0�/i09 �tL,/slide) in a cytocentrifuge (700g. 10

mm, room temperature; Shandon, Frankfurt, Germany).

Activation was performed by adding phorbol myristate acetate (PMA;

10 ng/mL) 24 h before or simultaneously with the addition of liposomes;

the length of the stimulation did not influence the outcome of the uptake

experiments.

Endothelial cells

For phagocytosis assays, the cells were trypsinized and placed in 16-

well chamber slides coated with 1% gelatin/PBS (10� cells/bOO

�t1Jwell). At the time the assays were performed, that is, at least 16 h

after trypsinization and passage into the chamber slides, endothelial

cells are known to have recuperated their surface characteristics (I.

Hauser et al., unpublished observations). The incorporation of carbocy-

anine-labeled empty PEG-S liposomes was assessed 10 mm and 3, b3,

24, and 72 h following addition of 5 �tL (50 j.tg of lipid) of the liposome

suspension to each well.

Cells were activated for 12 h prior to the addition of liposomes with

5 ng/mL human recombinant TNF-a (kind gift of Knoll AG, Ludwig-

shafen, Germany, or Dr. R. Voll, University of Erlangen-Nuremberg,

Germany).

Fibroblasts

Incubation with empty PEG-S liposomes was performed as for endothe-

hal cells, but without precoating the chamber slides with gelatin. Con-

fluent cells from passages 1 to 5 were used. At the time of the assays,

that is, at least 16 h after trypsinization and passage into the chamber

slides, fibroblasts are known to have recuperated their surface charac-

teristics [28].

Cells were activated with 17 ng/mL human recombinant interleukin-

11� (IL-1�3; kindly donated by Dr. U. Feige, Amgene, Thousand Oaks,

CA) for 24 h prior to the addition of liposomes.

Assessment of cell viability

Five microliters of either PEG-S or PC clodronate-liposomes (50 �ig of

lipid; final concentration of intraliposomal clodronate 0.044 mM) or

negative controls, that is, medium (RPMI/1O% FCS), free clodronate

(final concentration 80 p.tg/mL; 0.22 mM), empty liposomes (50 jig of

lipid), or empty liposomes in combination with free clodronate at theabove amounts or concentrations, were added to resting or stimulated

cells (10 cells/bOO j.tLlwell) in 16-well chamber slides. Gliotoxin (1

Monocytes

Resting

1,25 (OH)2D3

LPS

PMNs

Resting

LPS

T cells

Resting

PMA

Endothelial cells

Resting

TNF--a

n.d. n.d.

n.d. n.d.

Schmidt- Weber et al. Cellular effects of 233

�tM), known to induce cell death in monocytes [29], was used as a

positive control in the monocyte assays. Ten minutes or 3, 13, and 24 h

after addition of the substances (for T cells, endothelial cells, and

fibroblasts also 72 h), the supernatant was removed from the wells and

trypan blue added. In the case of PMNs, the time points were 10 mm,20 mm, and 1, 3, and 24 h. In control experiments, monocytes were also

incubated with 1 mL of PBS, empty PEG-S liposomes, or PEG-S do-

dronate-liposomes in 4 mL of whole blood for 24 h. In all cases, the

percentage of dead cells was evaluated by counting trypan blue-positive

cells in a minimum of 100 cells in each well.

In the case of T cells, an additional evaluation of long-term do-

dronate-liposome cytotoxicity was performed in two clones of a human

Herpesvirus saimiri-transformed T cell line [30], kindly provided by Dr.

B. Broker, Hamburg, Germany. Briefly, iO’� cells/bOO �tUwell in

RPMI/CG medium (50%-50%, v/v) (Gibco; Vitromex, Vilshofen, Ger-

many, respectively) were exposed to IL-2 (10, 30, or 100 U/mL; Euro-

cetus, Frankfurt a.M., Germany) and to high-dose PEG-S

clodronate-liposomes, free clodronate, or empty PEG-S liposomes at the

above amounts or concentrations for 3 days in a 96-well round bottom

culture plate. Eighteen hours before the end of the experiment, 1 �tCi of

[3H]thymidine was added to each well for the assessment of [3H]thymid-

inc incorporation.

Transmission electron microscopic analysis

Five hundred microliters of empty or high-dose PEG-S clodronate-

liposomes (in both cases 5 mg of lipid) were added to purified mono-

cytes (1-5 x 1O� cells/lO mL per Petri dish; size 100 x 20 mm),

prestimulated with 10 ng/mL 1,25(OH)2D3 for 24 h; the cells were

incubated for 4, 8, 12, and 24 h, trypsinized (0.25% trypsin, 0.02%

EDTA; Gibco), and centrifuged. In a separate experiment, bO mL of

whole blood containing no or 100 j.tg LPS/mL was incubated for 24 h

with 1 mL of empty or high-dose PEG-S clodronate-liposomes and sub-

sequently purified as described above. In both cases, samples were fixed

twice with cacodylate-buffered 2.5% glutaraldehyde (Roth, Karlsruhe,

Germany). Subsequently, the samples were postfixed for 20 mm in 1%

aqueous osmium tetroxide (Paesel, Frankfurt am Main, Germany), de-

hydrated with a graded ethanol series completed by acetone, cytocentn-

fuged at each step, and embedded in Epon 812 (Roth). Semithin

sections (0.5 jim) were cut in a plane sagittal to the surface and stained

for light microscopic survey. Subsequently, thin sections were cut and

counterstained with 10% uranyl acetate followed by 2.8% lead citrate

(both Merck, Darmstadt, Germany). For each experiment, three thin

sections were investigated using a Zeiss EM 902 transmission electron

microscope.

Gel electrophoretic analysis of nuclear DNAfragmentation

Five hundred microliters of either high-dose PEG-S clodronate-

liposomes or negative and positive controls in the amounts or concen-

trations used for the preceding experiments were added to monocytes

(1-5 x b06 cells/1O mL per Petri dish; size 100 x 20 mm), prestimu-

lated with either 10 ng/mL 1,25(OH)2D3 for 24 h or LPS (10 ng/mL)

simultaneously with the addition of the liposomes; cells were then incu-

bated for 7 h and trypsinized (0.25% trypsin, 0.02% EDTA).

