(CANCER RESEARCH 46, 4330-4335, September 1986]

In Vitro Activation of Rat Liver Macrophages to Tumoricidal Activity by Free orLiposome-encapsulated Muramyl Dipeptide1

loos Daemen,2 Aletta Veninga, Frits H. Roerdink, and Gerrit L. Scherphof

Laboratory of Physiological Chemistry, University of Groningen, Bloemsingel 10, 9712 KZ Groningen, The Netherlands

ABSTRACT

We investigated the in vitro activation of rat liver macrophages to atumoricidal state with free and liposome-encapsulated immunomodula-

tors. The cytolytic activity of liver macrophages was determined by aradioactivity release assay using murine B16 melanoma cells, labeledwith |/m'/A.i7-'I I|tli\niidim-. Exposure of the liver macrophages to con

centrations of 50 MUof free, nonencapsulated, mummy I dipeptide (MDP)per ml resulted in maximal levels of tumor cell lysis of approximately20%. Encapsulation of the MDP within liposomes (multilamellar vesicles, 0.3 to 0.5 urn in diameter, consisting of egg phosphatidylcholine,cholesterol, and dicetylphosphate, 4:5:1) not only caused a 500-fold

reduction in the amount of MDP required to obtain the same levels ofcytolysis but also increased the maximally obtainable level of cytolysismore than 2-fold.

A synergistic effect of lipopolysaccharide and free or encapsulatedMDP on cytolytic activity was observed when the macrophages wereexposed to a combination of the two agents simultaneously.

Besides causing tumor cell lysis, activated macrophages were also ableto suppress tumor cell proliferation by 80 to 90% as determined by a|/m'/Ar/- 'I l|tli> mulini- incorporation assay.

With a fixed amount of MDP, encapsulated in different amounts ofliposomal lipid, the extent of macrophage activation was found to increasewith a larger amount of encapsulating lipid. This increase in macrophageactivation may be the result of a sustained intracellular release of encapsulated MDP from the liposomes. Liposome structure and composition»•illthus be important parameters in the in vivo application of liposomes

as carriers of immunoactive substances.

INTRODUCTION

The liver is a major site of metastatic growth derived fromprimary colorectal carcinoma. The average survival time ofpatients with liver métastasesis 6 mo. Surgical resection ispossible in only 5 to 10% of all patients, and available chemo-and/or radiotherapeutic treatments fail to improve survival timeby more than a few months at most (1); thus, there is a greatneed of alternative methods for the treatment of liver métastases. Activation of the host immune system appears to be apromising approach towards that purpose (2). In vitro exposureof monocytes and alveolar or peritoneal macrophages to avariety of immunomodulators such as lymphokines (3), -,-interferon (4), and MDP3 (5, 6) has been shown to render these

cells tumoricidal. After in vivo administration of free MDP,however, no enhancement of cell-mediated immunity could bedemonstrated, since more than 90% of the drug is excretedfrom the body within 60 min (7); this calls for the design of anefficient drug delivery system for these agents. Studies on theuse of liposomes as drug carriers have shown that these particles

Received 2/24/86; revised 5/28/86; accepted 5/30/86.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Research supported by the Dutch Foundation for Cancer Research, the

Koningin Wilhelmina Fonds.2To whom requests for reprints should be addressed.3The abbreviations used are: MDP, muramyl dipeptide; I .I'S. lipopolysaccha

ride; MLV, multilamellar phospholipid vesicles; PC, egg phosphatidylcholine;DCP, dicetylphosphate; PS, phosphatidylserine; FCS, heat-inactivated fetal calfserum; GBBS, Gey's balanced salt solution; HN-buffer, buffer containing NaCIand 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; PBS, phosphate-buffered saline; | 'I l|d I luí.|mrí/i.r/-'I l|ilivmi<lini-.

are primarily taken up by liver and spleen macrophages (8).Immunomodulator-containing liposomes may thus be exploitedto achieve efficient activation of liver macrophages, thus preventing or eradicating metastatic tumor growth in the liver.

