ELSEVIER Journal of Immunological Methods 174 (1994) 83-93

JOURNAL OF IMMUNOLOGICAL METHODS

Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications

Nico Van Roo i jen *, Annemar ie Sanders

Department of Cell Biology, Division of Histology, Faculty of Medicine, Free University, Van der Boechorststraat 7, 1081 BT Amsterdam, Netherlands

Abstract

Selective depletion of macrophages from tissues in vivo can be used to investigate whether these cells are playing a role in defined biological processes. This question is particularly relevant to various host defense mechanisms. We have developed a macrophage 'suicide' technique, using the liposome mediated intracellular delivery of dichloromethylene-bisphosphonate (CI2MBP or clodronate). The method is specific with respect to phagocytic cells of the mononuclear phagocyte system (MPS) for the following reasons: (1) The natural fate of liposomes is phagocytosis. (2) Once ingested by macrophages, the phospholipid bilayers of the liposomes are disrupted under the influence of lysosomal phospholipases. (3) CI2MBP intracellularly released in this way does not easily escape from the cell by crossing the cell membranes. (4) C12MBP released in the circulation from dead macrophages or by leakage from liposomes, will not easily enter non-phagocytic cells and has an extremely short half life in the circulation and body fluids. In the present review, the preparation of CI2MBP-liposomes has been described in detail. Furthermore, the mechanism of action of the new approach and its applicabilities are discussed.

Keywords: Macrophage; Function of macrophages; Depletion of macrophages; Liposome; Dichloromethylene- bisphosphonate (C12MBP, or clodronate).

1. Introduction

Apart from their role as scavengers and their role in immune and non-immune defense mecha- nisms, macrophages play a role in numerous bio- logical phenomena. Macrophage effects are often mediated by their products (e.g., cytokines). Macrophages may play a crucial role in a particu- lar biological process or they may have a potenti-

* Corresponding author.

ating or inhibiting influence. Depletion of macrophages followed by functional studies in such macrophage-depleted animals can be used to confirm the involvement of macrophages. Ear- lier methods for depletion of macrophages were based on the administration of silica, asbestos (Kagan and Hartmann, 1984), carrageenan (Shek and Lukovich, 1982) or by other treatments (Pinto et al., 1989). However incompleteness of deple- tion as well as unwanted effects on non-phago- cytic cells were obvious disadvantages. For that reason we have developed a more sophisticated approach (Van Rooijen and Van Nieuwmegen,

0022-1759/94/$07.00 1994 Elsevier Science B.V. All rights reserved SSDI 0022-1759(94)00129- K

84 N. Van Rooijen, A. Sanders/Journal of Immunological Methods 174 (1994) 83-93

1984; Van Rooijen, 1989) based on the liposome mediated intracellular delivery of dichloromethy- lene-bisphosphonate (C12MBP or clodronate). This approach is now used in an increasing num- ber of fundamental studies on macrophage func- tions and might become interesting from a clini- cal point of view. In the present review, the mechanism of action and selectivity of the lipo- some mediated macrophage 'suicide' approach, as well as some applications are discussed. Fur- thermore, technical details of the preparation of ClaMBP-liposomes are given.

2. Liposomes

Liposomes are artificially prepared spheres, consisting of concentric phospholipid bilayers separated by aqueous compartments. They form, when phospholipids, e.g., phosphatidylcholine molecules, are dispersed in water. The phospho- lipid molecules will find a conformation in which their hydrophobic fatty acid chains are prevented from making contact with water. For that reason, phospholipid bilayers are formed in which the relatively hydrophilic head groups are making up both of the outer parts of each bilayer, whereas the hydrophobic fatty acid groups are located directly opposed to each other in the inner side of the bilayer. Part of the aqueous solution to- gether with hydrophilic molecules (such as C12MBP), dissolved in it, will be encapsulated during the formation of the liposomes (Fig. 1). Lipophilic molecules will be associated with the phospholipid bilayers themselves. The hydropho- bic parts of amphipathic molecules will be in- serted in the bilayers, whereas their hydrophilic parts extend into the aqueous compartments or are exposed on the outer surfaces of the lipo- somes. The numbers of concentric phospholipid bilayers (unilamellar and multilamellar lipo- somes), the phospholipid composition and the charge of the liposomes can be varied. Targeting of liposomes may be achieved by the insertion of target molecules (e.g., monoclonal antibodies) in their outer (surface) bilayer (Gregoriadis, 1988). Apart from liposomes that have been developed to avoid their uptake by macrophages (so called