TABLE 1 . Incorporation of Empty, Carbocyanine-Labeled Polyethyleneglycol-Stearate (PEG-S) Liposomes and PC Liposomes”

Synovial fibroblasts

Empty PEG- S liposomes

10mm 3h 13h 24h

+11 +/1 ++/3 +++/3

+11 +11 ++/3 +++/3

+11 +11 +++/3 +++/3

10mm 20 mm 1 h 3 h 24 h

++/3 ++/3 ++/3 ++/3 ++/3

++/3 ++/3 ++/3 ++/3 ++/3

10mm 3h 13h 24h 72h

- n.d. n.d. - +11

- n.d. nd. - +11

10mm 3h 13h 24h 72h

- +++/1 +13 +13 +/3

- +++/1 +13 +13 +/3

10mm 3h 13h 24h 72h

Em pty PC liposomes

10mm 3h 13h 24h

- - +/1 ++/3

- - +/2 +/2

- +/1 ++/3 +++/3

10 mm 20 mm i h 3 h 24 h

++/3 ++/3 ++/3 ++/3 ++/3

n.d. n.d. n.d. n.d. n.d.

10mm 3h 13h 24h 72h

Resting - - - +13

IL-43 - - +/3

“Activated monocytes (LPS 10 ng/mL) were more efficient than resting monocytes in incorporating either liposomal preparation. In general, however, PEG-Sliposomes were internalized more efficiently than PC liposomes. LPS was a more potent stimulus for endocytosis than 1,25(OH)2D3, in that it halved to 13 h the time

required for maximal incorporation of PEG-S liposomes and rendered maximal the uptake of PC liposomes at 24 h. PMNs in whole blood (see Materials and Methods),whether at rest or stimulated with LPS (100 �tg/mL), incorporated liposomes veiy quickly, regardless of their composition, but more moderately than monocytes.Endothelial cells, in contrast, incorporated PEG-S liposomes weakly from 13 h onward, but single cells were strongly positive at 3 h and thereafter (see Results andFig. 1 for details). Stimulation with TNF-a did not enhance phagocytosis in these cells. T-cells, whether at rest or following stimulation with PMA (10 ng/mL), did notinternalize PEG-S liposomes until 72 h. Synovial fibroblasts showed a very weak signal only at 72 h. In all cases, except for endothelial cells and rheumatoid synovial

fIbroblasts, cells were obtained from three normal donors; determinations were performed in triplicate; a minimum of 100 cells was counted in each assay. -, negative;+, weak; + +, moderate; + + +, strong intracellular fluorescence signal; nd., not determined; 1, �10%; 2, approximately 50%; 3, �90% of the cells.


In separate experiments, 4 mL of whole blood containing no or 100

jig/mL LPS was incubated with 1 mL of empty or high-dose PEG-S

clodronate-liposomes; PMNs were purified after 7 h as described above.

In both cases, DNA gel analysis was performed as previously reported

L31] with minor modifications. To minimize the influence of different

amounts of DNA on gel electrophoresis, equal numbers of cells were

cl�

z

.-

used in each preparation. The cells were only lysed and digested with

proteinase K and RNase in order to minimize the loss of cellular mate-

rial. Briefly, cells were centrifuged (338 g, 10 mm, 4#{176}C),the pellets

resuspended in 20 IlL of 10 mM EDTA, 50 mM Tris-HC1 (pH 8)

containing 0.5% sarkosyl (Serva, Heidelberg, Germany) and 1 mg/mL

proteinase K (Boehringer Mannheim), and incubated at 50#{176}Cfor 1 h.

Fig. 1 . Phase contrast (left) and fluorescence (right) images of cells incorporating empty, carbocyanine-labeled polyethyleneglycol-stearate (PEG-S)

liposomes. LPS-stimulated (10 nglmL) peripheral blood monocytes (A and B) incorporated liposomes more strongly than PMNs (C and D) or than the

majority ofendothelial cells (E and F), at time points at which all cell types have already reached maximal incorporation ofthe vesicles (13, 3, and 13

h, respectively; Table 1). In the case of PMNs and endothelial cells, the degree of uptake was independent of the resting or stimulated status. Original

magnification for all pictures x186.

Monocytes

Empty PEG-S liposomes

Schmidt-Weber et a!. Cellular effects of 235

TABLE 2. Comparison of the Incorporation Rates of Empty Versus Clodronate-Laden Polyethyleneglycol-Stearate (PEG-S) Liposomes in Monocytes (High-DosePreparation; Final Concentration 0.044 mM)#{176}

10mm 3h 13h 24h

Clodronate PE C-S liposomes

10mm 3h 13h 24h

Resting +/1 +/1 + +/3 + + +/3 +/1 +/1 + +/3 + + +/3

LPS � +/1 +/1 +++/3 +++/3 +/1 +/1 ++/3

“Liposomes were labeled with carbocyanine (see Materials and Methods). The uptake of empty liposomes was similar to that shown in Table 1 for both resting andLPS-activated monocytes (10 ng/mL), with maximal incorporation at 13 h in the case of LPS. In the case of clodronate-laden liposomes, in contrast, the uptake uponLPS stimulation remained moderate at both 13 and 24 h in the fraction of cells that had remained attached. Considerable numbers of monocytes had detatched fromthe culture dish at these time points. Monocytes were obtained from three normal donors; determinations were performed in triplicate; a minimum of 100 cells wascounted in each assay. -, negative; +, weak; + +, moderate; + + +, strong intracellular flourescence signal; 1, �, 10%; 3, � 90% of the monocytes.