Fidler et al. (9) achieved significant reduction of experimentallung métastasesby i.v. injection of liposomes containing MDPin mice inoculated with syngeneic B16 melanoma cells. Thiseffect was obtained despite the fact that only 6.6% of theadministered dose of liposomes was found associated with lungmacrophages. Xu and Fidler (10, 11) recently demonstratedalso that murine liver macrophages can be activated to a tumoricidal state after exposure to free, soluble immunomodulators. In addition, Thombre and Deodhar (12) reported on theinhibition of liver métastasesfrom murine colon adenocarci-noma by the systemic administration of liposome-encapsulatedC-reactive protein or crude lymphokines. The therapeutic effectis likely to involve activation of liver macrophages (Kupffercells).

As an extension of our investigations on uptake and processing of liposomes by liver macrophages (13, 14), we recentlyinitiated a study on the activation of rat liver macrophages totumor cytotoxicity by means of liposome-encapsulated immunomodulators. In this paper we demonstrate that cultured livermacrophages can be rendered cytostatic and cytolytic againsttumor cells by incubation with immunomodulators such as LPSand MDP. Moreover, we found that encapsulation of MDPwithin liposomes substantially augments the MDP-induced cytotoxicity.

MATERIALS AND METHODS

Animals. Specific-pathogen-free female Wistar rats, weighing 160 to180 g, were used in all experiments.

Media and Reagents. All cultures were grown in KI'M I 1640 fromGibco and supplemented with 2 DIML-glutamine (Flow Labs.), penicillin G (100 units/ml), streptomycin (100 /¿g/ml)(both from Gist-Brocades), and heat-inactivated FCS (Gibco). MDP was a generous giftfrom Ciba Geigy, Ltd. MDP was stored desiccated at 4°C.Lipopoly

saccharide B from Escherichia coli 0127:B8 (LPS) was purchased fromDifco Lab. and dissolved in medium immediately before use. | 'I Ijlnulin(specific activity, 1 Ci/mmol) and [3H]dThd (specific activity, 5 Ci/

mmol) were from Amersham, Ltd. DNase (Grade II) was purchasedfrom Boehringer-Mannheim. Media and reagents used in this studywere checked for endotoxin content by a Limulus amebocyte lysateassay with a detection limit of O.SOng/ml and found negative. Occasionally when liposome preparations were found to have higher endotoxin levels, due to a too high endotoxin level in the HN-buffer,experiments using these preparations were excluded from the resultsgiven in this paper.

Tumor Cell Culture. B16-B6 melanoma, syngeneic with CS7BL/6mice, was grown as a monolayer in culture medium containing 10%FCS and IO"5 M /3-mercaptoethanol.

Lipids and Preparation of Liposomes. Egg PC (L-a-lecithin from eggyolk), DCP and cholesterol (type CH-S) were purchased from SigmaChemical Co., Ltd. The lipid solutions in chloroform:methanol (8:2)were stored under nitrogen at —20°C.^V-(Lissamine Rhodamine B

sulfonyl)phosphatidylethanolamine was from Avanti Polar-Lipids, Inc.MLV were prepared as follows. Lipids (PC:cholesterol:DCP in a molarratio of 4:5:1) were mixed, dried under reduced nitrogen pressure,

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ACTIVATION OF LIVER MACROPHAGES BY LIPOSOMAL MDP

dissolved in benzene, and lyophilized. The lipids were then hydrated in135 HIM NaCMO mM 4-(2-hydroxyethyl)-I-piperazineethanesuIfonicacid pH 7.4 (HN-buffer), or in HN-buffer containing MDP or [3H]-

inulin and vortexed for 10 min at room temperature. The vesiclesformed were sized by extrusion through a series of polycarbonatemembranes (Nucleopore) (15) of l-fttn to 0.8-/im to 0.6-/jm to 0.4-^mpore diameter, respectively. Vesicles containing MDP or [3H]inulinwere freed from nonencapsulated material on a Sephadex G-100 column (Pharmacia). Approximately 3% of the initially dissolved agentwas encapsulated, as determined by the amount of lipid-associated| 'I I |imilin. Liposome preparations were stored under nitrogen at 4 (