~ = Hydrolahi l ic group = Hydrolahobic fat ty acid chains OH CI OH

I I = O=P~ -P=O

I I I OH CI OH

Fig. 1. C12MBP-liposomes. Liposomes are artificially pre- pared spheres, consisting of concentric phospholipid bilayers, separated by aqueous compartments. They form, when phos- pholipid molecules are dispersed in water. Part of the aque- ous solution, together with hydrophilic molecules which have been dissolved in it, such as the bisphosphonate CIEMBP (black squares; see also structural formula) or the chelator molecules EDTA and DTPA, are encapsulated during the formation of the liposomes.

'stealth' liposomes), their usual fate is ingestion and digestion by macrophages. For this reason, liposomes form a suitable tool to manipulate macrophage function (Van Rooijen, 1992a).

3. Mechanism of action

CI2MBP (in a somewhat modified form: Ostac, a product of Boehringer Mannheim) is one of the bisphosphonates used for treatment of osteolytic bone diseases. It is not a toxic drug in itself and liposomes (if, e.g., prepared of phosphatidyl- choline and cholesterol) are also not toxic. The free drug does not easily cross cell membranes and has an extremely short half life in circulation and body fluids (Fleisch, 1988). The implication is that CI 2 MBP, once delivered into phagocytic cells using liposomes as vehicles, will not escape from the cell. On the other hand free CI2MBP, re- leased by leakage from liposomes or released from dead macrophages, will not enter ceils in amounts that are able to disturb their metabolism. After disruption of the phospholipid bilayers of the liposomes under the influence of the lysoso- mal phospholipases in the macrophage, the drug, which is dissolved in the aqueous compartments between the bilayers, is released into the cell. The more of the concentric bilayers are affected,

N. Van Rooijen, A. Sanders/Journal of lmmunological Methods 174 (1994) 83-93 85 I%. Fig. 2. Macrophage 'suicide'. Liposomes, encapsulating the CI2MBP molecules (squares), are ingested by macrophages via endocytosis. After fusion with lysosomes (L) containing phospholipases (arrowheads), the latter disrupt the bilayers of the liposomes. The more concentric bilayers that are dis- rupted, the greater, the CI2MBP release within the cell. The mechanism by which CI2MBP , EDTA and DTPA affect the metabolism of the cells is currently under investigation. (N = nucleus of the macrophage).

the more of the drug is accumulating in the cell (Fig. 2). The exact mechanism by which the re- suiting high intracellular concentration of CI 2- MBP affects the cellular metabolism has still to be unravelled. It may be based on the depletion of intracellular iron (Van Rooijen, 1993a; M6nkk6nen and Heath, 1993) or on a direct effect on the ATP metabolism in the cell (Peleorgeas et al., 1992; Rogers et al., 1992; Van Rooijen, 1993a). Using various alternative chela- tor molecules and chelator-metal ion complexes we have ruled out the possibility that intracellular calcium is playing a crucial role (Van Rooijen and Poppema, 1992).

4. Preparation of liposomes

Several methods for the preparation of lipo- somes have been described. In our laboratory, a conventional method is used for the production of multilamellar Cl2MBP-liposomes. A detailed description of the various steps in their prepara- tion follows.

(1) A stock solution of phosphatidylcholine (egg lecithin) in chloroform (100 mg/ml) is made. It can be stored at -20C.

(2) 8 mg cholesterol is dissolved in 10 ml chloro- form in a 500 ml round bottom flask.

(3) 0.86 ml of the phosphatidylcholine stock so- lution (containing 86 mg phosphatidyl- choline) is added.

(4) The chloroform phase is removed by low vacuum (gradually reducing from 200 mbar to 150 mbar) rotation (150 rpm) evaporation. At the end a thin milky white phospholipid film is formed against the inner wall of the flask.