After short heat denaturation (90#{176}C), 1 �.tL of RNase Ti (1 mg/mL;

Boehringer Mannheim) was added to each sample; incubation followed

for 1 h at 50#{176}C.Subsequently, the probes were heated to 65#{176}Cand 6 pi

of 10 mM EDTA (pH 8) containing 0.25% bromphenol blue and 15%

Ficoll (type 400; Pharmacia) was added to each sample. Gel electropho-

resis of the entire sample was performed on a 1 .5% agarose gel contain-

ing 10 �ig/mL ethidium bromide and carried out in a 0.26 mM

Na2HPO4, 33 mM NaH2PO4, 10 mM EDTA buffer for 2 h at 70 V.

Statistics

The nonparametric Mann-Whitney U test was applied to analyze differ-

ences for all parameters examined using the StatViewll program (Aba-

cus Concepts, Berkeley, CA). The effects of clodronate-liposomes and

those of the combination of empty liposomes plus free clodronate were

tested against empty liposomes. The effects of individual components or

gliotoxin were compared with those of medium. Significant differences

were accepted for P � 0.05.

RESULTS

The capacity of cells to incorporate either PEG-S or PC

liposomes was determined with PBS-containing (i.e.,

empty) liposomes to avoid having the effects of clodronate-

liposomes on cell viability differentially interfere with the

phagocytic capacity of the different cell types. The incor-

poration of free clodronate was also not taken into consid-

eration, since this was previously characterized: because of

its highly hydrophilic features, only a very small percent-

age of clodronate enters phagocytic or nonphagocytic cells

[32].

In the case of monocytes, the incorporation assay was

repeated with PEG-S liposomes laden with high-dose do-

dronate to determine how the clodronate content affected

the intrinsic capacity of these cells to incorporate

liposomes.

Incorporation of empty polyethyleneglycol-stearateliposomes

The results obtained with the PEG-S preparation of

liposomes are shown on the left in Table 1.

Monocytes

In most of the resting monocytes there was a moderate

fluorescent signal at 13 h, which reached a maximum at 24

h (Table 1).

Stimulation with 10 ng/mL 1,25(OH)2D3 for 24 h prior

to exposure to liposomes did not augment liposome incor-

poration in comparison with resting monocytes, whereas 10

ng/mL LPS added simultaneously with liposomes halved to

13 h the time required to reach maximal incorporation

(Table 1; Fig. 1A and B).

PMNs

These cells, both at rest and following stimulation with LPS

(100 �ig/mL in whole blood), incorporated PEG-S

liposomes to a moderate degree; maximal incorporation

was reached after 10 mm of incubation (Table 1; Fig. 1C

and D).

T cells

There was no incorporation of liposomes, whether at rest or

following stimulation with PMA (10 ng/mL), until 72 h,

when weak fluorescence was visible in a few cells (ap-

proximately 1%; Table 1). Other cells of different morphol-

ogy and size (approximately 5%), most likely

contaminating monocytes, incorporated PEG-S liposomes

to a higher degree.

Endothelial cells

Approximately 1% of these cells incorporated large

amounts of liposomes 3 h following the beginning of the

incubation (Table 1). From 13 h onward, cells incorporat-

ing liposomes (approximately 10%) formed clusters, whose

number and size increased with time (Table 1; Fig. 1E and

F). At 13 h, in addition to the cells forming clusters, most

of the remaining cells weakly internalized PEG-S

liposomes. At all time points, the fluorescent signal was

characteristically concentrated in the perinuclear area of

the cytoplasm (Fig. iF). Stimulation of endothelial cells

with TNF-a (5 ng/mL) for 12 h prior to the addition of

liposomes did not enhance liposome incorporation.

Synovialfibroblasts

These cells did not internalize liposomes until 72 h, when

a weak signal was observed in most of the cells (fable 1);

previous stimulation with IL-1�3 (17 ng/mL) for 24 h did

not increase incorporation.

80

60

40

20

00 6 13 24

80

60

40

20

00 6 13 24

LPS

0 6 13 24

Medium


Incorporation of empty phosphatidylcholineliposomes

The results obtained with the PC preparation of liposomes

are shown on the right in Table 1. In comparison with

PEG-S liposomes, the general features observed for mono-

cytes were (1) lower rates of incorporation and/or (2) lower

density of incorporated vesicles, both at rest and upon

stimulation. There were no differences between the two

liposome preparations in the case of PMNs and T cells.

Incorporation of empty versus clodronate-ladenliposomes by monocytes

80

60

40

20

0

1,25 (OH)�D3

time (h)

Fig. 2. Mortality rates of resting and stimulated peripheral blood mono-

cytes upon exposure to polyethyleneglycol-stearate (PEG-S) liposomes

containing two different doses of clodronate (HD, high-dose, 0.044 mM;

LD, low dose, 0.028 mM; see Materials and Methods fordetails). Negative

controls were medium, free clodronate (0.22 mM), empty liposomes, and

the combination of free clodronate (0.22 mM) plus empty liposomes.

Gliotoxin ( 1 �tM) was used as positive control. Resting monocytes showed

no sensitivity to PEG-S clodronate-liposomes (A). Stimulation with

1,25(OH)2D3 (B) enhanced the cytotoxicity of the high-dose PEG-S

clodronate-liposomes. In the case of LPS stimulation (C), the degree of

the effects depended on the intraliposomal dose of clodronate, with

high-dose PEG-S clodronate-liposomes approaching the efficacy of glio-

toxin. Values are means ± SEM; n 3 normal donors; determinations for

each time point were performed in triplicate. *� � 0.05 in comparison

with medium in the case ofgliotoxin; in comparison with empty liposomes

in the case of clodronate-liposomes.

The results of this assay are depicted in Table 2. Resting

monocytes incorporated high-dose PEG-S clodronate-

liposomes as efficiently as the empty counterpart through-

out the experimental time. In the case of activated

monocytes (LPS 10 ng/mL), in contrast, the enhancement

of liposome incorporation seen with empty PEG-S

liposomes (Tables 1 and 2; left) was no longer visible with

clodronate-laden liposomes; of note, a considerable

number of monocytes detached and the degree of internali-

zation in the fraction of cells that was still attached re-

mained moderate at both 13 and 24 h (Table 2, right).