and used within 2 days after preparation.Isolation of Rat Liver Macrophages. Liver macrophages were isolated

by Pronase digestion of the liver and purified by centrifugal elutriationas first described by Knook and Sleyster (16) for BN/BiRij rats and asmodified by Dijkstra et al. (17). Briefly, the liver was preperfused withGBSS to remove blood and perfused for 3 min with 0.2% Pronase(Boehringer-Mannheim). The organ was removed, cut in small pieces,and incubated in 0.2% Pronase for 50 min in the presence of DNase.The cell suspension was washed once, and nonparenchymal cells wereseparated from nonviable parenchyma! cells and remaining erythrocytesby centrifugation on a metrizamide gradient (analytical grade; Nye-gaard) (0.7%) for 15 min, 1500 x g. The top layer containing thenonparenchymal cells was washed once and resuspended in 5 ml ofGBSS containing DNase. These cells were separated into macrophagesand endothelial cells by elutriation centrifugation (Beckman; type .116 elutriation rotor). The cell suspension was flushed into the rotor at aflow rate of 20.5 to 21.0 ml/min, rotor speed at 2500 rpm, at 4'C. At

this flow rate lymphocytes, including natural killer cells, and endothelialcells were flushed out in 250 ml of GBSS. The macrophages werecollected at a flow rate of 46.5 ml/min in 150 ml of GBSS, concentratedby centrifugation at 700 x g for 10 min, and resuspended in culturemedium containing 20% FCS. Liver macrophages (2.5 x 10s per well)in 200 n\ of culture medium were seeded in 96-well microtiter plates(Costar) to obtain a monolayer of liver macrophages.

Phagocytosis of Liposomes. One day after cell isolation, macrophagemonolayers were incubated with MLV, in RPMI-1640 (10% PCS),containing l'I Ilimititi as a metabolically inert marker. At different time

points the medium was removed, the wells were washed 4 times withcold PBS, and the cells were lysed with 0.5 M NaOH. Aliquots weretaken for the determination of protein content and for radioactivitymeasurement.

In Vitro Cytolytic Assay. Macrophage-mediated cytolysis was assessed by a radioactivity release assay. One day after isolation, isolatedliver macrophages in monolayer culture were incubated with free orliposome-encapsulated immunomodulators. Target tumor cells in exponential growth phase were radiolabeled by a 20-h incubation inmedium containing 0.3 //<'i of [3H]dThd per ml. The cells were then

washed free from radioisotope and cold pulsed by incubation in freshmedium for 3 to 4 h, to deplete cytoplasmic pools of | 'I l|d I lui and

minimize spontaneous release of label. Subsequently the cells werewashed twice with PBS, 37*C, to remove unbound radiolabel, harvested

by short trypsinization (0.05% Difco trypsin-0.2% EDTA, for 45 s at37*C), washed 4 times with PBS, and resuspended in culture mediumat a concentration of 10s cells/ml. Four h after the addition to the

macrophage monolayers of 100 >¡\of culture medium (10% FCS)containing the immunomodulator(s), 100 //I of medium containing to'[3H]dThd-labeled tumor cells were added per well. At this ratio of

macrophage to target cell (25:1) untreated macrophages were notcytotoxic to neoplastic cells, indicating the absence of natural killercells. Radiolabeled target cells were also plated alone, as an additionalcontrol. Forty-eight h after the addition of tumor cells, the supernatantswere collected, and the radioactivity was measured in a liquid-scintillation counter. Total dpm added per well were determined by measuringthe radioactivity of IO4 tumor cells in 200 n\ of medium mixed with

200 ill of 1% sodium dodecyl sulfate. Specific cytolysis was calculatedas follows.

in which a is dpm in the supernatant of tumor cells cocultured withtest macrophages, b is dpm in the supernatant of tumor cells coculturedwith control macrophages, and c is dpm in the total amount of tumorcells added per well.