(5) The phospholipid film is dispersed in 10 ml of aqueous solution (A or B) by gentle rota- tion (max. 180 rpm) at room temperature (RT), during 10-20 min (solution A) or 5-10 min (solution B). Solution A: for empty lipo- somes, 10 ml of phosphate buffered saline (PBS) is used. Solution B: for C12MBP-lipo- somes, 10 ml of a 0.6 M C12MBP solution (2.5 g C12MBP in 10 ml aqua dest.) is used. (Store at - 20C.)

(6) The milky white suspension is kept at RT for 1.5-2 h under nitrogen (N 2) gas. (N 2 is used to prevent denaturation of phospho- lipid vesicles. This is particularly important in the case of C12MBP-liposomes, since the latter are floating on the aqueous phase after their preparation; PBS liposomes form a pellet on the bottom of the tubes.)

(7) After gentle shaking of the suspension, it is sonicated in a waterbath sonicator for 3 min.

(8) The suspension is kept under N 2 for 2 h at RT (or overnight at 4C) for swelling of the liposomes.

(9) Before using the liposomes which can be stored in the original CI2MBP solution at 4C, the non-encapsulated C12MBP should be removed by centrifugation (10 000 g) of the liposomes. At the end (after about 15 min) the C12MBP-liposomes form a white band at the top of the suspension, whereas the suspension itself will be nearly clear. Since the relatively large C12MBP-liposomes are especially efficacious with respect to de- pletion of macrophages, there is no problem

86 N. Van Rooijen, A. Sanders/Journal of Immunological Methods 174 (1994) 83-93

when the suspension is not completely clear, since the remaining liposomes will be very small.

(10) Using a Pasteur pipette, the CI2MBP solu- tion under the white band of liposomes is removed carefully. In order to prevent spoil- ing of C12MBP (only about 1% will be en- capsulated), the non-encapsulated CI2MBP can be reused after filtration through a 0.45 /zm filter. This recycling procedure should not be repeated for more than five times.

(11) The CI/MBP liposomes are washed 2-3 times using sterilized PBS. (Centrifugation at 25 000 g for 30 min.)

(12) Finally the pellet is resuspended in 4 ml sterilized PBS. The suspension must be shaken gently before administration to ani- mals or before dividing into several portions, in order to warrant a homogeneous distribu- tion of the liposomes over the suspension.

(13) Under N 2 (in sealed tubes) the CleMBP- liposomes can be stored in PBS at 4C for up to 2 weeks. If they are kept in the origi- nal CleMBP solution, they can be stored for longer periods of time. In that case however, before using the liposomes, the procedure should be continued from step 7 (including sonication).

(14) We are despatching Cl2MBP-liposomes to several laboratories for collaborative studies on macrophage functions. These are shipped by Express Mail (Europe) or Federal Ex- press air courier (USA). There is no indica- tion that the macrophage-depleting activity of the C12 MBP-liposomes is affected during transport.

The amount of CI/MBP encapsulated in the liposomes has been determined using methods based on murexide-C12MBP competition for cal- cium (Claassen and Van Rooijen, 1986) and more recently on measurements of 99mTc-labelled CI2MBP (Buiting et al., manuscript in prepara- tion). About 1% of the CI2MBP appears to be encapsulated in the liposomes and the final C12MBP-liposomes suspension (4 ml) contains about 20 mg of C12MBP. For intravenous admin- istration (to deplete splenic macrophages) about 0.1 ml of the suspension is injected per 10 g body

weight. However macrophages in the liver (Kupffer cells) were completely eliminated 24 h after intravenous administration of 0.02 ml of the suspension per 10 g body weight. See the relevant literature for detailed information on the doses of Cl2MDP-liposomes required to deplete different macrophage (sub)populations in various organs.