Effects of polyethyleneglycol-stearateclodronate-liposomes on cell viability

The results obtained with monocytes are shown in Figures

2 and 3. For all cell types investigated, including mono-

cytes, a common feature was the inefficacy of medium, free

clodronate, and empty liposomes in inducing cell death; in

addition, the combination of free clodronate plus empty

liposomes remained ineffective in both resting and LPS-

stimulated monocytes (Fig. 2), confirming the clinical in-

efficacy of this particular control following in vivo

treatment of rat adjuvant arthritis (C.B. Schmidt-Weher et

al., unpublished results). Of note, cell activation without

exposure to PEG-S clodronate-liposomes did not enhance

cell death, showing that, at the doses used, the stimuli used

did not per se affect cell viability.

Monocytes

PEG-S liposomes containing either low-dose (0.028 mM)

or high-dose (0.044 mM) clodronate had no effects on the

cell viability of resting monocytes throughout the 24-h ex-

penimental period. In contrast, the positive control glio-

toxin (1 tiM) was highly effective at both 13 and 24 h (Fig.

2A).

Monocytes prestimulated with 1,25(OH)2D3 for 24 h

showed significantly increased rates of cell death at 24 h

of incubation with PEG-S liposomes containing the high-

dose clodronate (Fig. 2B). The low-dose preparation was

ineffective.

Activation with LPS (10 ng/mL) and simultaneous expo-

sure to high-dose PEG-S clodronate-liposomes induced

B:j�

� ftr�.n#{225}

Schmidt- Weber el al. Cellular effects of clodi .liposomes 237

cell death in purified monocytes at 24 h of incubation in a

dose-dependent fashion (Fig. 2C).

Incubation of activated monocytes with high-dose PEG-

S clodronate-liposomes in whole blood (LPS 100 �.tg/mL for

24 h) reduced the mortality of monocytes by 20% in com-

panison with the maximal effect observed under cell cul-

ture conditions (Figs. 2C and 3A). This suggests that whole

blood partially protects monocytes from the effects of do-

dronate-liposomes and that activation by LPS itself, in

spite of the large dose used, does not significantly contnib-

ute to cell mortality.

PMNs

Resting PMNs remained insensitive to the effects of high-

dose PEG-S clodronate-liposomes throughout 24 h (data

not shown; Fig. 3B for the 24-h time point), in spite of their

capacity to incorporate liposomes in whole blood as early

as 10 mm (Table 1). Strong and protracted activation with

LPS (100 �.tg/mL for 3 or 24 h) did not influence the

refractoriness of PMNs to the effects of high-dose PEG-S

clodronate-liposomes (Fig. 3B for the 24-h time point).

Functional properties of PMNs, such as degranulation,

were also apparently spared (data not shown).

T cells

No trypan blue positivity could be observed until 72 h of

incubation with PEG-S liposomes containing high-dose

clodronate (0.044 mM), whether T cells were at rest or had

been prestimulated for 24 h with 10 ng/mL PMA (data not

shown).

T cell clones

Simultaneous exposure of Herpesvirus saimiri-immontal-

ized T cell clones to IL-2 (10, 30, or 100 U/mL) and

high-dose PEG-S clodronate-liposomes for 3 days did not

affect the [3H]thymidine incorporation of these cells (data

not shown). Free clodronate and empty PEG-S liposomes

were also ineffective.

Endothelial cells

Resting or TNF-�-stimulated (5 ng/mL) endothelial cells,

incubated with high-dose PEG-S clodronate-liposomes, re-

mained viable throughout 72 h (data not shown), a time in

which there was some incorporation of liposomes in virtu-

ally all cells (Table 1). At this time point there were some

holes in the cell monolayer, and cells harvested from the

supernatant showed disrupted membranes; however, these

changes were observed regardless of whether the cells had

been exposed to empty or clodronate-laden PEG-S

liposomes.

Synovialfibroblasts

Fibroblasts remained trypan blue negative as long as 72 h

following incubation with PEG-S liposomes containing

high-dose clodronate, whether at rest or after prestimula-

tion with IL-1� (data not shown).

Effects of phosphatidylcholineclodronate-Iiposomes on cell viability

Monocytes (Fig. 4), PMNs, and synovial fibroblasts (data

not shown) were less sensitive to PC than to PEG-S do-

dronate-liposomes. Monocytes, in particular, were signifi-

cantly sensitive to PC liposomes only in the case of

stimulation with LPS at 24 h (Fig. 4C), that is, the only

circumstance in which maximal PC liposome incorporation

had been achieved (Table 1, right).

Ultrastructural effects ofpolyethylenegiycoi-stearate

clodronate-liposomesMonocytes

The vast majority of 1,25(OH)2D3-stimulated monocytes

incubated with empty PEG-S liposomes did not exhibit

major ultrastructural changes (data not shown); in particu-

lan, there were no signs of cell death, whether necrotic or

;1I

T-cell depletedPBMC

PMN

I - 11 +ILPSI - II +1

0 PBS

D PEG-S PBS-Liposomes

. PEG-S Clodronate-Liposomes

Fig. 3. Comparison ofthe cytotoxic effects of polyethyleneglycol-stearate

(PEG-S) clodronate-liposomes in monocytes (A) and PMNs (B) following

incubation in whole blood for 24 h; LPS was given in excess (100 j.tg/mL)

compared to cell culture conditions (10 ng/mL; Figs. 2 and 3), to coun-

teract possible neutralizing effects of blood [27]; isolation was performed

thereafter as described in Materials and Methods. At rest (LPS -), there

were no appreciable differences for the two cell types. When cells were

activated with LPS (LPS +), there was no increase ofmortality in the case

of PMNs (B), whereas cell death following exposure to PEG-S clodronate-

liposomes increased significantly in the case of monocytes (A). Compara-

ble results were obtained after3 h ofincubation (data not shown). Of note,

LPS itself, or the parallel presence of LPS and empty liposomes, was a

weak inducer of monocyte mortality (approximately 10% [LPS +] versus

5% [LPS -] in (A); in the case ofPMNs (B) LPS was completely ineffective.