In Vitro Cytostatic Assay. Macrophage-mediated inhibition of theproliferative capacity of tumor cells was assessed by measuring | '111

dThd incorporation into the DNA of cocultured tumor cells. After a 4-h incubation of the macrophage monolayers with 100 /<!of culturemedium containing immunomodulators, 100 //I of medium containingII)4tumor cells were added per well. Tumor cells alone were plated asan additional control. After 24 h of cocultivation 0.01 ¿iCiof [3H]dThdwas added per well. Twenty-four h after the addition of label the cultureswere washed 3 times with PBS (4°C),and the adherent viable cells were

lysed with 0.2 ml of 0.5 M NaOH. The radioactivity of the lysate wasmeasured in a liquid scintillation counter, and the inhibition of tumorcell proliferation was calculated as follows.

of inhibition = 100 x 11 —

in which x is dpm in tumor cells cocultured with test macrophages, andy is dpm in tumor cells cocultured with control macrophages.

RESULTS

The Isolation of Rat Liver Macrophages. The average yield ofmacrophages from a Pronase-perfused rat liver was 6.3 ±0.8x IO6cells per g of wet liver (n = 16). The purity of these cellpreparations was 85 to 90%, based on peroxidase-positive staining of the macrophages. When plated in culture medium containing 20% FCS, the macrophages attach to the bottom of theculture plates within 4 h and stretch within 20 h, at which timepoint the cells are able to endocytose liposomes as is demonstrated in the following experiment.

In Vitro Uptake of Liposomes by Rat Liver Macrophages. Inthese studies we used negatively charged liposomes containingSO mol percent of cholesterol since we earlier found the uptakeof these vesicles by liver macrophages to be much higher thanthat of neutral or positively charged liposomes (14), and sinceleakage of encapsulated material is low by virtue of the cholesterol content. Fig. 1 shows the association of negatively chargedMLV with cultured liver macrophages at 37°Cand at 4°C.TheMLV are radiolabeled with [3H]inulin, entrapped within the

aqueous phase of the vesicles. Following an incubation with 25nmol of liposomes at 37°Cfor 2, 4, and 24 h, respectively,

values of 1.2%, 2.1%, and 8.7% of the added liposome-label

300,-

of specific cytolysis = 100 xa-bc-b

12 16incubation time (hi

Fig. 1. Uptake of [3H]inulin-containing liposomes. Multilamcllar vesicles composed of egg POcholesterohDCP (in a 4:5:1 molar ratio) contained I'll limitili.Twenty-five nmol (•,O) or 50 nmol (A, A) of liposomal lipid were added tomacrophage monolayers and incubated at 37'C (•,A) or 4'C (O, A) in medium

containing 10% FCS. At different time points the wells were washed 4 times withcold PBS, and the cells were lysed with 0.5 M NaOH. Aliquots were taken for thedetermination of protein content and radioactivity measurement. Given is themean uptake in nmol of lipid per mg of protein of duplicate determinationsagreeing within 5%.

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ACTIVATION OF LIVER MACROPHAGES BY LIPOSOMAL MDP

were found associated with the macrophages, respectively. Following an incubation with 50 nmol of liposomes, values of1.0%, 1.8%, and 5.7% were found associated, respectively. At4°Cno more than 0.5% of the added label became associated

with the cells in 24 h, indicating that the uptake process isenergy dependent. Therefore, most of the uptake is assumed tobe mediated by phagocytosis. By fluorescence microscopy, usingrhodamine-phosphatidylethanolamine as a fluorescent liposo-mal marker, the association of the liposomes with cells couldbe visualized (not shown). At 37°Cthe liposomes were inter

nalized by the macrophages, as fluorescence was localizedwithin discrete cytoplasmic vacuoles, whereas at 4°Cthe fluo

rescence was spread around the cells indicating mere bindingof the vesicles to the cell surface without internilii/ation.