5. Characteristics of liposomes: influence on in- gestion by macrophages

In our experience, large multilamellar lipo- somes have proved to be more efficient in the elimination of macrophages than their smaller unilamellar counterparts. Such multilamellar li- posomes can be prepared from one single phos- pholipid, e.g., phosphatidylcholine (egg lecithin) and cholesterol or from mixtures of different phospholipids. By adding negatively charged phospholipids such as, e.g., phosphatidic acid, phosphatidylserine or dicethylphosphate or by adding positively charged stearylamine to the phospholipid mixtures, the charge of the resulting liposomes can be influenced correspondingly. Macrophages in general and peritoneal macro- phages in particular, seem to prefer negative lipo- somes (Nishikawa et al., 1990; Fadok et al., 1992). It has been shown that orally administered dis- tearoylphosphatidylcholine liposomes, containing phosphatidylserine, were preferentially taken up by Peyer's patches in the lower ileum. If such liposomes are relatively large (> 374 nm), they might offer a suitable carrier for drugs and anti- gens to the so-called M cells in the Peyer's patches (Aramaki et al., 1993). It should be possible to investigate the ability of such liposomes to de- plete the M cells in order to study their role in gut-associated immune responses and to find out whether they are suitable target cells for antigen containing liposomes in a vaccine. The choles- terol contents of liposomes may influence their distribution over various macrophage populations (Patel, 1992) and the uptake of liposomes by macrophages can be enhanced by incorporation of, e.g., mannose in their bilayers (Gregoriadis, 1992).

Such molecules have to be anchored in the

N. Van Rooijen, A. Sanders/Journal of Immunological Methods 174 (1994) 83-93 87

bi layers by conjugat ion to strongly hydrophobic molecules. The best approach is through conjuga- t ion with the phosphol ip ids themselves (e.g., phosphat idy lethanolamine) . The result ing amphi- pathic conjugates are incorporated in the bilay- ers. A l though monoclonal antibodies, recognizing surface determinants on part icular subsets of macrophages may be exposed on the l iposomal bilayers, such target molecules will not prevent the uptake of the l iposomes by other macrophages (Van Rooi jen et al., 1992). Since l iposomes are not able to cross capi l lary walls and other vascu- lar barr iers, considerable di f ferences in the de- plet ion of various macrophage populat ions can be achieved through the use of di f ferent administra- t ion routes.

6. Access of liposomes to macrophages in various tissues

Res ident macrophages can be found in most organs in the body whereas local inf lammatory react ions may attract macrophages from the cir- culation. In several organs, l iposomes cannot be

targeted to macrophages, because they are not able to cross the vascular barr iers formed by capi l lary walls. In other organs, l iposomes can be targeted to def ined macrophage (sub)populat ions provided that they are given along appropr ia te administrat ion routes (see Table 1). Intravenous administrat ion must be chosen when l iposomes have to be targeted to macrophages in the liver (Kupffer cells) and/or spleen (Van Rooi jen et al., 1990).

Subcutaneous administrat ion permits target ing of the l iposomes to the draining lymph nodes (De lemarre et al., 1990), and intratracheal admin- istration of l iposomes causes their uptake by alve- olar macrophages in the lung (Thepen et al., 1989). Direct local injection of l iposomes in the tissues can be used to inf luence macrophages in organs with a loosely woven structure such as the testes (Bergh et al., 1993). A l though it was ini- tially postu lated that mannosylated l iposomes are able to pass the blood brain barr ier (BBB, Umezawa and Eto, 1988) later studies did not confirm the entry of such l iposomes into brain tissue (Micklus et al., 1992). Nevertheless, man- nosylated C12MBP-l iposomes were able to sup-

Table 1 Depletion of macrophage (sub)populations in different organs

Organ/macrophages i.v. s.c. i.t. loc. i.a. i.p. inc. References

Spleen/three different macrophage * - - - * - subpopulations

Liver/Kupffer cells * . . . . * - Lymph nodes/two different - * . . . . .

subpopulations Lung/alveolar macrophages - - * . . . . Testis - - - * - _ _ Knee joint/phagocytic synovial . . . . * - -

lining cells In vitro/macrophages in cell . . . . . . *

suspensions and macrophage tumor cells

Mouse: Van Rooijen et al., 1984, 1989b Rat: Van Rooijen et al., 1990 Rat: Van Rooijen et al., 1990 Mouse: Delemarre et al., 1990

Mouse: Thepen et al., 1989 Rat: Bergh et al., 1993 Rat: Van Lent et al., 1993, 1994

Splenic macrophages: Claassen et al., 1990; RAW 264 macrophage tumor cells: Van Rooijen et al., 1988

Influence of the route of administration of Cl2MBP-liposomes. Key: i.v. = intravenously into tail vein; s.c. = subcutaneously into draining area of lymph node; i.t. = intratracheally; loc. = locally into testis; i.a. = intraarticularly; i.p. = intraperitoneally; inc. = incubation during culture of cells; * = macrophage depletion.