The integrity of liposomes thus appears preserved upon incubation in

whole blood; more important, PMNs are refractory to the effects of PEG-S

clodronate-liposomes even upon strong activation; finally, activation also

seems a decisive factor for the susceptibility of monocytes to PEG-S

clodronate-liposomes in whole blood. Values are means ± SEM; n 3normal donors; determinations were performed in triplicate. Sf) � 0.05 in

comparison with empty liposomes.

60

0 6 13 24

1,25 (OH)�D3

0 6 13 24

LPS80

60

40

20

00 6 13 24

Medium


80-a-- Medium

-#{149}0-#{149}PC PBS-Uposomes

� -�- PEG-S PBS-llposomes

-ar- PEG-S Clo.llposomes (HD)

-.- PC Clo-liposomes (HD)

40’

time (h)

Fig. 4. Comparison of the mortality rates of peripheral blood monocytes

upon exposure to polyethyleneglycol-stearate (PEG-S) or phosphatidyl-

choline (PC) liposomes, in both cases containing the high-dose of do-

dronate (HD; 0.044 mM). The sensitivity of monocytes to PC

clodronate-liposomes increased only upon stimulation with LPS at 24 h

(C), that is, the only circumstance in which PC liposomes were maximally

incorporated by monocytes (Table 1, right); the effects of PC clodronate-

liposomes remained significantly lower than those of PEG-S clodronate-

liposomes (B and C). Values are means ± SEM; n 3 normal donors;

determinations were performed in triplicate. �1� � 0.05 in the comparison

between empty and clodronate-laden PC liposomes. §P � 0.05 in the

comparison between PEG-S clodronate-liposomes and PC clodronate-

liposomes.

apoptotic. At 12 and 24 h some cells showed lysis, indica-

tive of necrotic cell death (data not shown).

Exposure of 1,25(OH)2D3-stimulated, purified mono-

cytes to PEG-S liposomes laden with high-dose clodronate

resulted in a series of morphological changes typical of

apoptotic cell death (Fig. 5), that is, vacuolization, chro-

matin condensation, and formation of membrane-bound cy-

toplasmic bodies. The clodronate-liposomes could be

easily identified as membrane-enclosed bodies with elec-

tron-lucent contents (Fig. 5). After 12 h many cells, and

after 24 h most of the cells, showed ruptured cell mem-

branes and disintegrating cytoplasm, indicative of secon-

dary necrotic cell death, a well-known in vitro

phenomenon [33, 34]. The number of internalized vacuoles

per cell appeared to decrease with time (A through D);

although no quantitative conclusions can be drawn from

this morphological analysis, it is conceivable that cells

undergoing apoptosis have a reduced phagocytic capacity,

as suggested by the results in Table 2. This is different

from the results in Table 1, in which empty liposomes were

used: since the latter do not induce apoptosis or cell death

in general, the cells were conceivably still capable of fur-

ther vesicle incorporation at 13 and 24 h.

LPS-stimulated monocytes exposed to high-dose PEG-S

clodronate-liposomes in whole blood showed both vesicle

internalization and clear signs of apoptosis (data not

shown); stimulation with 100 �ig/mL LPS per se, with or

without the addition of empty liposomes, did not induce

apoptosis but only vacuolization as a sign of cell activation

(data not shown). In whole blood, interestingly, apoptosis

could still be observed at 24 h, a time at which secondary

necrotic cell death had already ensued in purified mono-

cytes under cell culture conditions (Fig. 5). Incubation in

whole blood, thus, may slow down the apoptotic process

and/or prevent secondary necrosis, as already suggested by

the 20% decrease in cell death rates between purified

monocytes (Fig. 2C) and monocytes exposed to clodronate-

liposomes in whole blood before purification (Fig. 3A).

Whether this effect is to be ascribed to interaction of whole

blood components with LPS [27] or to the influence of

blood components on the surface characteristics of

liposomes remains to be elucidated.

PMNs

Exposure of resting or stimulated PMNs (LPS 100 j.tg/mL)

to PEG-S clodronate-liposomes for 24 h in whole blood did

not result in apoptosis, in spite of successful internaliza-

tion of the vesicles (Fig. 6), and in agreement with the

tiypan blue negativity shown in Figure 3B. Also in this

case, LPS induced vacuole formation as a result of cell

activation. Several cells showed signs of lytic necrosis.

Effects of polyethyleneglycol-stearateclodronate-liposomes on DNA fragmentation

Monocytes stimulated either with 1,25(OH)2D3 (Fig. 7A)

or LPS (Fig. 7B) and exposed for 7 h to PEG-S liposomes

A

a

4h*

..r � “ #{149}:�-��‘ � � #{149}D� � ‘..

‘ S

_4

.� F

0’�

24h


laden with high-dose clodronate (0.044 mM) displayed

fragmentation of nuclear DNA in multiples of approxi-

mately 200 bp. This ladder pattern is typical of apoptotic

cell death, as also indicated by the analysis of nuclear

fragments obtained using the positive control gliotoxin

(Fig. 7A), a substance known to induce apoptosis in mono-

cytes [29]. Exposure of resting or activated monocytes to

medium, free clodronate, or empty PEG-S liposomes did

not result in any DNA fragmentation (Fig. 7A and B).

Nuclear extracts of resting or activated PMNs exposed to

high-dose PEG-S clodronate-liposomes for 7 h did not

show any apoptotic DNA fragmentation (data not shown).