Macrophage-mediated Cytolysis with Free and Liposome-en-capsulated MDP. Fig. 2 shows that rat liver macrophages inmonolayer culture can be rendered cytolytic against B16 mela-

100

80

"85 60-

Õ40(10)

10 10'ng MOP/well

TO3 10'

Fig. 2. Macrophage-mediated cytolysis with free and liposome-encapsulatedMDP. Per well of a 96-well microtiter plate 25 x IO4 liver macrophages were

incubated with medium (control), free MDP (O), or liposomes containing 1 ng ofMDP per nmol of liposomal lipid (•).After 4 h. 10* [JH)dThd-labeled B16melanoma cells were added per well. After another 48 h 3H release into the

supernatant was determined. Control liposomes induced no cytolysis. Points,mean percentage of the normalized cytolysis; bars, SD. Numbers in parentheses,number of experiments. Percentages of specific cytolysis ("Materials and Methods") were normalized by setting the percentage of specific cytolysis induced by

the combination of MDP (50 jig/ml) and LPS (50 ng/ml) at 100. The meanpercentage of specific cytolysis induced by this combination was 53.8 ±14.1% (n= 26).

noma cells after exposure of the cells to free as well as liposome-encapsulated MDP. For each experiment fresh liver macrophages were isolated and liposomes prepared while also thetarget tumor cells were freshly labeled for each separate experiment. This caused the induced percentages of specific tumorcytolysis to vary substantially between experiments. In order toallow better comparison of the results of different experimentswe normalized the percentages of specific cytolysis obtainedwithin each experiment by setting the percentage of specificcytolysis induced by the combination of MDP (50 jig/ml) andLPS (50 ng/ml) at 100. The percentages of specific cytolysisinduced by this combination (MDP/LPS) varied between 38%and 80% for the whole series of 26 experiments, and the meanvalue was 53.8 ±14.1% (SD). Fig. 2 shows that the normalizedpercentage of cytolysis induced by incubation of 2.5 x 10s

macrophages with free MDP reached an optimal level of 46%with 10,000 ng of MDP per 100 /il of medium. As little as 10ng of MDP were sufficient to obtain the same result withliposome-encapsulated MDP (1 ng of MDP per nmol of lipid).The results furthermore show that entrapment of the drugwithin liposomes significantly increased its cytolytic potency.Whereas free MDP induced cytolysis to a maximal normalizedlevel of 46%, incubation of the macrophages with 100 ng ofliposomal MDP resulted in 85% of the cytolysis induced by thecombination MDP/LPS.

Macrophage-mediated Cytolysis versus Cytostasis. Light-microscopic screening of the macrophage-tumor cell coculturesrevealed that, besides causing tumor cell lysis, activated livermacrophages profoundly inhibited tumor cell proliferation. Table 1 shows the percentage of inhibition of tumor cell proliferation (cytostasis plus cytolysis) compared to the percentage oftumor cell lysis (cytolysis). Tumor cell lysis induced by liposomal MDP was 31 to 56% as determined by the amount of 3H

released from disintegrated tumor cells. In the remaining tumorcells DNA replication was strongly inhibited. Inhibition due totumor cell lysis and tumor cell stasis was 86 to 99%, asdetermined by the amount of | 'I I |d I lui incorporation after a24-h coculture period. Control liposomes, not containing MDP,did not induce any significant cytolysis or cytostasis. The incorporation of [3H]dThd in control or activated liver macro

phages, cultured alone, was negligible (not shown). To determine any toxic effect of liposomes and/or MDP on tumor cells,IO4melanoma cells were incubated for 48 h with either control

Table 1 Effect of liposome-encapsulated MDP on macrophage cytolytic and cytostatic activity

Macrophagetreatment*MDP-liposomes(l

ng MDP/nmollipid)Control

liposomesMediumNone,

B16 cells aloneLiposomal

lipid(nmol/well)200

10050

25200100

5025Cytolysis

(dpm released insupernatant)*1306

±3' (56)

1082 ±84(41)929 ±64(31)938 ±78(31)485

±42(1)455 ±28 (0)439 ±11 (0)485±45(1)463

±43420

±21P*<0.001

<0.01<0.01<0.05Cytostasis

(dpmincorporated)*'121

±16(99)220 ±79 (98)962 ±21 (89)

1303 ±29(86)9367

±56 (0)9734 ±508 (0)9092 ±868 (0)9483 ±385(0)9105

±5728668

±556P<0.001

<0.001<0.001<0.001

" Per well 25 x 10* liver macrophages were incubated with medium, control liposomes, or liposomes containing 1 ng of MDP per nmol of liposomal lipid.b After 4 h. I(I*|'H|dThd-labeled melanoma cells were added per well. After another 48 h, 'II release into the supernatant was determined in triplicate experiments.