88 N. Van Rooijen, A. Sanders/Journal of lmmunological Methods 174 (1994) 83-93

press the expression of clinical signs of experi- mental allergic encephalomyelitis (EAE), whereas empty mannosylated liposomes or normal (non- mannosylated) C12MBP-liposomes were ineffec- tive (Huitinga et al., 1990). This effect might be mediated by a mechanism in which the mannosy- lated liposomes and precursors of brain macro- phages (monocytes) are both fixed to the same elements of the BBB (Huitinga et al., 1992). Recently it has been postulated that systemically administered liposomes can cross the BBB when the latter has been altered as a result of the presence of tumor tissue (Gennuso et al., 1993). Liposomal uptake in tumors of the CNS ap- peared to be enhanced after manipulation of the BBB with intracarotid etoposide. The administra- tion routes that have to be chosen to deplete particular subsets of macrophages are shown in Table 1

7. Heterogeneity of macrophages in lymphoid or- gans

Macrophages isolated from various tissues manifest large differences in shape, metabolic and functional activities (see review by Stein and Keshav, 1992) and in the expression of macrophage specific markers (for mice, see re- view by Gordon et al., 1992; for rats, see review by Dijkstra and Damoiseaux, 1993). It has been postulated that macrophage heterogeneity is gen- erated through differentiation-related mecha- nisms and that macrophage phenotypes are then maintained by systemic environmental constraints (Witsell and Schook, 1991). In the spleen, red pulp macrophages (RPM), marginal zone macrophages (MZM), marginal metallophilic macrophages (MMM), white pulp macrophages (WPM) and tingible body macrophages (TBM) all show a clear compartment-restricted localization pattern. They can be found in the red pulp, marginal zone, outer periphery of the white pulp, inner parts of the periarteriolar lymphocyte sheaths (PALS) and follicular germinal centres respectively (Van Rooijen et al., 1989a). Within one day after a single administration of liposome encapsulated clodronate, RPM, MZM and MMM

have been depleted from their compartments. Considerable differences between the repopula- tion kinetics of these three macrophage subpopu- lations were observed in the spleen of mice (Van Rooijen et al., 1989b) and rats (Van Rooijen et al., 1990). Using these differences in repopulation kinetics, functional specialization of the splenic macrophages can be studied (Van Rooijen, 1992b). WPM and TBM may be affected by treat- ment with liposome encapsulated C12MBP, but they are not depleted from their respective com- partments. There are no impermeable vascular barriers between these macrophages and the lipo- somes entering the spleen. However WPM and TBM are not adjacent to the main bloodstream in the spleen and densely packed lymphocytes in PALS and follicles may well prevent the lipo- somes from reaching the macrophages in suffi- cient numbers.

In popliteal lymph nodes, macrophages lining the subcapsular sinus as well as those located in the medulla were eliminated by a single subcuta- neous injection with ClzMBP-liposomes in the footpads (Delemarre et al., 1990). Macrophages in the cortex and TBM in germinal centres were not depleted. Here again the fact that they are not adjacent to the main lymph flow and the fact that lymphocytes are densely packed in the cortex and germinal centres may hinder access of the liposomes to the macrophages.

8. Control experiments

Although investigators generally use PBS or saline liposomes for control experiments, one should be aware that administration of PBS or saline liposomes does not represent the right control experiment in most cases. Compared to an experiment in macrophage depleted animals, the control animals should have normal healthy, non-blocked, non-suppressed and non-activated macrophages. Liposomes, however, in common with most other particulate compounds, may block phagocytosis for certain periods of time (Juliano, 1982; Proffitt et al., 1983; Dave and Patel, 1986). Moreover it is not known whether other macro- phage functions are suppressed or, in contrast,

N. Van Rooijen, A. Sanders/Journal of lmmunological Methods 174 (1994) 83-93 89

are activated and for what duration. As a result, the effects of macrophage depletion may be less than expected, if compared with those in control animals, when the latter have been treated with PBS liposomes. Macrophage function in the lat- ter can be influenced to some extent. To mimic depletion treatments without affecting macro- phages, sham injections of, e.g., NaC1 are suffi- cient. However if there is a chance that the observed effects are not due to macrophage de- pletion, both PBS or saline liposomes and free CI2MBP injections should be incorporated as control groups.