Effects of polyethyleneglycol-stearateclodronate-liposomes on endothelial cells

Because a minority of endothelial cells had shown promi-

nent liposome uptake as early as 3 h following exposure to

empty fluorescent PEG-S liposomes (Table 1), it was de-

termined whether these particular cells underwent apop-

totic cell death upon exposure to PEG-S liposomes laden

with high-dose clodronate (0.044 mM). The TUNEL assay

[18] was applied in this case, with a munine CTLL cyto-

toxic T cell line, which undergoes apoptosis upon IL-2

deprivation in virtually all cells (Dr. R. Voll, personal

Fig. 5. Transmission electron microscopy images of 1,25(OH)2D3-stimulated human peripheral blood monocytes incubated with

polyethyleneglycol-stearate (PEG-S) clodronate-liposomes (high-dose preparation; 0.044 mM). At 4 h the majority of the cells contained

numerous cytoplasmatic vacuoles of variable size (arrowheads, A). The arrows in A, C, and D indicate internalized liposomes. After 8 h

the nucleus showed chromatin condensation along its periphery (white arrowheads) and the vacuolization of the cytoplasm became more

pronounced (B). Note the remaining intact nucleolus (arrow in B). At this time point, the majority of the cells expelled membrane-bound

cytoplasmic bodies (arrowheads, C). At 24 h, most ofthe cells displayed ruptured cell membranes and disintegrating cytoplasm, indicative

of secondary necrotic cell death, superseding apoptosis at late time points; the formation of membrane-bound cytoplasmic bodies was no

longer observed (D). None of these changes followed exposure of monocytes to empty PEG-S liposomes (data not shown). Magnification

xll,664 (A), xlO,500 (B), x12,390 (C), and x12,250 (D).

Fig. 6. Transmission electron microscopy images ofresting PMNs exposed to medium (A) or LPS-stimulated PMNs (LPS 100 j.tg/mL) exposed to empty

polyethyleneglycol-stearate (PEG-S) liposomes (B) or to PEG-S liposomes laden with high-dose clodronate (0.044 mM; C) for 24 h in whole blood. The

white arrows in (B) and (C) indicate an internalized liposome. Black arrows in (C) indicate extracellular liposomes. Stimulated PMNs show distension

of the perinuclear endoplasmic reticulum and considerable vacuolization (white arrowheads in B and C), conceivably a consequence of LPS stimulation;

however, cells show no signs of apoptotic cell death (C), in spite of successful liposome internalization. At the edge of the pictures several erythrocytes

derived from the incubation in whole blood can be seen (black arrowheads in B and C). Magnification x8100.


communication), serving as positive control. A fraction of

endothelial cells incorporated carbocyanine-labeled do-

dronate-liposomes very efficiently, similarly to the results

of Figure iF. However, individual resting or activated cell

showing a high fluorescent signal were consistently nega-

tive for apoptotic breakage of DNA (data not shown).

DISCUSSION

Clodronate-liposomes preferentially affect cells ofthe M4 lineage

The present results confirm the main hypothesis of the

study; that is, in vitro cells of the m4 lineage are preferen-

tial targets of PEG-S clodronate-liposomes in comparison

with PMNs, endothelial cells, T cells, and fibroblasts.

These findings thus strongly support in vivo observations

that clodronate-liposomes display high selectivity for M�s

[3-10, 13]. In fact, monocytes in culture, particularly fol-

lowing activation, showed the highest degree of incorpora-

tion of liposomes and, more important, the highest degree

of cell death upon exposure to clodronate-liposomes.

PMNs and endothelial cells also incorporated liposomes,

but they were insensitive to the cytotoxic effects of do-

dronate-liposomes, consistent with their sparing in vivo [4,

7, 10, 13]. T cells and fibroblasts, in turn, were resistant

to cytotoxicity, as expected on the basis of their weak

liposome incorporation (Table 1). A high degree of incor-

poration thus appears necessary for PEG-S clodronate-

liposomes to exert their effects. This conclusion is also

supported by preliminary results showing that sodium az-

ide dose dependently decreases endocytosis in parallel

with rates of mortality in activated monocytes (data not

shown).

The results obtained with PMNs and endothelial cells

demonstrate, on the other hand, that mere incorporation of

clodronate-liposomes is not sufficient to induce cell death

and that at least two further requirements need to be met:

(1) that a high intracellular threshold of clodronate is

reached; in our system this requirement seems fulfilled

only in activated monocytes exposed to PEG-S or PC

liposomes (Table 1; Figs. 2 and 4); and (2) that cells

possess the biochemical machinery to cleave liposomal

membranes and release their contents [14, 16]; this crite-

non may be particularly relevant for the few endothelial

cells that take up as many liposomes as activated mono-

cytes but remain refractory to cell death.

Although the present experiments seem to favor the hy-

pothesis that significant cell death is observed as a conse-

quence of high vesicle incorporation, it cannot be excluded

that clodronate is delivered to m4s not via endocytosis of

intact vesicles but rather through fusion between liposome

and cell membranes [21] or, alternatively, through passive

entry of extraliposomal clodronate into the cells; the latter

modality may result from entrapment of the highly hydro-

philic clodronate within the hydrophilic head groups of the

PEG-S on the outer liposomal membrane. This possibility

seems unlikely, however, since the combination of free

clodronate plus empty liposomes, which should represent

an extreme of such interaction, was unable to induce

monocyte cell death in vitro (present results) as it was

ineffective following in vivo treatment of experimental an-

thritis (C.B. Schmidt-Weber, unpublished results). Also,

the degree of monocyte cell death is dose dependently

related to the intraliposomal dose of clodronate (Fig. 2),

indicating that the clodronate content of intact vesicles,

rather than surface mechanisms, dictates the extent of the

cytotoxic effects.

Activated but not resting monocytes die viaapoptosis

The clearest indication that activated monocytes are tar-

geted by clodronate-liposomes in a unique fashion was that

8

bp

I636

396

344

1,25 (OH)2D3 stimulated monocytes

0,

0

8I

II

bp

1636

LPS stimulated monocytes

Schmidt- Weber et al. Cellular effects of clods - 241

they undergo apoptotic cell death, as shown by ultrastruc-

tural and gel electrophoretic analyses (Figs. 5 and 7). This

phenomenon was not dependent on activation per se, as

reported with PMA [34], since monocytes activated with

LPS or 1 ,25(OH)2D3, but exposed only to medium or empty

liposomes, did not undergo apoptosis (data not shown; Fig.