Numbers in parentheses, percentage of specific cytolysis; see "Materials and Methods."' Statistical significance compared to medium control (Student's i test).'Afteran incubation of 4 h with the immunomodulators 10' melanoma cells were added per well. After 24 h of cocultivation [3HJdThd was added to the wells for

another 24 h. At this time (48 h of cocultivation) the cultures were washed 3 times with PBS, and the cells were lysed with 0.5 M NaOH. Radioactivity of the lysatewas determined in triplicate experiments. Numbers in parentheses, percentage of inhibition; see "Materials and Methods."

' Mean ±SD.

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Table 2 Synergism ofLPS and free or liposome-encapsulated MDP on macrophage cytolytic activity

dpm released insupernatant"First

macrophagetreatment"LPSMDPMDP-liposomesLPS

+MDPLPS+MDP-liposomesLPSMDPLPSMDP-liposomesMediumNone,

BI6 cells aloneSecond

macrophagetreatment"MDPLPSMDP-liposomesLPSWithoutremovingthe

firstimmuno-modulator(s)464

±36°(13)'425

±8(11)858±54(36)1264±24(60)'1409±6(68)«795

±28(32)*799

±82(33)871±70(37)799+10(33)237

±39220±12Removing

thefirstimmunomodulator(s)287

±13(3)440±32(12)572±34(19)870±19(37/754±75(30)660

±30(25)*704

±70(27)664±62(25)683±55 (26)

"Twenty-five x IO4 rat liver macrophages were incubated with medium (control) or the indicated immunomodulators, LPS (5 ng/well), MDP (5 ng/well), andMDP-liposomes (50 ng of MDP per well encapsulated in 50 mimi of liposomal lipid). After 4 h, medium or a second immunomodulator, together with IO4l'I I|<li ini

labeled melanoma cells, was added to the wells with or without removing the first immunomodulator(s) by washing.* Cytolytic activity was determined after a 48-h coculture of macrophages and [3H|dThd-labeled melanoma cells. 'II release into the supernatant was determined

in triplicate experiments.c Mean ±SD.d Numbers in parentheses, percentage of specific cytolysis; see "Materials and Methods."' Statistical significance compared to the sum of cytolysis induced by both immunomodulators alone (Student's i test), /' < 0.005.fp< o.oio.'P< 0.025.* P < 0.050.

liposomes, or with free, or liposome-encapsulated MDP. Neither cytolysis nor cytostasis was observed (not shown).

Synergism of MDP and LPS. As has been shown for alveolarmacrophages (18), peritoneal macrophages (19), and humanblood monocytes (20), a synergistic effect on cytolytic activitycan be obtained by applying a combination of immunomodulators. In Table 2 a similar synergistic effect of LPS and MDP,either free or liposome encapsulated, on the activation of livermacrophages is shown. While 5 ng of LPS per 2.5 x 10* cells

induced only 13% cytolysis, and 5 ng of free MDP in the sameexperiment induced only 11%, the combination of the twoagents activated the macrophages to cause 60% cytolysis. Thisaugmentation of cytolysis was also obtained with the combination of LPS and liposomal MDP. The 36% cytolysis causedby 50 ng of liposomal MDP was enhanced to 68% by theaddition of 5 ng of LPS.