Since multilamellar liposomes are exclusively ingested by phagocytic cells (depending on their phospholipid composition), and CI2MBP, once it has been released in the circulation, has a very short half life and does not cross cell membranes, selective depletion of phagocytic cells by Cl2MBP-liposomes was not surprising. Indeed the approach permitted the selective removal of mononuclear phagocytes from heterogeneous spleen cell populations in vitro. No effect was observed on non-phagocytic spleen cells as mea- sured by growth, protein production, antigen pre- sentation and antigen specific T cell proliferation (Claassen et al., 1990). Dendritic cells (DC) ap- peared not to be removed from spleen tissue after intravenous treatment with CI2MBP-lipo- somes (Van Rooijen et al., 1989b). In addition, the number of DC isolated from animals treated with Cl2MBP-liposomes was similar to that of DC obtained from control animals. Moreover, DC from animals treated with empty (PBS) lipo- somes were equally effective at inducing in vitro primary CTL responses (Nair et al., 1994). An important question concerns the possible deple- tion of neutrophil granulocytes as a consequence of treatment with CI 2 MBP-liposomes. These cells are largely responsible for the phagocytosis of many foreign microorganisms. Neutrophils, how- ever, appeared neither morphologically, nor func- tionally to be affected by C12MBP-liposomes in vivo (Qian et al., 1994). In vitro experiments have confirmed that neutrophils cultured in the pres- ence of Cl2MBP-liposomes were not affected. In contrast, it has been postulated that the neu- trophils in the cultures cause clustering of the

liposomes by a still unknown mechanism (Van Lent et al., 1994). After overnight culture of human neutrophils with Cl2MBP-liposomes (at a concentration comparable to that in the circula- tion of mice after intravenous administration of C12MBP-liposomes) no effects on the neutrophils could be demonstrated (unpublished results). Further studies showed that the failure of C12 MBP-liposomes to affect the neutrophils could be explained by a low level of ingestion of the liposomes. Both DiI labelled Cl2MBP-liposomes and DiI labelled PBS-liposomes were ingested by a very small minority of the neutrophils only (unpublished results). Apart from macrophages themselves, only their direct precursors, the monocytes, appeared to be affected by CI2MBP- liposomes (Huitinga et al., 1992).

9. E l iminat ion versus activation

It is generally assumed that silica can be used to deplete tissues of macrophages in vivo, since phagocytosis is not be observed after treatment with silica. However, it is unwise to assume the abrogation of all macrophage functions, if only this parameter is taken into account. The disap- pearance of any particular characteristic of macrophages (Van Rooijen et al., 1989a,b), such as their internal markers (e.g., acid phosphatase activity and non-specific esterase activity), their external markers (e.g., cell surface antigens that can be demonstrated using macrophage specific monoclonal anti-bodies) or their phagocytosis ca- pability (demonstrated by the uptake of, e.g., carbon particles, latex spheres or labelled lipo- somes) does not imply the abrogation of all macrophage functions such as, for instance, cy- tokine production. To be sure that all functions of a particular macrophage population are really abrogated, the physical depletion of these cells from the tissues should be confirmed, e.g., at an ultrastructural level (Van Rooijen et al., 1985). Silica may well stimulate (non-depleted) macro- phages to produce interleukin-1 (Souvannavong and Adam, 1990) and interleukin-6 (Vreden et al., 1993). This might explain why, in several comparative studies, conflicting or even opposite

90 N. Van Rooijen, A. Sanders/Journal of Immunological Methods 174 (1994) 83-93

effects were observed after treatment with silica and treatment with Cl2MBP-liposomes (Vreden et al., 1993; Qian et al., 1994). However, even if Cl2MBP-liposomes are not efficacious in the elimination of particular macrophages, they might still have an opposite (i.e., activating) effect. In contrast to alveolar macrophages in the lungs of mice, which could be selectively depleted by C12MBP-liposomes (Thepen et al., 1989), alveolar macrophages in the lungs of guinea pigs could be eliminated only partially. Moreover, non-depleted macrophages appeared to be activated. Basal oxy- gen radical production by these alveolar macro- phages, as measured by chemiluminescence, was increased significantly (Folkerts et al., manuscript in preparation). We have also observed an in- creased (3 ) proliferation activity of non-de- pleted white pulp macrophages in the spleen after intravenous treatment with C12MBP- liposomes (Wijffels et al., 1994).