7) or cell death in general (Figs. 2, 3, and 4). Activated

PMNs and the minority of highly endocytic endothelial

cells, in turn, did not undergo apoptotic or lytic death in

spite of their capacity to incorporate substantial amounts of

vesicles (Table 1, Fig. 6; data not shown).

The fact that activated monocytes die via apoptosis

raises the question of whether this effect is triggered by

surface contact of liposomes with the Fas receptor without

the necessity of liposome internalization [35]. Our data do

not formally exclude this possibility; however, several fac-

tors do not support it: (1) the ultrastructural evidence of

internalization of intact liposomes was very striking, as

also reported in other studies [36]; (2) the necessity of

internalization, rather than the sufficiency of surface con-

tact, is supported by the lack of cell death if endocytosis is

inhibited by sodium azide (I. Schmidt et al., unpublished

observations); (3) a Fas ligation mechanism should also be

operative with empty liposomes, but these remained com-

pletely ineffective; (4) negatively charged liposomes such

as those used in the present study may preferentially attach

to monocytes via scavenger receptors [37, 38], which favor

internalization of particles but are not reported to trigger

apoptotic cell death.

The mechanisms underlying apoptotic death once do-

dronate is incorporated into monocytes remain elusive.

There is evidence that the enzyme responsible for chroma-

tin cleavage in apoptosis is a neutral endonuclease [39],

dependent on the coincident presence of Ca2+ and Mg�

ions [39, 40], and that alteration of the ionic environment

in the nucleus may be sufficient to activate this enzyme

[40, 41]. Accordingly, chelation of intracellular Ca2” pre-

vents both the activation of the endonuclease and the onset

of apoptosis in the case of T cells [40]. Because clodronate

is a chelator for bivalent cations [42], its property of induc-

ing apoptosis remains a paradox, which will require further

detailed investigation. Since clodronate interferes with the

ATP metabolism in amebae [43], it is possible that

changes in cell energy metabolism may cause or favor

apoptosis.

The influence of different stimuli on the inductionof cell death

1,25(OH)2D3 and LPS were both effective in promoting

clodronate-liposome--induced cell death, and this phe-

nomenon appeared proportional to the degree of enhance-

ment of phagocytosis. LPS was more potent than

1,25(OH)2D3 in promoting vesicle internalization (Table 1)

and was also more potent in enhancing cell death (Fig. 2);

also, monocytes had to be prestimulated for 24 h for

1,25(OH)2D3 to enhance cell death. These observations

are consistent with the notion that 1,25(OH)2D3 is a matu-

rational rather than an activating stimulus [15]; LPS, in

turn, while per se incapable of inducing apoptosis (Figs. 2,

3, and 7B; see also refs. 34 and 44), can enhance phago-

506396

344

220

I

�iI

Fig. 7. Inverted agarose gel image following electro�horesis of nuclear

extracts of equal numbers of monocytes (5 x 10 ) stimulated with

1,25(OH)2D3 (A) or LPS (B), in all cases after 7 h of incubation. bp

base pair standard. Polyethyleneglycol-stearate (PEG-S) clodronate-

liposomes (high dose, HD) induced a ladder pattern of multiples of 200

base pairs, similar to that obtained with gliotoxin (A), a substance known

to induce apoptosis in monocytes [29]. No such fragmentation was

induced by free clodronate (A, B) or empty PEG-S liposomes (A, B) in

activated monocytes. Similar analyses of nuclear extracts from resting or

LPS-stimulated PMNs revealed no apoptotic DNA fragmentation (data

not shown).


cytosis and/or strong cellular responses in monocytes [16,

45], for example, expression of proinflammatory cytokines

and induction of enzymes, that may be directly or mdi-

rectly relevant for the degradation of liposomes.

A third phagocytosis-enhancing stimulus was also tested

in this series of experiments (PMA, 10 ng/mL); this

strongly promoted phagocytosis within the first 4 h; how-

ever, it became per se capable of inducing apoptosis, as

already reported [34], regardless of whether monocytes

were exposed to empty or to clodronate-liposomes. It will

be of great interest to learn how pro- and anti-inflammatory

cytokines, stimuli that are present in vivo at sites of inflam-

mation and that differentially influence m4 function, affect

the sensitivity of M4s to PEG-S clodronate-liposomes.

Free clodronate is ineffective in inducing cell death

In the present study, clodronate as a free drug (0.22 mM)

proved ineffective in inducing cell death in monocytes

(Fig. 2). Free clodronate can become toxic for mouse peri-

toneal M�s, but only at concentrations considerably higher

than those used in the present study [46, 47]; at 3 mM and

after 48 h of incubation, that is, under much more extreme

conditions than in the present experiments, free clodronate

induces at most 30% cell death in M�-like cells [47]. Free

clodronate also appears incapable of inducing apoptosis

following in vivo administration of doses as high as 30

mg/kg to normal rats [48]. These findings suggest that the

clinical efficacy of free clodronate in the late phase of

arthritides [8, 10, 49] may be related not to depletion of

M4s but rather to effects on other cell types, for example,

osteoclasts [50]. Because only a very low percentage of

free clodronate enters M�s [32], it is conceivable that the

pharmacology of free clodronate, in both qualitative and

quantitative terms, differs radically from that of the encap-

sulated counterpart [32].

The suitability of PEG-S clodronate-liposomes forin vivo treatment

A biologically relevant finding of the present study was the

preferential toxicity of PEG-S clodronate-liposomes for ac-

tivated rather than resting monocytes (Figs. 2, 3, and 4).