Enhanced activation of macrophages was found to requiresimultaneous rather than sequential exposure to the two substances. Liver macrophages were incubated with LPS or MDP.After 4 h the second immunomodulator, MDP or LPS, andtumor cells were added to the wells. The results show that,whether or not the first immunomodulator was removed priorto addition of the second one, the percentage of induced cytolysis was not or barely significantly in excess of the sum ofthe percentages of cytolysis induced by LPS and MDP alone.A similar effect was observed with the combination of liposomalMDP and free LPS.

Effect of the Ratio of Liposomal Lipid to Encapsulated MDP.We prepared two liposome preparations containing differentamounts of MDP. Fig. 3 shows that, for each of the twopreparations, an increase in cytolysis was obtained with anincreasing amount of liposomes added. The preparation with ahigh ratio of liposomal lipid to MDP (i.e., 1 nmol of lipid perng of MDP, the "high-lipid" preparation, O) resulted in a

plateau level of 40% cytolysis, reached at 25 ng of MDP perwell, i.e., at 25 nmol of lipid. With the "low-lipid" preparation

(•)no plateau was reached within the concentration rangeapplied, i.e., up to 500 ng of MDP encapsulated in 50 nmol oflipid. The difference in effect between the two liposome preparations becomes particularly apparent upon comparing the cy-

ng Iposome-encopsuloted MDP/well

Fig. 3. Influence of the ratio of liposomal lipid to encapsulated MDP on livermacrophage cytolysis. Twenty-five x IO4 rat liver macrophages were incubatedwith medium (control) or liposome-encapsulated MDP. O, liposomes with 1 nmolof lipid per ng of MDP; »,liposomes with 0.1 nmol of lipid per ng of MDP.After 4 h, 104[3H]dThd-labeIed melanoma cells were added per well. After another48 h, 'H release into the medium was determined. Points, mean of the percentageof specific cytolysis of triplicate experiments; see "Materials and Methods." Bars,

SD.

totoxicities caused by identical amounts of MDP (Fig. 3); forexample, when 50 ng of MDP were added to the cells encapsulated within 50 nmol of lipid ("high-lipid" preparation, {),

the cytolytic activity of the macrophages was 3 times that whichwas obtained when only 5 nmol of lipid were used to encapsulatethe same amount of MDP (arrowhead). Control experimentsdemonstrated that the enhanced cytotoxicity was not caused bythe mere presence of high lipid concentrations, as a mixture of40 nmol of empty liposomes and 10 nmol of M DI' containingliposomes induced the same cytotoxicity as 10 nmol of MDP-liposomes alone (not shown). Apparently, exposure of the cellsto a fixed amount of MDP encapsulated in a relatively largenumber of liposomes results in a more efficient activation thanencapsulating the drug in relatively few vesicles.

DISCUSSION

In the present study we have shown that cultured liver macrophages can be activated in vitro to a tumoricidal state. Activation was achieved by the incubation of these macrophageswith free, nonencapsulated immunomodulators such as MDP

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ACTIVATION OF LIVER MACROPHAGES BY LIPOSOMAL MDP

and/or LPS. Tumoricidal capacity could be enhanced by encap- the activation of macrophages depends on a multistep reaction.

sulating the MDP in liposomes.Because of the relatively large number of cells required in the

type of studies described in this paper, we used rats as a sourceof liver macrophages, while applying xenogeneic murine B16melanoma cells to determine the induced cytotoxic activity. Ashas been reported by Sonéand Fidler (21) the in vitro treatmentof alveolar macrophages with liposomes containing MDP orlymphokines results in the induction of tumoricidal activitytowards syngeneic tumor cells and to a comparable extenttowards allogeneic or xenogeneic tumor cells.

Liposome-encapsulated MDP was 2 to 3 orders of magnitudemore potent in inducing liver macrophage cytotoxicity thanfree MDP.