Since (1) liposomes are immunologically inert, (2) free (released) C12MBP does not easily enter cells, and (3) free CI2MBP exhibits a very short half life in both circulation and body fluids, the present approach may be considered extremely specific in affecting phagocytic cells only. How- ever, one should bear in mind possible complica- tions attributable to the dying macrophages themselves. A variety of lysosomal enzymes and cytokines may be released from these cells as soon as the integrity of their cell membranes has been lost. As an example, the released enzyme acid phosphatase could be demonstrated extracel- lularly in splenic follicles (Laman et al., 1990). Most of the released enzymes and cytokines will lose their activity, due to alterations in pH, dilu- tion, or neutralizing molecules in the circulation. To date, we are not aware of any direct effect of such released molecules, and no measurements have been performed in the circulation and body fluids. As a possible indirect consequence of the release of such molecules, the numbers of lym- phocytes and granulocytes may be enhanced for some time (Pinto et al., 1991) although the quan- tity of effector molecules released will be deter- mined by the normal rates of their production, intracellular storage and secretion. Usually, we recommend that the experiments start two days

after treatment with Cl2MBP-liposomes in spite of the fact that macrophages are depleted within one day. Two days after intravenous treat- ment, cell remnants have been shown to be cleared from the spleen (Van Rooijen et al., 1989b) and serum concentrations of various re- leased molecules will have reached normal values (no experimental data available).

9. The macrophage 'suicide' approach in immu- nity

The present approach has been shown to be useful in studies of macrophage functions in vari- ous biomedical disciplines. Here, in an immuno- logical journal, its applications in immunology should be summarized. In a recent review (Van Rooijen, 1992b), the results of several studies were discussed, in which splenic macrophages were shown to play a role in humoral immune responses against intravenously injected particu- late antigens, but not in responses against soluble antigens. We have also compared two different macrophage mediated mechanisms by which lipo- somes may act as immunoadjuvants (Van Rooij- en, 1993b). On the one hand liposomes can be used to target antigens into marginal zone macrophages of the spleen, as a first step in the induction of an antibody response. On the other hand, liposomes can be used to block or deplete the activity of suppressor macrophages as in, for example, the alveolar macrophages in the lung. Liposomes containing CI2MBP, EDTA, DTPA or various calcium- or metal ion-complexes of these chelators have been shown to have the ability to deplete macrophages in vivo (Van Rooijen and Poppema, 1992). For this reason such liposomes should be considered as potential adjuvants, especially when given via the bronchia. Needless to say the same liposomes can be used as carriers of both antigens and drugs, as we (Claassen et al., 1987), and others (Shek et al., 1986) demonstrated several years ago. In this way, the present liposome mediated macrophage 'suicide' approach offers promising perspectives for a clinical application in the construction of

N. Van Rooijen, A. Sanders/Journal of lmmunological Methods 174 (1994) 83-93 91

vaccines, and we intend to investigate this possi- bility. Apart from the effects on humoral immu- nity, it has been shown that depletion of macrophages may impair the induction of cyto- toxic T lymphocyte (CTL) responses by antigens encapsulated in so called 'pH sensitive' liposomes (Huang et al., 1992; Zhou et al., 1992; Nair et al., 1994). These results confirm that macrophages may also have a role in the induction of cell mediated immunity against particulate antigens, in spite of the fact that it is generally assumed that dendritic cells are the crucial antigen pre- senting cells. The role of macrophages in both humoral and cell mediated immune responses is not believed to depend on the presentation of antigen, but on earlier so-called (pre)processing of antigens followed by their transfer to more specialized antigen presenting cells such as den- dritic ceils or B lymphocytes (Van Rooijen, 1992b).

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