The selectivity of PEG-S clodronate-liposomes for acti-

vated monocytes may explain the success of treatment of

rat experimental autoimmune disorders [8, 10], diseases in

which activated m�s play a major pathogenetic role, both

as immunocompetent cells [1 1] and as effectors of tissue

destruction [51, 52] through release of oxygen radicals

[53], nitric oxide [54], tissue-degrading enzymes [12, 55],

and/or proinflammatory cytokines [52, 56]. Of note, M4s

isolated from adjuvant arthritic rats show a clear enhance-

ment of phagocytosis [57]; in vivo, this feature conceivably

contributes to the susceptibility of activated M4s to PEG-S

clodronate-liposomes, because of higher incorporation of

this preparation. PEG-S clodronate-liposomes thus seem to

represent a means of preferentially targeting disease-rele-

vant M4s in experimental autoimmune disorders, in con-

cordance with the in vivo findings that M4s in immuno-

competent areas of spleen and draining lymph nodes were

preferentially depleted by systemic treatment of arthritic

rats with this formulation of clodronate-liposomes [10].

The fact that activated monocytes undergo apoptosis

upon exposure to PEG-S clodronate-liposome is particu-

larly interesting also in view of the safety of in vivo treat-

ment of inflammatory conditions. Side effects, such as

secondary damage in surrounding tissues by release of

lysosomal enzymes and nuclear material from necrotic

cells, may be greatly reduced by the fact that these compo-

nents remain encapsulated in apoptotic bodies [33]. In

fact, apoptosis in strongly activated M�s has been defined

as a physiological down-regulating mechanism to limit tis-

sue insult [34]. Confinement of the secondary effects in

tissues subjected to M� elimination may thus explain the

remarkable safety of clodronate-liposomes in the systemic

treatment of arthritic rats [8, 10].

Another factor that may contribute to the safety of in vivo

treatment with PEG-S clodronate-liposomes is the lack of

cytotoxicity for endothelial cells; this suggests that, in vivo,

no particular damage of vascular endothelium should be

expected upon systemic treatment with this liposome

preparation; uptake of liposomes by endothelial cells may

actually turn advantageous, as it may favor “shuttling” of

clodronate-liposomes into the interstitium of inflammatory

foci, as envisaged in the case of tumoral masses [21, 58].

Sterically stabilized PEG-S liposomes are superiorto conventional PC liposomes

PEG-S liposomes were superior to PC liposomes in induc-

ing cell death in activated monocytes (Fig. 4). The lack of

cytotoxicity of conventional PC liposomes in resting mono-

cytes thus confirms previous results obtained with m4-like

cell lines [47]. The substitution of PC with synthetic lipids

appears crucial in determining the preferential affinity of

PEG-S liposomes for activated monocytes, although the

nature and the modalities of this interaction remain to be

characterized. The effects of steric stability, surface

charge, and pH [19-21], as well as opsonization [59, 60],

for the particular formulation used in the present study

(PEG-S, sodium dodecyl sulfate, cholesterol; molar ratio

4: 1 :4) need to be clearly assessed in relation to the uptake

capacity of monocytes. In particular, the opsonization by

serum protein such as complement components may be

relevant, in that this feature largely influences uptake via

Fc receptors [60]. However, PEG-S liposomes are less sen-

sitive than PC liposomes to opsonization, and thus likewise

to incorporation via Fc receptors, inasmuch as (1) surface-

attached PEG creates a strong repulsive pressure [21] and

(2) the rates of cell death following incubation with PEG-S

liposomes do not change whether normal or heat-macti-

vated fetal calf serum (50-60#{176}C for 30-45 mm) is used in

the medium (present study; data not shown).

Suitable candidates for preferential uptake of PEG-Sliposomes by activated monocytes may prove to be the

scavenger receptors [37, 38], in that they probably undergo


activation-mediated up-regulation and apparently enhance

the incorporation of membranes carrying a net negative

charge [37]. The scavenger receptor may thus efficiently

capture PEG-S liposomes, which carry a negative charge

conferred by the sodium dodecyl sulfate component.

It is thought that the major advantage of the use of

sterically stabilized liposomes is their slow clearance by

M�s of the mononuclear phagocyte system, which leads to

prolonged circulation times in vivo [19-21]. The results of

the present study apparently contradict such a belief, in

that PEG-S liposomes are very efficiently phagocytosed by

cells of the M4 lineage. First, it is possible that fine chemi-

cal and physical differences among grossly similar formu-

lations yield marked differences in vivo; this may include

differential interactions with whole blood components,

which possess the capacity of slowing down and/or reduc-

ing the apoptotic process caused by clodronate-liposomes

(Fig. 3). A second possibility is that, in vivo, only a fraction

of M�s, those that are in a state of activation, avidly extract

circulating PEG-S liposomes, whereas the majority of m�s,

which are in a quiescent state, do not take up very many

liposomes (Table 1); as a consequence, enough vesicles

would remain in circulation for a prolonged time.

The present study provides evidence that sterically sta-

bilized liposomes laden with clodronate preferentially de-

plete activated monocytes in vitro. This preparation can

therefore be used for selective in vivo elimination of acti-

vated M�s in inflammatory disorders [4-10]. Encapsula-

tion of Mt-modulating agents, alternatively to

M�-eliminating toxins, may be further exploited for experi-

mental or therapeutic approaches.

ACKNOWLEDGMENTS

We thank Birthe Muller and B. Niescher for excellent

technical assistance; Dr. Zingsen, Blood Bank Erlangen,

for providing buffy coats; A. Hecht, Institute of Anatomy,

University of Erlangen-Nuremberg, for preparing transmis-

sion electron microscopy pictures; Boehringer Mannheim

for supplying clodronate; Dr. M. Meyer for advice on sta-

tistical analysis; Dr. R. Haliman for valuable advice; Dr.

B. Broker for providing Herpesvirus saimiri-immortalized T

cell clones; and Prof. K. Von der Mark and Prof. J.R.

Kalden for support. F. E. and R.W.K. were supported by

the German Ministry for Research and Technology (BMVI’;

FKZ O1VM9311 and 01VM8702); C.B. S.-W. and E. B. by

the Graduiertenkolleg Erlangen.

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incorporation of clodronate-liposomes

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