This difference in potency may be due to a number of factors,(a) The rate of internalization of liposomal MDP via adsorptiveendocytosis (17) will be substantially higher than the rate atwhich free MDP, dissolved in the extracellular medium, entersthe cell, presumably by fluid-phase endocytosis. (b) The mechanism of intracellular processing and/or intracellular compart-mentation of free versus encapsulated MDP may, conceivably,be different. Upon intralysosomal degradation of the vesiclesthe liposomal MDP will be gradually released. Subsequently,MDP or metabolic products are likely to cross the lysosomalmembrane into the cytosol. Free MDP, on the other hand, maystart to cross the endosomal/lysosomal membrane immediatelyafter being endocytosed and will thus not, or only for a relativelyshort time, be exposed to lysosomal enzymes and low pH.Results reported recently by Menta et al. (22, 23) suggest thatmost of the target sites ("receptors") for MDP are at intracel

lular locations and that relatively high intracellular levels ofMDP are required for activation. Our data show that also forrat liver macrophages cytolytic activity tends to increase withincreasing amounts of free MDP added to the cells. At noconcentration applied, however, free MDP, continuously present during coculturing of liver macrophages and tumor cells,was able to activate the macrophages to the level obtained withliposomal MDP. The relatively high intracellular level of MDPneeded for optimal activation can, presumably, not be attainedwith free MDP.

Interestingly, a higher load of lipid to carry the MDP intothe cells leads to a higher level of tumor cytotoxicity with thesame amount of MDP. Observations on protease secretion ofperitoneal macrophages stimulated with liposomal MDP showa comparable effect (23). These observations may be explainedby assuming a relatively short intracellular half-life of MDPonce this agent has become available to its target site. Asustained intracellular release of MDP from the liposomeswould provide conditions required for optimal activation, bymaintaining sufficiently high MDP levels at the target site(s)for a prolonged time. This view is compatible with studies bySchroit and Fidler (24) who studied the effect of liposomestructure and lipid composition on the activation of alveolarmacrophages. Their results show that MDP encapsulated indistearoyl POPS liposomes induced a higher level of cytotoxicity than MDP encapsulated in (egg) POPS liposomes. Theysuggest that distearoyl POPS liposomes may be more resistantto intralysosomal degradation than liposomes containing (egg)PC and thus produce a sustained activation of alveolar macrophages.

Our results show that liposomal MDP activates liver macrophages not only to a cytolytic state but also to a cytostaticstate. DNA replication in B16 melanoma cells was almostcompletely suppressed by activated macrophages. Most likely.

An intermediate step would render them cytostatic, while completion of a series of reactions may be needed to attain acytolytic state. By analyzing the activation of peritoneal macrophages from macrophage-defective mouse strains, Boraschiand coworkers (25) provided strong evidence for a dissociationbetween macrophage tumoricidal capacity and antiproliferativeactivity.

Recently several studies have been reported on the use ofvarious combinations of immunomodulators such as 7-inter-feron and LPS (26), 7-interferon and MDP (19), and lymphokines and LPS (18). In attempts to enhance liver macrophagecytotoxicity we incubated the cells with a combination of LPSand free or liposome-encapsulated MDP. Our data demonstratea synergistic effect of LPS and MDP on the tumoricidal potencyof the macrophages. The enhanced cytotoxicity induced by acombination of immunomodulators favors a two-step activationmechanism in which one immunomodulator functions to primethe macrophages, making them responsive to a second onewhich triggers them to become cytotoxic (27). In our assay wecannot determine whether LPS or MDP primes the macrophages, since a significantly enhanced cytotoxicity was onlyachieved by the simultaneous presence of the two agents. Ashas been reported by Kildahl-Andersen and Nissen-Meyer (28),both LPS and MDP most probably function partly as primaryinducers but are most efficient as secondary inducers.

ACKNOWLEDGMENTS

We thank CIBA GEIGY for generous supplies of MDP, Bert Dontjeand Jan Wijbenga for skillful technical assistance. Professor J. W.Oosterhuis, MD., for his stimulating interest in our work and forproviding the tumor cells, and Rinske Kuperus for preparing themanuscript.

REFERENCES

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1986;46:4330-4335. Cancer Res   Toos Daemen, Aletta Veninga, Frits H. Roerdink, et al.   Activity by Free or Liposome-encapsulated Muramyl Dipeptide

Activation of Rat Liver Macrophages to TumoricidalIn Vitro

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